Membrane domain structures of three classes

of histidine kinase receptors by cell-free

expression and rapid NMR analysis
Innokentiy Maslennikova,1, Christian Klammta,b,1, Eunha Hwangc, Georgia Kefalaa,b, Mizuki Okamuraa, Luis Esquiviesa,
Karsten Mörsd, Clemens Glaubitzd, Witek Kwiatkowskia, Young Ho Jeonc,2, and Senyon Choea,b,2
a
Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; bJoint Center for Biosciences, Songdo, Incheon 406-840, Korea;
c
Division of Magnetic Resonance, Korea Basic Science Institute and University of Science and Technology, Ochang, Chungbuk 363-883, Korea; and dCentre
for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe University, Frankfurt 60438, Germany

Edited* by Robert M. Stroud, University of California, San Francisco, CA, and approved April 30, 2010 (received for review February 11, 2010)

NMR structural studies of membrane proteins (MP) are hampered

by complications in MP expression, technical difficulties associated
with the slow process of NMR spectral peak assignment, and
limited distance information obtainable for transmembrane (TM)
helices. To overcome the inherent challenges in the determination
of MP structures, we have developed a rapid and cost-efficient
strategy that combines cell-free (CF) protein synthesis, optimized
combinatorial dual-isotope labeling for nearly instant resonance
assignment, and fast acquisition of long-distance information
using paramagnetic probes. Here we report three backbone struc-
tures for the TM domains of the three classes of Escherichia coli
histidine kinase receptors (HKRs). The ArcB and QseC TM domains
are both two-helical motifs, whereas the KdpD TM domain com-
prises a four-helical bundle with shorter second and third helices.
The interhelical distances (up to 12 Å) reveal weak interactions
within the TM domains of all three receptors. Determined conse-
cutively within 8 months, these structures offer insight into the
abundant and underrepresented in the Protein Data Bank class
of 2–4 TM crossers and demonstrate the efficiency of our CF com-
binatorial dual-labeling strategy, which can be applied to solve MP
structures in high numbers and at a high speed. Our results greatly
expand the current knowledge of HKR structure, opening the doors
to studies on their widespread and pharmaceutically important
bacterial signaling mechanism.

backbone NMR structure ∣ cell-free synthesis ∣

combinatorial selective labeling Fig. 1. Three classes of HKRs. (A) Schematic representation of TM domains
of three classes of HKRs and (B) ribbon representation of 3D structures of the
TM domains of E. coli HKRs ArcB, QseC, and KdpD.

H istidine kinase receptors (HKRs) are part of a two-compo-

nent system, in which an HKR in the bacterial inner mem-
brane transmits a signal to a response regulator located in the
cytoplasm (1). This signaling system constitutes the predominant
signal transduction mechanism by which bacteria interact with
their environment (1), however, many aspects of its signaling
mechanism are unknown. Based on the part of the receptor used
to sense environmental stimuli, HKRs are grouped into three
structural classes. Class 1, the largest class, is characterized by
the presence of an extracytoplasmic sensory domain that senses
external stimuli and transmitting the signal across the membrane.
Class 2 HKRs lack an apparent extracytoplasmic domain and the
stimuli-sensing region is believed to be in the membrane domain
itself. Class 3 is characterized by a cytoplasmic sensory domain.
For this study, we selected QseC (class 1, quorum sensor), ArcB
(class 2, aerobic respiratory control sensor), and KdpD (class 3,
K þ sensor) as representatives from each of the three classes.
HKRs are multidomain proteins with diverse domain structures
and topologies (Fig. 1): QseC has two transmembrane (TM)
helices with an intervening 130 amino acid periplasmic sensor do-
main, ArcB has two TM helices connected by a short periplasmic
loop, and KdpD has four TM helices with short interhelical
loops. The cytoplasmic regions of HKRs comprise a dimerization
domain and a kinase domain, several structures of which have
been reported (2–9), including that of ArcB. There are also
several reported periplasmic sensor domain structures (10–13).
However, due to the highly flexible multidomain nature of HKRs,
there is still no structure of a full-length receptor, nor has any
structural information on an HKR membrane domain been
reported to date. Such a structure is essential to understand
the mechanistic aspect of signal transduction.
Despite impressive progress in the structure determination of
membrane proteins (MPs) by X-ray crystallography and NMR
Author contributions: I.M., C.K., and S.C. designed research; I.M., C.K., E.H., G.K., M.O., L.E.,
K.M., C.G., W.K., and Y.H.J. performed research; I.M. and C.K. analyzed data; I.M., C.K.,
W.K., and S.C. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The NMR-derived data (atomic coordinates, chemical shifts, and
restraints) have been deposited in the Protein Data Bank, www.pdb.org, under accession
codes 2KSD [ArcB(1-115)], 2KSE [QseC(1-185)], and 2KSF [KdpD(397-502)].
1
I.M. and C.K. contributed equally to this work.
2
To whom correspondence may be addressed. E-mail: yhjeon@kbsi.re.kr or choe@salk.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1001656107/-/DCSupplemental.

10902–10907 ∣ PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1001656107

spectroscopy in recent years (see reviews in refs. 14 and 15), only
about 250 structures of unique integral MPs have been deter-
mined, representing less than 1% of known protein structures
(16). In addition to problems with expression, