doi:10.1006/jmbi.2001.4780 available online at http://www.idealibrary.com on J. Mol. Biol.

(2001) 310, 635±658

The Dimerization Domain of HNF-1a a: Structure and

Plasticity of an Intertwined Four-helix Bundle with
Application to Diabetes Mellitus
Narendra Narayana, Qing-xin Hua and Michael A. Weiss*

Department of Biochemistry
Case Western Reserve
University, 10900 Euclid
Avenue, Cleveland
OH 44106-4935, USA
*Corresponding author
Maturity-onset diabetes mellitus of the young (MODY) is a human gen-
etic syndrome most commonly due to mutations in hepatocyte nuclear
factor-1a (HNF-1a). Here, we describe the crystal structure of the HNF-
1a dimerization domain at 1.7 A Ê resolution and assess its structural plas-
ticity. The crystal's low solvent content (23 %, v/v) leads to tight packing
of peptides in the lattice. Two independent dimers, similar in structure,
are formed in the unit cell by a 2-fold crystallographic symmetry axis.
The dimers de®ne a novel intertwined four-helix bundle (4HB). Each pro-
tomer contains two a-helices separated by a sharp non-canonical turn.
Dimer-related a-helices form anti-parallel coiled-coils, including an
N-terminal ``mini-zipper'' complementary in structure, symmetry and
surface characteristics to transcriptional coactivator dimerization cofactor
of HNF-1 (DCoH). A con¯uence of ten leucine side-chains (®ve per pro-
tomer) forms a hydrophobic core. Isotope-assisted NMR studies demon-
strate that a similar intertwined dimer exists in solution. Comparison of
structures obtained in multiple independent crystal forms indicates that
the mini-zipper is a stable structural element, whereas the C-terminal
a-helix can adopt a broad range of orientations. Segmental alignment of
the mini-zipper (mean pairwise root-mean-square difference (rmsd) in Ca
coordinates of 0.29 A Ê ) is associated with a 2.1 A
Ê mean Ca rmsd displace-
ment of the C-terminal coiled-coil. The greatest C-terminal structural vari-
ation (4.1 AÊ Ca rmsd displacement) is observed in the DCoH-bound
peptide. Diabetes-associated mutations perturb distinct structural features
of the HNF-1a domain. One mutation (L12H) destabilizes the domain
but preserves structural speci®city. Adjoining H12 side-chains in a
native-like dimer are predicted to alter the functional surface of the mini-
zipper involved in DCoH recognition. The other mutation (G20R), by
contrast, leads to a dimeric molten globule, as indicated by its 1H-NMR
features and ¯uorescent binding of 1-anilino-8-naphthalene sulfonate. We
propose that a glycine-speci®c turn con®guration enables speci®c inter-
actions between the mini-zipper and the C-terminal coiled-coil.
# 2001 Academic Press
Keywords: diabetes mellitus; gene regulation; isotope-edited NMR; leucine
zipper; protein structure

Abbreviations used: ANS, 1-anilino-8-naphthalene sulfonate; DCoH, dimerization cofactor of HNF-1; DG, distance
geometry; DM, diabetes mellitus; 4HB, four-helix bundle; HNF-1a, hepatocyte nuclear factor-1a; HNF-p1,
dimerization domain of hepatocyte nuclear factor-1a (residues 1-32); LZ, leucine zipper; MODY, maturity-onset
diabetes mellitus of the young; MPD, methyl-2,4-pentanediol; MSe and Nle designate selenomethionine and
norleucine, respectively (amino acids are otherwise designated by standard single-letter code); NOE, nuclear
Overhauser enhancement; PDB, RCSB Protein Data Bank; SA, simulated annealing.
E-mail address of the corresponding author: weiss@biochemistry.cwru.edu

0022-2836/01/030635±24 $35.00/0 # 2001 Academic Press

636
Introduction
Maturity-onset diabetes mellitus of the young
(MODY) is a monogenic human syndrome
arising from impaired glucose-stimulated
insulin secretion by the pancreatic b cell in the
islet of Langerhans.1 Six MODY genes have
been identi®ed, ®ve of which encode
speci®c transcription factors. The most common
form (MODY3) is caused by heterozygous
mutations in hepatic nuclear factor HNF-1a.2 ± 8
Although HNF-1a is also an important regulator
of hepatic and renal gene expression, the
MODY3 phenotype is restricted to the pancreatic
b-cell{. Homozygous or compound heterozygous
mutations in human HNF-1a have not been
observed and are presumably incompatible with
post-natal life. Homozygous disruption of HNF-
1a in the mouse leads to massive liver failure,
Fanconi's renal dysfunction, and perinatal death.9
In the b-cell, HNF-1a functions as a weak activa-
tor of the insulin gene and a putative cassette of
genes required for regulated insulin secretion.10,11
This HNF-1a-regulated cassette is not well
characterized.
HNF-1a is composed of four functional
regions: an amino-terminal dimerization domain
(residues 1-32); a bipartite DNA-binding motif
with variant homeodomain (residues 98-280);
a carboxy-terminal transactivation domain
(281-631); and a ¯exible linker (residues 33-97)
that connects the dimerization and DNA-binding
domains.12 ± 15 Excepting this linker, MODY
mutations occur in each region.16,17 The present
study focuses on the N-terminal dimerization
domain,18,19 an autonomous module12,14,20,21
required for the protein's gene-regulatory
activity.8 This module also functions as a target
of transcriptional coactivator dimerization
cofactor of HNF-1 (DCoH).22 ± 30 An homologous
leucine-rich motif occurs in HNF-1b (also a
MODY gene)31 and mediates combinatorial
homo- and heterodimerization.32
The structure of the dimerization domain has
been investigated by circular dichroism (CD) and
1
H-NMR spectroscopy. Although initially proposed
to form a leucine zipper (LZ; parallel coiled-coil),
NMR studies have demonstrated the presence of a
helix-turn-helix secondary structure.21 Possible sol-
ution structures based on parallel or anti-parallel
{ Designation of HNF-1a as a hepatic factor re¯ects
the history of its discovery and role in liver-speci®c
gene regulation.15 Heterozygous mutations in the coding
region or promoter of the human HNF-1a gene have no
known effect on hepatic function in contrast to
impairment of b-cell function.10
{ In crystallographic studies of the HNF-p1/DCoH
complex,35 a-helical orientations in the intermolecular
4HB were described as opposite to those predicted.34
This is incorrect: predicted and observed helical
orientations are congruent (see the Supplementary
Material).
An Intertwined Four-helix Bundle in HNF-1
four-helix bundles (4HB) have been proposed.33,34
We have recently suggested that residues 8-19 and
190 -80 comprise an anti-parallel coiled-coil (``mini-
zipper'') along one face of the 4HB and that this
face mediates binding of HNF-1a to the saddle-
shaped coactivator DCoH.34 Recruitment of DCoH
(not itself a DNA-binding protein) into a speci®c
preinitiation complex, although not necessary for
HNF-1a/DNA recognition, enhances by HNF-1a-
directed transcriptional activation.30 Symmetry-
based modeling suggested that the HNF-1a mini-
zipper sits atop the saddle to form an intermolecu-
lar four-helix bundle{.34 Essential features of this
model have been veri®ed recently by X-ray crystal-
lography (Figure 1(a)).35
We describe here the crystal structure of the
dimerization domain of HNF-1a at 1.7 A Ê resol-
ution. Experimental phases were obtained by
multi-wavelength anomalous dispersion using
selenomethionine (MSe) as scatterer. Because the
native amino-terminal methionine residue was
not suf®ciently ordered, MSe was substituted for
one of the leucine side-chains. The structure
reveals an intertwined a-helical dimer anti-paral-
lel in orientation. ``Eisenberg''-like helix
swapping36,37 between protomers leads to a
novel intertwined topology. Because such swap-
ping was unanticipated by previous NMR mod-
els,33,34 we have reinvestigated the structure in
solution. Dimer-related nuclear Overhauser
effects (NOEs), de®ned by asymmetric 13C-isoto-
pic labeling,38 are consistent with the overall
orientation of protomers as observed in the crys-
tal structure. Flexibility among N and C-terminal
segments is observed in solution and rationalizes
patterns of thermal (B) factors. Recent indepen-
dent crystallographic analyses of the HNF-1a
domain by Alber and co-workers39 enable com-
parison of the peptide structure in different crys-
tal forms and lattice environments. Such
comparison suggests that the 4HB contains a
rigid element (the mini-zipper) and an adjustable
element (variably oriented C-terminal a-helices).
These elements are connected by a tight turn
unrelated to classical b- or g-turn families.40,41
Diabetes-associated mutations in this turn and
adjoining N-terminal a-helix2,5,42 destabilize the
4HB by distinct mechanisms. Whereas a destabi-
lizing mutation in the mini-zipper (L12H) pre-
serves the domain's architectural speci®city, a
mutation in the turn (G20R) leads to a molten
globule. We propose that a glycine-speci®c turn
architecture is required for speci®c packing of
side-chains of the C-terminal coiled-coil against
side-chains of the N-terminal mini-zipper. Studies
of the diabetes-associated variants suggest that a
frustrated turn alters the global topography of
the free-energy landscape.
An Intertwined Four-helix Bundle in HNF-1 637

Figure 1. Structure of the dimerization domain of HNF-1a. (a) Ribbon representation of co-crystal structure of
DCoH dimer (blue) complexed with HNF-p1 (red and green) (accession number 1F93).35 The arrow indicates 2-fold
rotation axis. (b) Crystal structure of isolated HNF-p1w dimer determined in this study. Red and green cylinders rep-
resent a-helices 1 and 2 in the two protomers. Each protomer comprises a helix-turn-helix secondary structure with
MODY mutation site G20 (represented as a yellow sphere) within a non-canonical turn. a-Helix 1 (residues 4-19) is
longer than a-helix 2 (residues 23-32). (c) Stereo view of a portion of 2Fo ÿ Fc electron density map calculated using
1.7 AÊ resolution data. The map is contoured at the 1.5s level and superimposed on the re®ned model. MSe denotes
selenomethionine residue.
638
Results
The crystal structure (HNF-1a residues 1-32 with
W33 extension{ and MSe13 substitution{; desig-
nated HNF-p1w) was determined by multiple
anomalous dispersion (MAD) phasing and re®ned
to 1.7 AÊ resolution (Rcrystˆ0.231 and Rfreeˆ0.245;
see Table 1). Electron density (2FoÿFc) correspond-
ing to residues 10-16 is shown in Figure 1(c). The
asymmetric unit contains two independent poly-
peptides and 55 ordered water molecules}. The
polypeptide molecules are not related by the phys-
iological dimer axis (see below). Molecule 1 is bet-
ter ordered than molecule 2; respective mean
thermal B-factors are 15.9 AÊ and 23.0 AÊ 2. The con-
®gurations of non-glycine residues are in the most
preferred region of the Ramachandran plot. Resi-
due G20 in each polypeptide is in the generously
allowed L-region adjoining the aL island. Mol-
ecules 1 and 2 each contain two a-helices con-
nected by a sharp reversal segment, rendering a V
shape to the protomer's overall architecture
(Figure 1(b)). The angle between a-helices is 37 in
each case. Alignment of the two polypeptides by
least-squares superposition of main-chain atoms
{ HNF-p1w differs from the peptide studied by Alber
and colleagues (HNF-p1)35 by the presence of an
additional C-terminal tryptophan-amide (W33; in place
of P33 in native HNF-1a sequence), N-terminal
substitution of methionine by norleucine, and
substitution of L13 by MSe for MAD phasing. W33
appears to stabilize the structure,34 presumably via side-
chain contacts observed in solution and not re¯ected in
the crystal structure (see the Supplementary Material).
Norleucine was employed to avoid methionine
oxidation. Se-Met at position 13 was also employed by
Rose et al. 39 for MAD phasing.
{ Substitution of L13 by MSe has no effect on the
a-helix content of the HNF-1a dimer, as inferred by CD.
However, the variant dimer is less stable;

Gu, as
estimated by CD-detected guanidine titration, is
2.4(0.2) kcal/mol (see the Supplementary Material).
The relative orientation of N and C-terminal a-helices in
the crystal structure of HNF-p1w differs from that seen
in the DCoH co-crystal structure. 35 These differences
are retained in a preliminary electron-density map of
native (L13) HNF-p1w peptide crystals (N.N. and
M.A.W., unpublished results) and so are unrelated to
the MSe13 substitution. Similar results have been
obtained by Rose et al.,39 who provide ®ve sets of
dimer-speci®c coordinates: (i) and (ii) two independent
dimers in the asymmetric unit in wild-type HNF-p1; (iii)
and (iv) two independent dimers of an MSe12 analogue
and (v) one MSe-13 analogue. Of these ®ve dimers, only
the MSe-13 structure exhibits exact 2-fold symmetry in
the dimer. Quantitative comparisons of these and the
present structure are described in Discussion (Figures 7
and 8, and Table 4).
} In deposited coordinates, residues in molecules 1
and 2 are labeled 1-33 and 36-63, respectively. Residues
34, 35, and 64-66 are disordered and hence absent from
the coordinate set. The 55 ordered water molecules are
numbered 67-121. In the text and Figures, primed labels
denote dimer-related residues.
An Intertwined Four-helix Bundle in HNF-1
(residues 3-30) yields a root-mean-square deviation
(rmsd) of 0.24 A Ê.
Because molecule 1 is better ordered than mol-
ecule 2, analysis focuses on molecule 1 unless
otherwise stated. Residues 1-3 form an extended
strand followed by a-helix 1 (residues 4-19), a
sharp turn (20-22), a-helix 2 (residues 23-32) and
W33. No interhelical interaction (<3.5 A Ê ) is
observed in the protomer. Within the asymmetric
unit, molecules 1 and 2 are connected loosely
through a-helices 2 and 20 (interface depicted in
Supplementary Material). The surface area buried
at this interface is only 846 AÊ 2. By contrast, mol-
ecules 1 and 2 each form extensive interactions
with another polypeptide in the unit cell related
by a crystallographic 2-fold symmetry axis
(Figure 1(b)). The surface area buried at this inter-
face is 1741 A Ê 2. The putative symmetry-related
dimer is consistent with prior analyses of NOEs in
solution33,34 and in accord with the co-crystal struc-
ture of a HNF-p1/DCoH complex (Figure 1(a)).35
We therefore regard this symmetry-related packing
as the biological dimer. The orientation between
protomers within dimers 1 and 2 in the unit cell
are essentially identical.
Dimer topology and interactions
between protomers
a-Helices 1 and 2 each form anti-parallel coiled-
coils with dimer-related mates to de®ne opposite
surfaces of the 4HB. Each coiled-coil remains con-
tiguous throughout its length. Helix 1 contacts
helix 20 , whereas helix 10 contacts helix 2. Despite
this novel topology, individual coiled-coils are sep-
arately superimposable on those of a conventional
4HB (see Supplementary Material). As
expected,43,44 the helices in HNF-p1w contain 3.6
residues per turn. Distances of closest approach
between helical axes are 10.9 A Ê (N-terminal coiled-
coil) and 9.3 A Ê (C-terminal coiled-coil). In accord
with classical expectations,45 the angle between
helices 1 and 10 is 30  and that between helices 2
and 20 is 23  . The crossing angle between N and
C-terminal coiled-coils is 26  . Although the overall
structure resembles that observed in a peptide-
DCoH complex35 and peptide dimers in different
crystallographic environments,39 the precise orien-
tation of coiled-coils differs (see Discussion and
Supplementary Material).
The dimer interface is stabilized by extensive
hydrophobic and van der Waal's interactions
between protomers and by symmetry-related
hydrogen bonds between the side-chains Q9 and
S190 (and likewise between Q90 and S19). Unlike
coiled-coils,44,46 there is no salt-bridges between
protomers. The dimer's hydrophobic core contains
a cluster of leucine side-chains. A prominent fea-
ture is the packing of L12 and MSe13 (L13 in the
native sequence) against L120 , MSe130 , L160 , L210 ,
and L260 (Figure 2). Eight leucine and two MSe
side-chains thus contribute to the intertwined inter-
face. The side-chains of L12 and L120 (a site of
An Intertwined Four-helix Bundle in HNF-1 639

Table 1. Statistics for data collection, phasing, and re®nement

A. Data collection and phasing
Space group P21212
Ê)
Unit cell (A aˆ28.42, bˆ42.19, cˆ42.43
Vm (AÊ 3/Da) 1.6 (two protomers/asymmetric unit; 23% solvent content)
Resolution (A Ê) 1.7 (MAD)
Data set (Se scatterer) Inflexion (li) Peak (lp) Remote (lr)
Wavelength (A Ê) 0.9800 0.9794 1.0030
Observed reflections 47,253 46,501 29,713
Unique reflections 5693 5669 5778
Completeness (%) 95.2 94.8 96.6
Average I/sa 26.8 (4.3) 26.1 (4.1) 23.9 (3.7)
Rmerge (%)a,b 3.8 (2.6) 4.0 (2.6) 3.7 (2.4)
Mean figure of merit (FOM)c 0.90

B. Refinement statistics
Resolution range (A Ê) 40-1.7
Rcryst (%) (F > 3sF)d 23.1
Rfree (%) (F > 3sF) 24.5
Reflections (F > 3sF; completeness)
Working set 4949 (83 %)
Test set 296 (5%)
Number of non-H atoms
Protein 458
Solvent 55
Average B-values (A Ê 2)
Protein 19.1
Solvent 28.1
Ramachandran plot (%)
Most favored 98.1
Additionally allowed 1.9
a
Values in parentheses indicate the highest resolution shell.
b
Rmerge ˆ hklijIi(hkl) ÿ hI(hkl)ij/hkliIi(hkl). Friedel-mates were collected at wavelengths li and lp.
c
MAD phases were computed using data between 20 and 2.5 A Ê resolution. FOM ˆ hcos (
a)i, in which
a is the error in phase
angle of a re¯ection.
d
Rcryst ˆ hkljFobs(hkl) ÿ Fcalc (hkl)j/hklFobs(hkl). The structure was re®ned using X-ray data collected at a remote wavelength.

MODY mutation; see Discussion) are contiguous
and contribute to the non-polar surface of the
mini-zipper. The N-terminal portion of helix 1 con-
tacts the symmetry-related turn as the side-chain of
Q9 contacts S190 , G200 , and L210 . The side-chains of
K23, L26, I27, A29, L30, E32, and W33 (and their
symmetry-related mates) mediate interactions
between helix 2 and helix 20 .
At the interface between helices 1 and 20 , key
contacts occur between the side-chains of MSe13
and L17 (helix 1) and L260 (helix 20 ). L17 also con-
tacts A290 and E320 . Preliminary studies of iso-
morphic crystals containing the native L13 peptide
reveal an essentially identical structure at 1.7 A Ê in the present structure is 37  , however, that in the
resolution with ten leucine side-chains in the core.
Of the two molecules in the asymmetric unit, elec-
tron density for W33 is seen only in molecule 1.
Ê ) by
The indole side-chain is surrounded (<4.5 A
residues 29 through 32 and L170 , L210 , S220 , K230 ,
and L260 (see the Supplementary Material) The
main-chain amide nitrogen atom of W33 forms a
hydrogen bond with the carbonyl oxygen atom of
G31 and so provides a C-cap to a-helix 2. Lattice
contacts to W33 in molecule 1 are observed from
symmetry-related (in addition to dimer-related)
polypeptides; these lattice contacts involve residues
1-5.
The intertwined 4HB contains a non-
canonical turn
a-Helix 1 is connected to a-helix 2 by a sharp
turn whose structure differs from that of classical b
or g-turn families.40,41,47 The turn (comprising resi-
dues 19-22) exhibits successive backbone (f,c)
angles of (78.7  , ÿ0.5  ), (97.0  , 23.7  ), (ÿ52.6  ,
136.4  ), and (ÿ85.5  , 156.3  ). Similar torsion
angles occur in diverse crystallographic
environments39 and in the peptide-DCoH com-
plex.35 Whereas the angle between helices 1 and 2
bound peptide is 65  .35 The conformation of the
turn's second residue (G20) lies in the right side of
the Ramachandran plot (i.e. positive f angle near
the aL island). Although G20 is invariant among
mammalian HNF-1a and HNF-1b sequences
(Figure 3), L-amino acids would not be excluded
on the basis of a Ramachandran criterion. Molecu-
lar modelling nonetheless suggests that the b car-
bon atom of an L-amino acid would clash with the
carbonyl oxygen atom of the preceding residue
(S19). The mutation G20R has been observed in a
MODY pedigree42 and causes a profound decre-
ment in the dimer's thermodynamic stability
(

Gu > 4 kcal/mol (1 cal ˆ 4.184 J); Table 2).34
640
Flexible regions and disorder
Electron density is well de®ned for all residues
in molecule 1, whereas in molecule 2 the N-term-
inal two and C-terminal three residues are disor-
dered. Mean main-chain B-factors for a-helix 1 in
molecules 1 and 2 are 12.6 and 16.5 A Ê 2, respect-
ively, and for a-helix 2, 14.1 and 29.2 AÊ 2, respect-
ively. Corresponding side-chain B-values for
a-helix 1 in molecules 1 and 2 are 18.0 and 19.5 A Ê 2,
respectively, and for a-helix 2, 21.8 and 30.5 A2, Ê
respectively. (Residue-speci®c B-factors are pro-
vided as Supplementary Material.) The apparent
¯exibility of residues 1-2 and 31-33, as inferred
from higher thermal values in molecule 1 and
absence of density in molecule 2, is corroborated
by 1H-NMR motional narrowing.34 Whereas the
An Intertwined Four-helix Bundle in HNF-1
Figure 2. Leucine-rich cluster in
HNF-p1w dimer. (a) and (b) Rib-
bon representations (related by
180o about the horizontal axis pas-
sing between L12 and L26, and
L120 and L260 ) of the HNF-p1w
dimer. CPK models of leucine side-
chains that form the leucine-rich
cluster in core. In one protomer,
the ribbon is shown in black and
the leucine residues in red (L12,
MSe13, L16, L21, L26). In the other
protomer, the ribbon is colored
gray and leucine side-chains green
(L120 , MSe130 , L160 , L210 , L260 ).
Each protomer contributes four leu-
cine and one selenomethionine resi-
due. (c) A stereo view of the
leucine-rich cluster shown in (a).
dimer's secondary structure in solution as de®ned
by NOEs comprises a-helices 8-19 and 22-29 (see
the Supplementary Material), the N-terminal
a-helices in the crystal extend from residues 4-19 in
both molecules in the asymmetric unit. This differ-
ence in helical length is unlikely to be due to the
presence of an organic cosolvent (25 % methyl-2,4-
pentane-diol; MPD) under conditions of crystalliza-
tion, as the circular dichroism (CD) spectrum of
the dimer is unaffected by the cosolvent (see the
Supplementary Material).
Previous NMR analyses employed NOE patterns
(such as strong dNN and nested (i,i ‡ 3) daN con-
tacts) to delimit a-helices.21,33,34 It is possible that
such analyses, although assigning stable helical
elements correctly, failed to discern a signi®cant
helical bias among an ensemble of populated
An Intertwined Four-helix Bundle in HNF-1 641

Table 2. Mutant domains: guanidine denaturation studies and NMR chemical shifts
m(kcal molÿ1
Analogue Conc. (mM)
Gu

Gu Cmid Mÿ1) gc
gu
A. Thermodynamic parametersa
wt 5 11.9  0.1 - 4.1  0.07 1.27  0.02 ÿ6.69 5.26  0.1
wt-KEK 5 11.8  0.1 ÿ0.1  0.2 3.9  0.06 1.32  0.02 ÿ6.69 5.10  0.1
L12H 5 8.2  0.2 ÿ3.7  0.3 6.1  0.20 0.25  0.01 ÿ6.69 1.52  0.2
G20R-KEK 5 6.9  0.4 ÿ5.0  0.5 0.4  0.05 0.67  0.08 ÿ6.69 0.24  0.4
G20R-KEK 60 6.8  0.6 ÿ5.1  0.7 2.0  0.30 0.74  0.10 ÿ5.33 1.45  0.6

B. 1H-NMR differences in chemical shift (L12H)b

Residue HN CaH CbH Other
Leu5 0.1
Gln9 0.23
Glu11 0.1 CgH2 0.1
Ala15 ÿ0.17, ÿ0.72
Leu16 0.4 CdH3 0.2
a
Guanidine denaturation data were ®tted according to a two-state model (see Materials and Methods). Rows 1-4 were published
in part by Hua et al.34
Gu indicates the change in free energy on unfolding as corrected to standard conditions (1 M peptide
concentration and 4  C).
Gu ˆ gc ‡
gu, where
gu is obtained by curve ®tting and gc is a correction factor that depends on dimer
concentration and temperature. Cmid is de®ned as the denaturant concentration at which 50 % of the protein is unfolded. The m
value is the slope d(
Gu)/d(M). KEK designates N-terminal extension Lys-Glu-Lys.
b
Chemical-shifts are given relative to 3,3,3-trimethylsilylpropionate (0 ppm) at pH 7.0 and 25  C. Only differences > 0.1 ppm in
magnitude are shown. Positive value present resonances that are up®eld in the native spectrum relative to the spectrum of the L12H
analogue.

N-terminal structures. To investigate this possi-
bility, nascent helical propensity was probed by
two additional and independent NMR parameters:
(i) 1Ha and 13Ca chemical shifts;48 and (ii) confor-
mationally sensitive 3JaN coupling constants.49,50
Whereas residues in the 8-18 and 23-30 segments
exhibit 3JaN values less than 5 Hz in accord with
their helical NOE patterns, 3JaN values of residues
4-6 are 6.7, 6.8 and 6.0 Hz, respectively (3JaN of
residue 7 could not be obtained due to line-broad-
ening). Further, whereas the 1Ha and 13Ca chemical
shift index for residues 7-18 and 23-30 is consistent
with a-helix, index values for residues 4-6 are
inconsistent with helix. These results suggest that
residues 4-6 do not exhibit a strong a-helical bias
in solution. Accordingly, we imagine that the
environment of residues 1-7 in the crystal lattice
favors N-terminal extension of a-helix 1. In the
crystal structure of the DCoH-bound peptide35
a-helix 1 begins at residue 6, intermediate between
the NOE-de®ned a-helix in solution and that
observed in the present structure.
de®ned by contacts between side-chains, L12-L160 ,
MSe13-MSe130 , and L13-L160 . Contiguity between
the C-terminal helices is established by contacts
between K23-L300 , K23-W330 , L26-L260 , L26-L300
and I27-W330 . The intertwined helix 1/20 relation-
ship is veri®ed by contacts between side-chains
MSe13-L260 and L17-W330 . Representative NOE
data and corresponding structural crystal models
are provided in Figure 4. Within interacting side-
chains, the details of which protons are in closest
proximity across the dimer interface can differ
between crystal and solution structure (unpub-
lished results). Dimer-related NOEs inconsistent
with the crystal structure are observed involving
the side-chains of L5, L8, and W33, presumably
due to ¯exibility in solution (see the Supplemen-
tary Material). Similarly, W33-speci®c ring-current
shifts predicted on the basis of the crystal structure
of molecule 1 are not observed in solution (see the
Supplementary Material) in accord with this resi-
due's apparent disorder in molecule 2.

Contrasting effects of clinical mutations

The solution structure resembles the
crystal structure
To investigate the relationship between the top-
ology of the dimer in the crystal and in solution, a
uniformly 13C-labeled peptide was prepared in
Escherichia coli and mixed with the unlabeled pep-
tide to obtain an equilibrium between isotopic
homodimers and heterodimers. NOEs were then
identi®ed between 13C-attached protons and
12
C-attached protons at the dimer interface. Dimer-
related NOEs diagnostic of an intertwined top-
ology are in overall accord with the crystal struc-
ture (Table 3). The phase of the mini-zipper is
Previous studies have established that mutations
L12H and G20R permit native or near-native helix
content (as inferred from CD) but perturb the stab-
ility of the dimer (Table 2).34 Thermodynamic par-
ameters derived from guanidine denaturation
studies by the two-state formalism suggest that the
G20R mutation causes a larger decrement in stab-
ility (

Gu ÿ 5.0 kcal/mol) than does L12H
(

Gu ÿ3.7 kcal/mol). The structural basis of this
difference is enigmatic, as the L12H substitution in
the dimer's non-polar interface would seem to be
less conservative. Because this conclusion depends
on the appropriateness of the two-state model, CD
642 An Intertwined Four-helix Bundle in HNF-1

Figure 3. (a) Sequence alignment of HNF-1a and HNF-1b and (b) models showing proposed volume compensation.
(a) Peptide sequences of HNF-1a dimerization domain (1-32) available at the SWISS-PROT annotated protein
sequence data base release 39.9 of 22-Nov-2000 (http://www.expasy.ch/sprot/sprot-top.html)84 are aligned. Invariant
and diabetes-related mutational sites L12 and G20 are shown in red. I27L polymorphism63 is indicated by letter L (in
green). In the lower part of (a), peptide sequences of HNF-1b dimerization domain (1-32) are aligned. Invariant resi-
dues L12 and G20 are in red. An asterisk (*) at position 18 indicates a difference in the type of amino acid relative to
HNF-1a. Valine residues are found at positions 21 and 25 in HNF-1b compared to L21 and A25 in HNF-1a (T25 in
chicken). These valine residues, proposed volume-®lling substitutions in HNF-1b, are bracketed. (b) The left
®gure represents a CPK model of a putative G21-G25 crevice obtained by modifying residues at position 21 and 25
in HNF-p1w protomer. The stereo view of the same protomer without modi®cation displays the native volume-®lling
side-chains of L21 (red) and A25 (pink) in HNF-p1w protomer.

spectra were obtained as a function of peptide con-
centratrion (in the range 5-75 mM) as an explicit
probe of the stability of the dimer. The spectrum of
the native dimer is invariant in this concentration
range in accord with its low dissociation constant
(of the order of 1 nM; a precise estimate requires
the assumption of no residual structure in the
monomer). By contrast, the inferred values of
Gu
predict that the spectrum of the G20R analogue
would exhibit marked concentration-dependent
changes in this range, whereas the L12H analogue
would exhibit slight but discernible changes. This
is indeed observed (Figure 5). These data thus cor-
roborate the order-of-magnitude estimates of

G
provided by the two-state model. Nevertheless, the
original CD titration studies by Hua et al.34 were
conducted at a peptide concentration of 5 mM, at
which the G20R analogue (unlike native and L12H
dimers) is substantially dissociated. To address the
possibility that the unfolding curve and apparent

Gu value (which presumably re¯ected only the
fraction of dimer) were unreliable at this concen-
tration, the guanidine titration was repeated at a
peptide concentration of 60 mM to ensure a predo-
minance of dimers. Although the unfolding curve
is shifted to more negative (native-like) values of
ellipticity (Figure 5), derived thermodynamic par-
ameters are essentially unchanged (Table 2). The
statistical r2-value is 0.9996.
Inspection of the ®t between the two-state theor-
etical curve and the G20R titration data points at
60 mM reveals that systemic deviations occur
An Intertwined Four-helix Bundle in HNF-1 643

Table 3. Dimer-related NOEs and distances diagnostic of intertwined topology

Proton(s) Proton(s) Ê)
Distance across dimer (A Ê)
Distance in protomer (A
A. Phase of helix 1/helix 10 mini-zipper
L12d2-CH3 L160 d2- CH3 3.4 7.4
L13d1-CH3 L130 d1-CH3 6.2 -
L13d1-CH3 L160 d1-CH3 5.3 5.4

B. Intertwined helix 1/helix 20 relationshipsa

L13d1-CH3 L260 d2-CH3 5.3 4.6
L17d2-CH3 W330 C4Hb 5.5 14.9

C. Phase of helix 2/20 contact

L26d2-CH3 L260 d2-CH3 3.92 -
Distances shown represent carbon-carbon distances in crystal structure. Corresponding hydrogen-hydrogen distances were
estimated using the Biopolymer facility of InsightII (Molecular Simulations, Inc.).
a
Dimer-related NOEs are observed between W330 C4H and a set of protons in helix 1 (T10 gCH3, L13 dCH3 and A14 bH3) that
are inconsistent with the present structure (see the Supplementary Material) but would be consistent with the bound conformation35
if extended to W33 by model building. A dimer-related NOE is also observed between L5 dCH3 and L210 dCH3. This contact,
involving the ¯exible N-terminal segment, is inconsistent with the present structure and that described by Rose et al.35 The methyl
resonances of L5 exhibit anomalous broadening rather than motional narrowing (as exhibited by residues 1-3), presumably due to
millisecond exchange among multiple conformations.
b
Dimer-related NOEs are observed between L17 d2-CH3 and W330 C4H, which were classi®ed correctly in the side-by-side model
and are in accord with the present structure.

despite the reassuring r2 value (i.e. close to unity).
Points fall beneath the line at guanidine concen-
trations 0-1 M, above the line at guanidine concen-
trations 1-3.5 M, beneath the line from 3.5-5 M,
and above the line from 5-7 M (see the Supplemen-
tary Material). Such systematic deviations, not
observed in ®tting the native and L12H transitions,
are distinct from random scatter and suggest that
the transition is not two-state. To investigate poss-
ible origins of the G20R analogue's anomalous
titration behavior, 1H-NMR spectra were obtained
(Figure 5(b)). Whereas native and L12H samples
give rise to a spectrum typical of a globular
domain (with sharp and well-resolved resonances;
spectra (b)a and (b)b, respectively), the G20R
dimer gives rise to an anomalous spectrum charac-
terized by limited chemical-shift dispersion and
differential line broadening (spectra (b)d and (b)e).
Sharp 1H-NMR resonances from the ¯exible N ter-
minus region are retained. The extent of line broad-
ening is not signi®cantly affected by peptide
concentration in the range 0.2-1.5 mM, suggesting
that broadening is due to conformational exchange
that is intermediate on the millisecond time-scale
of NMR chemical shifts. Gel-®ltration chromatog-
raphy at a peptide concentration of 0.5 mM under
NMR conditions demonstrates that the G20R pep-
tide is predominantly dimeric; no higher-order
aggregation is observed. Line-broadening and lim-
ited dispersion preclude conventional 2D-NMR
analysis (see the Supplementary Material). The
extensive perturbations manifest in the NMR spec-
trum of the G20R analogue contrast with the
{ ANS, a hydrophobic organic dye,86 exhibits little
binding to native globular domains but can enter the
¯uid non-polar interior of a molten globule.87 Such
binding shields the dye from solvent quenching and so
gives rise to enhanced ANS ¯uorescence.
domain's near-native helix content as probed by
CD.
The anomalous NMR features of the G20R
domain suggest that the variant dimer is an a-heli-
cal molten globule. Because G20 adopts a positive
f angle in the crystal structures, we imagine that
substitution by Arg could lead to frustration of the
turn and could, in turn, disallow the native interdi-
gitation of side-chains between N and C-terminal
coiled-coils. Interconversion of the G20R structure
among a range of non-optimal helix-packing
schemes would rationalize both the analogue's
non-two-state unfolding transition and anomalous
NMR features. To test this model, we employed
1-anilino-8-naphthalene sulfonate (ANS) as a probe
of the molten-globule state{. The ANS ¯uorescence
emission spectra of the native and L12H domains
are similar in magnitude to that of a control globu-
lar protein (hen egg-white lysozyme). By contrast,
enhanced ANS ¯uorescence emission is observed
in the presence of the G20R domain at a peptide
concentration of 100 mM. This enhancement is
dimer-speci®c, as its extent diminishes as the pep-
tide concentration is reduced, indicating, as
expected, little or no binding of the dye to the
monomer.
The quality of the NMR spectrum of the L12H
dimer, by contrast to that of the G20R dimer, per-
mits conventional sequential assignment (see the
Supplementary Material). The pattern of chemical
shifts is similar to that of the native spectrum.
Only ®ve residues (5, 9, 11, 15 and 16) exhibit
changes in chemical shift greater than 0.1 ppm in
magnitude (Table 2(b)), The largest change occurs
at the Ha position of A15 (0.72 ppm) and may
re¯ect the net ring currents of H12 and H120 . Small
perturbations are observed at neighboring protons
at positions 11 (0.1 ppm change in a g-methylene
resonance) and 16 (0.2 ppm change in a methyl res-
onance related to L12 by (i,i ‡ 4) and dimer-related
644 An Intertwined Four-helix Bundle in HNF-1

Figure 4. The dimer's intertwined topology is retained in solution. (a) and (b) Molecular models and (c) and (d)
corresponding NOE spectra. (a) Anti-parallel contacts in mini-zipper. Stereo view of stick (gray) representation of
N-terminal helices (residues 5-19 and 50 -190 ) in the HNF-p1w dimer. Hydrogen bonding interactions between Q9 and
S190 , and Q90 and S19 are shown by broken lines. The side-chains of Q9 and Q90 are colored in red, S19 and S190 in
green. The asterisk (*) represents the crystallographic 2-fold axis perpendicular to the plane of paper. The methyl
groups of MSe13 and MSe130 are shown as spheres. Residues L12 and L120 are near the 2-fold axis. Corresponding
dimer-related NOEs are shown in (c). (b) Dimer-related contacts re¯ecting intertwined topology. A signature of the
intertwined dimer is provided by key contacts between MSe13-L260 and L17-W330 . Stereo view of ribbon (gray) rep-
resentation of HNF-p1w protomer. The side-chains of L21, L26 and W33 are shown in gray, cyan and gray colors,
respectively. Residues 60 through 190 are shown as green sticks. The methyl groups in L21, L26, W33, MSe130 , L170
and amino group of Q9 are shown as spheres. (c) The 2D NOESY spectrum in H2O contains the following cross-
peaks: (a) Q9-He1-L210 -Ha, (b) Q9-He1-S190 -Hb, (c) Q9-He1-L210 -Hg, (d) Q9-He1-L210 -Hb1, (e) Q9-He1-L210 -Hb2, (f) W33-
H5-T100 -Ha, (g) W33-H6-T100 -Ha, (h) W33-H6-A140 -Hb, (i) W33-H7-A140 -Hb, (j) W33-H5-K230 -Hg and/or T100 -Hg, (k)
W33-H6-K230 -Hg and/or T100 -Hg, (l) W33-H7-K230 -Hg and/or T100 -Hg; (a0 ) Q9-He2-S190 -Ha, (b0 ) Q9-He2-S190 -Hb1, (c0 )
Q9-He2-L210 -Hg, (e0 ) Q9-He2-L210 -Hb2. Arrow indicates NOE between L21-NH and L17-Ha. (d) The 2D projection of
13
C intermolecular NOE 3D spectrum of 13C-HNF-p1w and unlabeled peptide dimer in 2H2O. The spectrum contains
the following inter-subunit NOEs: (m) L13-Hd2-L260 -Hd2, (n) L13-Hd2-L160 -Hd2, (o) L17-Hd2-L300 -Hd2, (p) L30-Hd2-L170 -
Hd2, (q) L30-Hd2-L130 -Hd2, (r) L16-Hd1-L130 -Hd2, (s) L26-Hd2-L130 -Hd2, (t) ambiguous, I27-H1-I270 -H1 and/or L12-Hd1-
L120 -Hd1, (u) L26-Hd-L260 -Hd2 and possibly I27-Hd-I270 -Hd at up®eld edge, (m0 ) L13-Hd1-L260 -Hd2, (n0 ) L13-Hd1-L160 -
Hd2, (o0 ) L17-Hd1-L300 -Hd2, (r0 ) L16-Hd1-L130 -Hd1 and (s0 ) L26-Hd-L130 -Hd1.

contacts). A similar correspondence is observed in
comparison of NOESY spectra: diagonal plots of
inter-residue contacts are essentially identical
(Figure 6). Although isotope-directed NMR studies
of the L12H dimer have not been performed, the
overall similarities between chemical shifts and
NOEs suggests strongly that the variant side-chain
is accommodated within a native-like fold. Surpris-
ingly, the pK51
a of the H12 imidazole ring (7.2) is
not shifted to low values, indicating that the
variant structure is compatible with protonation of
the imidazole side-chains.
An Intertwined Four-helix Bundle in HNF-1 645

Figure 5. Diabetes-associated G20R mutation induces a molten-globule state. (a) CD and ¯uorescence studies of
peptide analogues. ((a)a and (a)b) CD studies of G20R and L12H domains at successive peptide concentrations:
75 mM (®lled circles), 50 mM (open circles), 25 mM (®lled triangles), 15 mM (®lled squares), and 5 mM (open triangles).
Explicit dissociation of the G20R dimer at this concentration range corroborates the estimate of its thermodynamic
destabilization (

Gu) by two-state modelling of denaturant-induced unfolding. (a)c Denaturant-induced unfolding
studies of the G20R domain at high and low peptide concentrations (60 and 5 mM) in each case demonstrate non-sig-
moidal transitions and yield similar estimates of
Gu. The unfolding curve of the L12H analogue by contrast retains
a sigmoidal character and exhibits a less marked
gu, in accord with the concentration-dependence of its CD spec-
trum. (a)d ANS ¯uorescence studies of HNF-1 peptide analogues. Binding of the hydrophobic dye to the G20R ana-
logue is indicated by enhanced ANS ¯ourescence quantum yield, whereas residual ANS ¯uorescence in the presence
of the native and L12H domains is similar to that observed in the presence of a control globular protein (hen egg-
white lysozyme). (b) 1D 1H-NMR spectra of the HNF-1 peptide and analogues: a, native HNF-p1W; b, L12H ana-
logue of HNF-p1W; c, KEK-extended HNF-p1W; d, G20R analogue of KEK-extended HNF-p1W at 1 mM peptide
concentration; and e, G20R analogue of KEK-extended HNF-p1W at 0.2 mM peptide concentration. The attenuation
of chemical-shift dispersion and differential resonance broadening in the G20R dimer provides evidence of a molten-
globule state.
646
Figure 6. Diagonal plots of (a) inter-residue NOEs in
native dimer and (b) L12H variant dimer demonstrate
structrural similarity. Contacts between side-chains are
shown in lower right-hand triangles; contacts between
main-chain atoms or between main-chain and side-chain
atoms are shown in upper left-hand triangles. Red boxes
in (a) indicate dimer-related NOEs, including those
given in Table 2B; green boxes in (a) indicate contacts
likely to include both intraprotomeric and dimer-related
contributions. Blue boxes in (b) highlight contacts by
mutant H12 side-chain. No distinction is made in (b)
between intraprotomeric and dimer-related contacts as
isotope-edited spectra were not obtained. Contacts a-e
in (a) are present in native spectrum but not in that of
L12H variant, whereas contact a in (b) is present in the
variant spectrum but not in the native spectrum. Assign-
ments are: a, 30-Ha/12-CdH3; b, 14- Ha/23-Hb2; c, 23-
Ha/16- CdH3; d, 17- HN/24- Cb; e, 23- Ha/17- Cd1, a, 3-
Hb/29- Cb. (Corresponding distances in crystal struc-
tures are given in the Supplementary Material.) Open
squares indicate possible contacts rendered ambiguous
by resonance overlap. An asterisk in (a) indicates
dimer-related NOEs inconsistant with crystal structure
(molecule 1)
An Intertwined Four-helix Bundle in HNF-1
Discussion
4HBs are ubiquitous structural motifs exhibiting
a rich diversity of tertiary structures.52,53 Such
motifs provide favorable models for studies of pro-
tein folding and design.54,55 Parallel dimeric 4HBs
are of particular importance in transcription factors
containing a helix-loop-helix DNA-binding
domain.56 ± 58 To our knowledge, the anti-parallel
HNF-1a domain represents the smallest 4HB motif,
a novel intertwined dimer. Mutations in this
domain are associated with diabetes mellitus in
humans.16,42,59,60
Comparison of HNF-p1 peptides in different
crystal forms
Crystal structures of related HNF-p1 peptides
(see footnote to page 638) were described recently
in independent studies by Alber and co-workers.39
While the manuscript was in review, multiple coor-
dinate sets were deposited in the RCSB Protein
Data Bank, permitting detailed comparison of
structures. The coordinates were of the native
HNF-p1 peptide and two Se-Met analogues at pos-
itions 12 and 13. Crystallization conditions and
crystal forms in each case differ from those
described here (a summary of crystallographic
information is given in the Supplementary
Material). The availability of multiple independent
structures provides an opportunity to evaluate the
conformational repertoire of the peptide structure
and effects of crystal packing.
Comparison of the present structure with those
described by Rose et al.39 was performed by least-
squares superposition of equivalent atoms. Two
alignment schemes were investigated. The ®rst
employed an overall alignment of all main-chain
atoms in the dimer. Residue-speci®c mean rmsd
values for main-chain and side-chain atoms are
shown in Figure 7(a) and (b). Whereas low main-
chain rmsd values are observed for two structures
(wild-type dimer bd and MSe-13 dimer; superim-
posed ®lled green and blue triangles, respectively),
large values are observed throughout the sequence
for one MSe12 dimer (designated ac; open red dia-
monds). For the other wild-type dimer (designated
ac; open green triangles) and the other MSe12
dimer (designated bd; ®lled red diamonds), low
rmsd values are observed in the mini-zipper and
larger values in the C-terminal a-helices. In each
case, side-chain rmsd values are lower in the
interior than among solvent-exposed residues.
Structural variability among C-terminal segments
can give rise to extremely large side-chain rmsd
values (3-8 A Ê ). Of the ®ve independent dimers
described by Rose et al.,39 the two most similar to
that obtained here are the wild-type bd dimer and
MSe-13 dimer (mean main-chain rmsd of 0.25 A Ê
and 0.24 AÊ , respectively, for residues 5-28 and 50 -
280 ).
The overall main-chain alignment described
above suggests an asymmetry in degree of struc-
An Intertwined Four-helix Bundle in HNF-1 647

Figure 7. Comparison of HNF-p1 peptide structures by rmsd in two alignment schemes. (a) and (b) Alignment
according to the main-chain atoms including all four helices in the dimer (residues 5-28 and symmetry-related mates)
suggests the extent of overall structural variation. (c) and (d) Alignment according to the main-chain atoms of mini-
zipper only (residues 5-19 and symmetry-related mates) highlights the segmental mobility of the C-terminal coiled-
coil and invariance of N-terminal mini-zipper. Five peptide dimers determined by Rose et al.39 are compared to mol-
ecule 1 of the present study. Abbreviations used for peptide dimers are the same as in Table 4, Wt-ac (open green tri-
angles), wt-bd (®lled green triangles), MSe12-ac (open red diamonds), MSe12-bd (®lled red diamonds), and MSe-13
(®lled blue triangles). Main-chain rmsd values are shown in (a) and (c), whereas side-chain rmsd values are shown in
(b) and (d). In each panel, positions of the four helices are indicated by ®lled or open bars at top. Because HNF-p1
dimers except MSe-13 lack exact 2-fold symmetry39 (unlike the present crystal form), rmsd values for residues 1-32
and 10 -320 are depicted separately. Values for residues whose positions were not de®ned in the electron density in the
corresponding structure are omitted.

tural similarity or difference: larger rmsd values
tend to occur in the C-terminal a-helix than in the
N-terminal a-helix. To explore this apparent asym-
metry further, we aligned the structures according
to Ca atoms in (a) the N-terminal a-helix of an iso-
lated protomer (residues 9-19) and (b) the mini-zip-
per (residues 9-19 and 90 -190 ). These alignments are
illutrated in Figure 8. The protomer-speci®c align-
ment (Figure 8(a)) demonstrates close superposi-
tion of helix 1 with variable orientation of helix 2.
Converse protomer-speci®c alignment on helix 2
(not shown) likewise results in skew positions of
helix 1. Dimer-speci®c alignment on the mini-zip-
per (Figures 7(c) and 8(b)) reveals invariance of the
mini-zipper and variable positioning of the C-term-
inal coiled-coil. The converse alignment on the
C-terminal coiled-coil (residues 23-28 and 230 -280 )
by contrast fails to yield a consistent view of the
aligned helices (not shown). We therefore regard
the mini-zipper alignment as providing physical
648
insight into the modular composition of the
domain. Residue-speci®c rmsd values in this align-
ment are shown in Figure 7(c) and (d). An asym-
metry in rmsd values between N and C-terminal
helices is consistent and striking. An overview is
provided by mean rmsd values for Ca atoms by
segment (Table 4A); corresponding interhelical
angles in HNF-p1 peptides are given in Table 4B.
Whereas the angle between mini-zipper helices is
nearly invariant, large variations are observed in
all other inter-helical angles: the angle between N
and C-terminal helices in a protomer, angle
between C-terminal helices, and crossing-angle
between mini-zipper and C-terminal coiled-coil.
The variable tertiary structure of the C-terminal
helices is reminiscent of a molten globule, a col-
lapsed state containing ordered elements of sec-
ondary structure of variable orientation.61 The
An Intertwined Four-helix Bundle in HNF-1
Figure 8. Least-squares superpo-
sition of HNF-p1 peptides. (a) Indi-
vidual protomers of HNF-p1
dimers35 were aligned with equiv-
alent Ca atoms of residues 9-19 of
the present HNF-p1w protomer
shown in red. The wild-type proto-
mers are in cyan (PDB ID: 1g39),
MSe12 protomers are in green
(PDB ID: 1g2y), the MSe-13 proto-
mer is in blue (PDB ID: 1g2z). Only
Ca atoms are displayed in (a)-(c).
The rmsd of superposition ranges
between 0.1 and 0.25 A Ê . (b) The
HNF-p1 dimers from Rose et al.35,39
were aligned with equivalent Ca
atoms of the mini-zipper segment
(residues 9-19 and 90 -190 ). The col-
oring scheme is the same as in (a).
The HNF-p1 dimer in DCoH-HNF-
p1 complex (PDB ID 1f93) is shown
in black. There is an excellent
agreement in each case with rmsd
of superposition less than 0.4 A Ê
indicating rigid mini-zipper.
(c) Same superposition as in (b) but
a different view from that seen in
(b) showing the variable helices 2.
The protomers are colored as in
(b). For clarity, only the C-terminal
helices 2 of different dimers are
shown except for the reference
HNF-p1 dimer (red), where all its
Ca atoms are shown.
precision of packing of the mini-zipper is, by con-
trast, characteristic of a native state.
The HNF-1a dimerization domain provides a
recognition surface for binding transcriptional
coactivator DCoH.30 Differences between structures
of free and bound peptides suggest that the HNF-
1a dimer undergoes a limited but detectable
change in conformation upon binding to DCoH.
In the absence of additional co-crystal structures,
it is not known whether the distinctive structural
features of the bound HNF-p1 dimer are a general
consequence of DCoH binding or simply one of
several possible bound conformations. The extent
of induced ®t is thus an unresolved issue.
Together, observations of higher thermal factors
for helix-2 and different relative orientations
between helix 2 and 20 in isolated and bound struc-
tures suggest that helix 2 is adjustable by rigid-
An Intertwined Four-helix Bundle in HNF-1 649

Table 4. Least-squares superposition and interhelical angles in HNF-p1 peptides

Residues MSe13* Wt-ac Wt-bd MSe12-ac MSe12-bd MSe-13 DCoH-HNF
A. rmsd (AÊ) of least-squares C superposition of mini-zipper
a a

9-19 90 -190 0.20 0.21 0.27 0.30 0.35 0.21 0.37

23-28 230 -280 0.27 2.40 0.18 3.30 2.50 0.29 4.10
B. Interhelical angles in HNF-p1 peptidesb
Angles (deg.) MSe13 Wt-ac Wt-bd MSe12-ac MSe12-bd MSe-13 DCoH-HNF
y1 37 39 37 42 43 37 65
y2 30 28 29 28 26 30 22
y3 23 12 16 3 20 18 9
y4 ÿ26 ÿ42 ÿ29 ÿ41 ÿ38 ÿ28 ÿ63
a
rmsd is the root-mean-square standard deviation calculated with reference to the better-ordered molecule (MSe13) in the present
structure. MSe13* is the less-ordered dimer in the present study. Wt-ac and Wt-bd represent wild-type dimers of subunits A and C,
and B and D, respectively (PDB ID: 1g39). MSe12-ac (subunits A and C) and MSe12-bd (subunits B and D) are HNF-p1 dimers with
Se-Met at position 12 (PDB ID: 1g2y). MSe-13 (HNF-p1 with Se-Met at position 13) dimer consists of subunits A and its 2-fold
related molecule (PDB ID: 1g2z). DCoH-HNF (subunits E and F of HNF-p1 peptide; PDB ID: 1f93).
b
y1 is the angle between helix 1 and helix 2 in the protomer. y2 is the interhelical angle between the mini-zipper helices. y3 is the
interhelical angle between interprotomer C-terminal helices 2 and 20 . y4 is the crossing angle between the C-terminal and N-terminal
helices.

body movement. We propose that helix 2 can
adopt different relative orientations without per-
turbing helix 1 and 10 (Figures 7 and 8), and is in
agreement with the recent crystal structures of
HNF-p1 peptides in different crystal forms.39
Relationship of structure in crystal and
in solution
The crystal structure of HNF-p1w differs from
either parallel or anti-parallel 4HB models pro-
posed on the basis of NMR studies.33,34 The anti-
parallel model successfully predicted the existence
of an N-terminal coiled-coil (mini-zipper) in accord
with dimer-related NOEs (9-190 and 9-210 ;
Figure 4(a) and (c)). Nevertheless, the putative 4HB
was envisaged as consisting of side-by-side proto-
mers rather than intertwined a-helices.34 A major
role in the NMR analysis was played by prominent
long-range NOEs involving the indole resonances
of W33 (Supplementary Material in Hua et al.).34
Although contacts between W33 and L170 were
correctly interpreted in the original analyses, a
side-by-side model was suggested by multiple
additional NOEs from the indole ring to the side-
chains of residues 10, 13, and 14. These are incon-
sistent with the crystal structure (see the Sup-
plementary Material) but presumably arise due to
¯exibility in solution leading to multiple confor-
mations. The criterion of consistency employed in
the original distance-geometry (DG) modelling
procedure34 did not consider possible confounding
effects of multiple W33 conformations. Analysis of
inferred interproton distances in the crystallo-
graphic dimer emphasizes the importance of NOEs
between degenerate (symmetry-related) 1H-NMR
resonances, unobservable in the absence of isotopic
labeling. Asymmetric 13C labeling allows such con-
tacts to be de®ned. Key examples are provided by
strong NOEs between methyl resonances of
MSe13-MSe130 and L26-L260 . These contacts, incon-
sistent with a side-by-side model, de®ne the phase
of the helix 1/10 and 2/20 contacts. A detailed
description of the dimer0 s solution structure and
dynamics is in preparation. It is important to note
that the confounding features of W33 in the orig-
inal NMR analysis did not arise due to inclusion of
a non-native C-terminal residue with aberrant
properties. Rather, the anomalous NMR behavior
of W33 provides a faithful re¯ection of the anoma-
lous molten character of the C-terminal coiled-coil
as revealed by comparison of multiple crystal
structures (see Figures 7 and 8, and above discus-
sion). It will be of future interest to investigate
which crystal form or subset of crystal forms best
represents the ensemble of structures in solution.
That 33 unique NMR spin systems are observed
indicates that interconversion among structural
substates is fast on the time-scale of chemical shifts
(<1 ms).
Symmetry-based modelling retrodicts
peptide-DCoH complex
Rigid-body docking of the HNF-p1w structure
and transcriptional coactivator DCoH provides an
opportunity to evaluate the strengths and limi-
tations of symmetry-based modelling.34 The recent
availability of a co-crystal structure35 enables
evaluation of previous predictions and retrodic-
tions based on the present structure. We ®rst dis-
cuss overall features of the HNF-1a/DCoH
complex and then molecular details of the inter-
face. As predicted,34 HNF-1a/DCoH recognition is
mediated by formation of an intermolecular 4HB
employing the HNF-1a mini-zipper and convex
(``top'') surface of the DCoH saddle (Figure 1(a))35
(see also the Supplementary Material). Such bind-
ing utilizes corresponding symmetry axes. The
HNF-1a dimer has exact 2-fold symmetry, whereas
the DCoH tetramer is a dimer of saddle-shaped
dimers with a 4HB at its interface; each protomer
contributes one helix to the 4HB with approximate
dihedral (222) symmetry. The interface superim-
650 An Intertwined Four-helix Bundle in HNF-1

Figure 9. Modeling of HNF-p1/DCoH complex. (a) Anti-parallel N-terminal helices 1 and 10 in HNF-p1 dimer are
shown as red ribbons. The rest of the HNF-p1 molecule is omitted for clarity. DCoH dimer (subunits A and B from
PDB code 1DCO) is shown as green ribbon. The 2-fold symmetry axes of DCoH dimer and the HNF-p1 dimer are
aligned. This results in the formation of a 4HB at the interface of DCoH and HNF-p1. The crossing angle between the
HNF-p1 helices and the DCoH helices is ÿ40  compared to ÿ27  in the co-crystal structure.35 (b) A diagram showing
relative orientaions of helices in the 4HB formed in the DCoH tetramer and DCoH/HNF-p1 complex. The left dia-
gram shows the dihedral symmetry of helical orientations in the DCoH tetramer. The right diagram shows the pre-
dicted 2-fold symmetry of the orientations of helices at the interface of the DCoH dimer and the HNF-p1 dimer,34
veri®ed in the DCoH/HNF-p1 crystal structure.35 Although the helical directions seen in the DCoH tetramer can
occur in the DCoH/HNF-p1 complex, it is predicted not to be favorable due to steric clashes and smaller buried sur-
face area upon dimerization (see the text). (c) The modeled interactions between the L12 residue (red) in HNF-p1 and
residues F47 (green) and T51 (violet) in DCoH are displayed by a dotted van der Waal's surface. Thick and thin rib-
bons correspond to DCoH and HNF-p1 helices, respectively. The stereo diagram depicts the ®t of L12 and L120 into
the hole formed by F47, T51 and their symmetry-related residues (F470 and T510 ).

poses these symmetry axes. Our previous docking
exercise, although employing a side-by-side model
of the anti-parallel HNF-1a 4HB, nonetheless cap-
tured essential features of the intermolecular 4HB
interface (see the Supplementary Material). In light
of the present crystal structure, we revisit the
docking problem to consider how reliably would
symmetry-based docking predict the actual
structure of the complex.35
Alignment of symmetry axes
The importance of symmetry in HNF-1a/DCoH
recognition has long been recognized. A previous
model of the HNF-1a dimer as a parallel 4HB33
thus posed a structural dilemma: the mismatch of
symmetries appeared to preclude any simple
mechanism of DCoH binding.25 Characterization of
an anti-parallel mini-zipper, although incorrect in
An Intertwined Four-helix Bundle in HNF-1
its details, resolved this incongruity and led to cor-
rect positioning of HNF-1a atop the DCoH
saddle.34 In this model, as in the co-crystal struc-
ture (Figure 1(a)),35 DCoH recognition is mediated
by HNF-p1w helices 1 and 10 , whereas helices 2
and 20 are distal to the interface. The model cor-
rectly de®ned the relative orientations of individ-
ual
a-helices in the intermolecular 4HB (Figure 9(a)
and (b)), which differ from that in the DCoH
homotetramer.23
The above features are retained on docking of
the present crystal structure. Signi®cantly, the
improved resolution of helical phases makes poss-
ible retrodiction of possible side-chain contacts.
Fortuitously, the length of a-helix 1 in HNF-p1w is
the same as those of contact helices in the DCoH
4HB (15 residues). Formation of the HNF/DCoH
4HB is predicted to bury a total surface area of
1820 A Ê 2. These values are similar to those observed
in the co-crystal structure, wherein a total surface
area of 1960 A Ê 2 is buried.35 The rmsd of super-
posed C atoms of helix-1 and helix-10 in HNF-p1w
a
and equivalent helices in the DCoH tetramer is
1.1 AÊ . Symmetry-based docking of the present
crystal structure thus yields the correct overall fea-
tures of the complex: N-terminal helices (1 and 10 )
are ``sandwiched'' between C-terminal helices (2
and 20 ) of the HNF-p1w dimer and a pair of DCoH
helices atop the saddle resulting in a six-helix
bundle.
An alternative possible mode of recognition
employing HNF-1a helices 2 and 20 was evaluated
as a control. An intermolecular 4HB may be
obtained readily by inversion of the HNF-1a
domain atop the saddle. Such an ``upside-down''
model in fact yields helical orientations whose
symmetry is similar to that of the DCoH homote-
tramer. Because a-helices 2 and 20 are shorter than
a-helices 1 and 10 (eight versus 15 residues), how-
ever, this alternative model exhibits a 32 %
reduction in buried surface area (to 1135 A Ê 2) In
addition, the side-chains exposed on the surface of
helices 2 and 20 are less compatible (with respect to
polarity and charge) with the juxtaposed surface of
the DCoH saddle than are the side-chains at the
surface of the mini-zipper. Helices 1 and 10 in the
present crystal structure are longer (by two resi-
dues) than in the peptide-DCoH complex, wherein
residues 1-6 assume a different conformation. In
the model, residues 1-5 of HNF-p1w exhibit a ster-
ic clash with DCoH. Because these residues are
¯exible in solution, the potential clash could be
avoided readily.
Retrodicted side-chain environments
The side-chains on the mini-zipper's surface
nearest to the HNF-1a dimer axis are L12 and L120 ,
a site of diabetes-related mutation. Alignment of
HNF-1a- and DCoH symmetry axes automatically
positions the methyl groups of L12 and L120 within
a hydrophobic pocket formed by DCoH residues
651
F47 and T51 and their 2-fold-related mates
(Figure 9(c)), as observed in the complex.35 The
model further retrodicts contacts from Q9, A15 and
S19 in HNF-p1 to L55, T51-R52, and L55 in DCoH,
respectively, also in accord with the co-crystal
structure. Farther from the symmetry axes,
however, several intermolecular interactions are
not retrodicted immediately. These include S6-E58,
Q7-R45, L8-E58, E11-R45, E18-R52, and S19-K59.
This limitation is due to a combination of factors.
(i) Rigid-body modelling neglects local induced ®t
or plasticity of side-chains in either protein. DCoH
residues N44, R45, R52, E58, and K59, each
involved in interactions with HNF-p1, assume
different side-chain conformations in the peptide-
DCoH complex35 relative to the DCoH homo-
tetramer.23 Relocation of the DCoH side-chain of
E58, for example, is necessary to relieve a potential
steric clash with HNF-p1 residue Q7. (ii) Whereas
differences in interhelical angles across the inter-
molecular 4HB yield negligible errors at the center
of symmetry (i.e. near L12 and L120 ), progressively
larger errors occur farther from the center.
These comparisons suggest that in the absence of
signi®cant conformational changes, symmetry-
based modelling provides a robust method for pre-
diction of the overall features of a complex and
some of its key contacts. Rigid-body docking is
insuf®cient, however, to predict all the details of
the contact surfaces. Predictions may be improved
by explicit consideration of side-chain electrostatic
and functionalities. Inspection of electrostatic sur-
faces in the present case strongly suggests the
possibility of the observed ionic interactions (such
as E11-R45 and E18-R52)35 with modest local
changes in side-chain con®guration.
Comparison of HNF-1a
a and HNF-1b
b
HNF-1a and HNF-1b belong to a family of
homo- and heterodimeric transcription factors reg-
ulating expression of many genes.15,22,62 Each binds
to speci®c DNA sites as a dimer and forms hetero-
dimers in vitro and in vivo. Alignment of HNF-1a
and HNF-1b sequences is shown in Figure 3(a).
Diabetes-related mutational sites L12 and G20 are
invariant (highlighted in red). In HNF-1b positions
21 and 25 contain valine residues in place of L21
and A25 in HNF-1a domain (brackets, lower panel
of Figure 3). The paired valine residues in HNF-1b
presumably function as compensating substitutions
to ®ll the volume occupied in the present structure
by adjoining L21-A25 side-chains. Molecular mod-
elling of L21G (red) and L25G (pink) creates a cre-
vice, as depicted on the left side of Figure 3(b). In
the crystal structure, this crevice is ef®ciently ®lled
by the close-packing of side-chains L21 and A25
(right side of Figure 3(b)). Given the conservation
of the remaining residues lining the HNF-1b's
putative DCoH-binding surface, we expect that an
homologous complex would indeed be formed. In
HNF-1b, position 18 has a serine residue in place
of the glutamic acid residue in HNF-1a (asterisk in
652
Figure 3(a), lower panel). E18 is not involved in
interactions between protomers, suggesting that
this subsitution is neutral with respect to dimeriza-
tion. However, because the E18 side-chain carboxy-
late group forms an ionic interaction with R52 of
DCoH,35 it is possible that the presence of S18 in
HNF-1b would weaken binding to DCoH. To our
knowledge, binding of HNF-1b or the HNF-1a/
HNF-1b heterodimer to DCoH has not been
demonstrated.
Environment of clinical mutation sites
I27L polymorphism
The substitution I27L (highlighted in green in
Figure 3(a), upper panel) is a common polymorph-
ism in diabetic and non-diabetic populations unas-
sociated with MODY.2 The I27 side-chain is
exposed on the surface of helix 2; its interactions
differ in detail between the present structure and
the peptide-DCoH co-crystal structure (see above).
Surprisingly, this polymorphism has been shown
recently to be an independent correlative determi-
nant of insulin sensitivity due to hepatic or periph-
eral insulin-resistance.63 It is not known whether
the mutation alters the regulation of glucose
metabolism directly or exists in linkage disequili-
brium with another polymorphic gene regulating
metabolism.
L12H MODY mutation
L12 is involved in van der Waal's interactions
with symmetry-related L120 and L160 side-chains
and projects to the surface of the mini-zipper
(Figures 2 and 10). The side-chain thus contributes
to both the stability and functional surface of the
dimer. The side-chain is partially buried upon
dimerization; in the extracted monomer, the frac-
tional exposed surface is 0.55, whereas in the
dimer it is 0.26 (a complete set of residue-speci®c
accessibilities is provided in the Supplementry
Material). L12 is invariant among mammalian
HNF-1a and HNF-1b sequences (Figure 3(a)). Mol-
ecular modelling suggests that substitutions L12G
or L12A would result in signi®cant packing defects
(cavities of respective volumes 20 and 15 A Ê 3).
MODY-associated mutation L12H effects a signi®-
cant reduction in thermodynamic stability (

G
value 3.7(0.3) kcal/mol; Table 2)34 and accord-
ingly weakens dimerization. L12 makes buried
contacts with dimer-related residues of HNF-p1
and the DCoH dimer in peptide-DCoH complex.35
Consistent with this location, L12H disrupts bind-
ing of HNF-1a to DNA and DCoH.35 Overexpres-
sion of the variant intact protein restores activation
of a reporter gene to native levels.8
Crystals of an L12H analogue of HNF-p1w could
not be obtained; however, the analogue's NMR
spectrum is nonetheless similar to that of the native
dimer. Although isotope-assisted NMR studies of
the L12H analogue were not performed, overall
An Intertwined Four-helix Bundle in HNF-1
similarities in chemical shifts and NOEs suggest
that a native-like fold is maintained. To explore
possible structural consequences of the L12H substi-
tution and their implications for DCoH recognition,
an homology model was constructed based on the
native crystal structure. The modelling procedure
employed the distance-geometry protocol of Havel
& Snow,64 wherein NOEs were simulated based on
the crystal structure (see Materials and Methods).
(Restraint information and statistical parameters are
provided as Supplementary Material.) A consistent
DG/SA model was obtained, suggesting that the
adjoining H12 side-chains are accommodated
readily in the mini-zipper interface with only local
conformational adjustments. The overall fold and
structure of the variant mini-zipper are shown in
Figure 10(c) and (d). Although L12 has been
described as a core side-chain, the methyl reson-
ances in fact contribute to the ¯at hydrophobic sur-
face of the native dimer (Figure 10(a) and (b)). The
L12H model predicts a striking remodelling of this
surface in which the contiguous imidazole rings
give rise to a weakly polar protuberance
(Figure 10(e)). Exposure of the titratable Ne edge of
the imidazole ring to solvent is supported by the
robustness of the fold to low pH.
G20R MODY mutation
G20 lies in a non-canonical turn (Figure 1(b))
and appears to direct the relative orientations of
helices in the peptide's ``active'' conformation. Its
main-chain atoms are hyperexposed in both an
extracted protomer and actual dimer with a frac-
tional exposed surface of 0.64. Although the pat-
tern of torsion angles in the turn differs from those
of classical turns, a similar pattern is observed in
helix-turn-helix motif of the arc repressor (PDB
code 1BDT).65 The structures are otherwise unre-
lated. MODY-associated substitution G20R is
associated with a profound thermodynamic desta-
bilization (Table 2; and see Hua et al.34) and loss of
architectural speci®city. Although the variant
dimer retains near-native helix content as inferred
from CD studies, a combination of features (non-
two-state unfolding, binding of ANS, loss of
chemical-shift dispersion with conformational
broadening) indicate that the variant dimer forms
a molten globule. We speculate that local frustra-
tion of the sharp turn precludes ef®cient packing
of the C-terminal coiled-coil against the mini-zip-
per. A possible mechanism of frustration is
suggested by the crystal structure: given the pre-
sent backbone torsion angles at residues 19 and 20,
any L-amino acid substitution at position 20 would
not be sterically favorable. The distance between
the carbonyl oxygen atom of residue 19 (ith pos-
ition) and the Cb of residue 20 (position i ‡ 1)
would be less than 2.4 A Ê , which predicts a steric
clash; by contrast D-amino acids should be accom-
modated readily. The proposed mechanism pre-
dicts that the G20R perturbation is not side-chain
speci®c; i.e. G20A and other analogues would exhi-
An Intertwined Four-helix Bundle in HNF-1 653

Figure 10. Surface representation of HNF-p1w dimer and L12H homology model. (a) Electrostatic potential of the
DCoH-binding surface of HNF-p1w (stereo pair). The coloring code is red, <ÿ10 kT/e; white, ÿ10kT/e to 10kT/e;
and blue, >10kT/e. Residues E11 and E18 are involved in ion-pair interactions with DCoH residues.35 The exposed
concave surface is complementary in shape to the top of the DCoH saddle as predicted by model building studies
(see the text) and observed in the recent co-crystal structure.35 (b) The DCoH-binding surface of HNF-p1w dimer has
a leucine-rich stripe (leucine residues 5, 8, 12, 13, 16 and 21, and their symmetry-related residues) shown in green.
The orientation of molecular surface (white) of the HNF-p1w dimer is same as in (a). L12 is located in the middle of
the leucine-rich stripe. Residues 1-4 and 10 -40 have been omitted for clarity. (c)-(e) Homology model of the L12H var-
iant dimer. (c) The variant side-chain is compatible with a native overall main-chain fold. One protomer is shown in
cyan and the other in red; terminal residues 1-4 and 10 -40 are disordered in accord with NMR evidence of ¯exibility.
(d) The structure of the mini-zipper is predicted to accommodate a pair of histidine side-chains (yellow) with
exposure of polar Ne ring protons. Neighboring side-chains are in positions similar to those in crystal structure.
(e) The surface of the variant mini-zipper is predicted to contain a weakly polar protuberance (yellow) due to the
edges of the two His side-chains. This change in surface character would be expected to perturb DCoH binding. (a),
(b) and (e) Generated using the program GRASP;85 (c) and (d) (and other Figures) were made using InsightII (MSI,
Inc., San Diego).

bit similar instabilities. It is unclear why the puta-
tive steric clash cannot be relieved by local confor-
mational adjustments to give an altered but
architecturally speci®c structure.
Concluding remarks
The dimerization domain of HNF-1a provides
an opportunity to compare structures at high resol-
ution in multiple crystallographic environments.
The structure consists of a novel intertwined four-
helix bundle with non-canonical turn. Multiple
structural comparisons suggest strongly that the
domain consists of a rigid element, an N-terminal
anti-parallel mini-zipper, and a plastic element, the
C-terminal coiled-coil. Two types of plasticity are
observed. (i) The immediate N and C-terminal seg-
ments can be a-helical or disordered depending on
packing environment. Corresponding ¯exibility in
solution has been demonstrated by 1H-NMR
654
motional narrowing, chemical-shift patterns,
selected main-chain coupling constants and, in the
case of W33, multiple inter-residue NOEs inconsist-
ent with a single unqiue structure. (ii) Plasticity
exists at the interface between the mini-zipper and
the overlying C-terminal a-helices leading to differ-
ent patterns of side-chain packing. Although the
non-canonical turn and intertwined topology are
maintained consistently, striking differences occur
in possible inter-helical angles. The collection of
HNF-p1 crystal structures is reminiscent of insulin,
whose many high-resolution crystal structures like-
wise exhibit a combination of invariant and plastic
features.66,67 The most divergent HNF-1a structure
is that observed in a speci®c complex with DCoH.
Because recognition is mediated by the invariant
mini-zipper and the molten C-terminal coiled-coil,
such induced ®t appears to involve a transmission
of conformation change. Transmission of confor-
mational change among crystal structures of insu-
lin has been highlighted by the late D. C. Hodgkin
and colleagues68 and, as in the HNF-1a/DCoH
complex, may facilitate receptor binding.69
The present study suggests new directions for
future study. First, given the collection of equival-
ent crystal structures, which subset of confor-
mations best represents the structure in solution?
Although the present isotope-edited studies verify
the overall intertwined topology and phase of the
mini-zipper, more detailed heteronuclear NMR stu-
dies of structure and dynamics will be required to
assess the inter-helical packing scheme(s) and esti-
mate the range of possible conformational change.
Laboratory-frame approaches based on residual
dipolar couplings are likely to be informative.
Second, what is the role of the non-canonical turn
in enabling a speci®c architecture to be obtained?
The role of turns in general in 4HBs has been the
subject of debate.70,71 We have suggested a speci®c
structural mechanism for the invariance of glycine
(G20) and predict that any L-amino acid (but not
D-amino acids) at position 20 would block speci®c
folding to create a novel molten globule. If so, the
HNF-1a motif would provide an example of an
energy landscape72 whose shape and depth
depend on a particular turn orientation. Finally,
what is the biological signi®cance of the domain's
asymmetric plasticity? A possible clue is provided
by the unexplained G31D diabetes-associated
mutation. Unlike L12H and G20R, the G31D var-
iant would seem not to affect the domain's struc-
ture, stability or interaction with DCoH.35 An
intriguing possibility is that in vivo HNF-1a inter-
acts with DCoH and with other transcriptional co-
activators or co-repressors and that such (to date)
uncharacterized interactions are disrupted by the
G31D mutation. This possibility in turn suggests a
structural model in which the plastic portion of the
HNF-1a domain adopts one orientation on binding
to DCoH and another conformation on binding to
a putative second regulatory cofactor. Plasticity in
the packing of the mini-zipper and C-terminal
a-helices may thus extend the domain's repertoire
An Intertwined Four-helix Bundle in HNF-1
of interactions as part of a structural switch
between functional states. An example of such a
dynamic switch has been characterized recently in
a cytoplasmic signalling pathway.73
Materials and Methods
Peptide synthesis
The HNF-1a domain with MSe substitution at position
13 was prepared by solid-phase synthesis (Gryphon
Sciences, Inc., South San Fransisco, CA). Sample purity
was greater than 98 % as estimated by analytical reverse-
phase HPLC. Fidelity of synthesis was veri®ed by
ion-spray mass spectrometery. The dimeric status of
the peptide was veri®ed by analytical gel-®ltration
chromatography.
Crystallization and X-ray data collection
The peptide was crystallized by the hanging drop/
vapor diffusion method. The peptide was made 3 mM in
50 mM Tris-HCl (pH 7.0), 2 mM DTT. Crystals were
grown at 4  C by mixing equal volumes of peptide sol-
ution (3 ml) and reservoir solution (3 ml) containing 20 %
(v/v) methyl-2,4-pentanediol (MPD), 50 mM Tris-HCl
(pH 7.5), and 1 mM DTT. Crystals mounted on nylon
loops were transferred to stabilization buffer (100 mM
Tris-HCl (pH 7.5), 30 % MPD or 30 % PEG400) and ¯ash-
frozen in liquid nitrogen before data collection at 100 K.
X-ray data were collected at beamline 19-ID at the
Advanced Photon Source (APS), Argonne National Lab-
oratory. Multiple anomalous dispersion (MAD) data sets
were collected at three wavelengths: remote (lr), peak
(lp), and at the inversion point (li) of selenium atom
(Table 1). The crystals belong to orthorhombic space
group P21212 with a ˆ 28.42 A Ê , b ˆ 42.19 A Ê , and
Ê . There are two molecules in the asymmetric
c ˆ 42.43 A
unit with a solvent content of 23 % (v/v). Raw data were
processed and scaled using the HKL2000 package.74
Data collection statistics are presented in Table 1.
Structure determination and refinement
The two selenium positions corresponding to two
independent molecules (molecules 1 and 2) in the asym-
metric unit were determined by computing anomalous
and dispersive- difference Patterson maps using program
CNS.75 Heavy-atom positions were re®ned against MAD
data with remote wavelength as reference using data
between 40 and 3.0 A Ê resolution. Further density modi®-
Ê resolution using
cation and phase extension to 2.5 A
CNS yielded a high-quality MAD-phased electron den-
sity map. It was possible to trace the two helices in mol-
ecule 1 and one helix in molecule 2. A polyalanine
model was constructed for molecule 1 and for one helix
in molecule 2 using program O76 and re®ned using data
collected at remote wavelength (lr). Structure re®nement
was done using CNS. Overall anisotropic scale factors
and a bulk solvent corrections were applied and a maxi-
mum likelihood amplitude-based target function were
employed.77 Simulated-annealing (SA) torsion-angle
re®nement78 and individual B-factor re®nement was per-
formed as implemented in CNS. Several rounds of alter-
nate SA re®nement and model rebuilding were
performed with gradual increments in the data resol-
ution. Each residue in molecule 1 was seen in the elec-
tron density map, whereas helix 2 in molecule 2 was
An Intertwined Four-helix Bundle in HNF-1
poorly de®ned and built residue-by-residue in iterative
re®nement cycles. Water molecules that are at a potential
hydrogen bonding distance from the protein or another
water molecule were added in ®nal rounds of re®ne-
ment. SA omit maps were computed by omitting two
residues at a time for both molecules 1 and 2, and the
model rebuilt. In molecule 2 the ®rst two N-terminal
residues and the last three C-terminal residues are not
seen in the map and are considered disordered. The ®nal
model consists of molecule 1 (33 residues), molecule 2
(28 residues), and 55 water molecules. The structure was
re®ned to a ®nal crystallographic R of 23.1 % using F > 3
sF between 40 and 1.7 A Ê resolution. The Rfree for the
re®ned model was 24.5 %. Some of the ®nal re®nement
statistics are given in Table 1. Average B-factors for mol-
ecule 1, molecule 2, and water molecules are 15.9 A Ê 2,
23.0 AÊ 2, and 28.1 A Ê 2, respectively. The ®nal structure
was validated using PROCHECK.79
HNF-DCoH model building
The crystal structure of HNF-p1w was used to dock
against the crystal structure of the DCoH dimer (sub-
units A and B of PDB code 1DCO) using the InsightII
molecular graphics package (MSI, Inc.). The 2-fold axis
of HNF-p1w was aligned on the 2-fold axis of DCoH,
superposing dimer-related anti-parallel helices of HNF-
p1 and the equivalent helices (without considering the
helix direction) in the DCoH tetramer (subunits C and
D). Superposition employed sheer rotation about the
aligned axis (Figure 9(a)). This procedure results in a
model for the HNF-p1w/DCoH complex with a 4HB
interface. The model predicts different relative helical
orientations for this 4HB compared to the DCoH tetra-
mer: the direction of the helices in HNF-p1w dimer are
reversed compared to the helices in DCoH tetramer
(Figure 9(b)) in accord with modelling studies by Hua
et al.34 and the co-crystal structure.35 Cavity volumes
were computed using the program surfnet.80
Expression and labeling of HNF-1a
a peptide
A DNA segment encoding HNF-p1w was prepared
by polymerase chain reaction (PCR) using the human
HNF-1a cDNA as template. The PCR product was
cloned into BamHI and EcoRI sites of plasmid pMW1
previously employed to express phage l N peptides.81
The HNF-1a peptide was expressed as a thrombin-
cleavable fusion protein with staphylococcal nuclease
as described.82 The expressed protein contained an
N-terminal hexa-His tag to enable af®nity puri®cation.
Biosynthetic 13C labeling was accomplished in an overex-
pressing strain of Escherichia coli by growth in M9 mini-
mal medium containing [13C]glucose as sole carbon
source. The biosynthetic peptide contained 36 residues,
including three non-native N-terminal residues (GSE);
the C terminus was not amidated.
Isotope-assisted NMR studies
The following spectra were obtained for the 13C-
labeled peptide homodimer: 13C-1H HSQC, 13C-1H 2D
HSQC-TOCSY (mixing time of 45 ms), 13C-1H 3D
HCCH-TOCSY, 13C-1H 3D NOESY-HSQC (mixing time
of 250 ms). Complete assignments were obtained. To
evaluate intermolecular NOEs, an equimolar mixture of
the synthetic peptide HNF-p1w and the labeled biosyn-
thetic peptide was prepared. The following spectra were
655
obtained for the equilibrium mixture: 13C-1H HSQC,
13
C-1H 2D HSQC-TOCSY (mixing time of 45 ms), and
13
C-1H intermolecular 2D NOESY.83
Circular dichroism
Spectra were obtained using an Aviv spectropolari-
meter equipped with thermister temperature control.
Samples were placed in a 1 mm path-length quartz
cuvette. Peptide concentrations in the presence of MPD
were corrected for volume change of solvent miscibility.
The buffer solutions corresponded to crystallization
conditions.
L12H homology modelling
A molecular model of the L12H variant dimer was
constructed using the DG/SA protocol as described:64
1212 homology distance restraints were imposed, sup-
plemented by one key H12-speci®c NOE (H12-L160 , pre-
sumed to be inter-protomeric). In brief, inter-proton
distances were inferred from the crystal structure (using
the HBUILD facility in InsightII (Molecular Simulations,
Inc.)) for residues 5-33, excepting the side-chain of resi-
due 12; no restraint was included for residues 1-4 or 10 -40
in light of NMR evidence of ¯exibility. Distance bounds
were assigned to conventional NMR ranges (i.e. strong
(<2.7 AÊ ), medium (<3.4 A Ê ) or weak (<5 A Ê )) based on
inferred values in the crystal structure. a-Helix-related
hydrogen bonds were imposed by pairs of distance
restraints (between the carbonyl oxygen atom of residue
i and the amide proton of residue i ‡ 4 (2.3-2.6 A Ê ) and
between the carbonyl oxygen atom of residue i and the
amide nitrogen atom of residue i ‡ 4 (2.7-3.3 A Ê )) for resi-
dues 10-19 and 26-30. Dihedral restraints on main-chain
f values were assumed for helical residues (residues 6-
19 and 22-30; f ˆ ÿ 65(40)  ); both f and c values
were imposed for G20 and G200 (f ˆ 70(40)  and
c ˆ ÿ 150(40)  ); and side-chain w1 and w2 dihedral
angles were imposed for residues 12 and 13 (40  of the
value in the present crystal structure). The main-chain
rmsd between the present structure and the L12H model
Ê . No signi®cant restraint
(residues 5-28 and 50 -280 ) is 1.1 A
violation is observed in the ®nal homology ensemble
(see the Supplementary Material, where a summary of
restraint information is provided).
Atomic coordinates
Atomic coordinates (1JB6) and X-ray data (Y1JB6Sp)
have been deposited with the RCSB Protein Data Bank..
Acknowledgments
We thank M. Shoham for allowing us to use his beam
time to perform our MAD experiment at the Advanced
Photon Source (APS). Use of the Argonne National Lab-
oratory structural biology center beam line 19-ID at the
APS is supported by the US Department of Energy,
Of®ce of Biological and Environmental Research under
contract W-31-109-ENG-38. We thank V. Srajer at the
BIOCARS beam line 14-bm-D at the APS for the initial
stages of this study; S. Soleiman for help in using O pro-
gram on their workstation; N. Phillips and M. Hake for
gel-®ltration experiments; A. Stern for ring-current shift
calculations; S. Korolev and R. Immormino for help in
656
data collection at APS; W. Jia for CD experiments and
related Figures; and S. Nakagawa (The University of
Chicago) and J. Wilken (Gryphon Sciences, Inc.) for
peptide synthesis. M.A.W. thanks the faculty of the Cold
Spring Harbor course on macromolecular crystallogra-
phy for their encouragement. This work was supported,
in part, by the Diabetes Research & Training Center at
The University of Chicago and by a grant from the NIH
to M.A.W.
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Edited by M. F. Summers
(Received 18 January 2001; received in revised form 10
May 2001; accepted 10 May 2001)
http://www.academicpress.com/jmb
Supplementary Material comprising 11 Figures
and six Tables is available on IDEAL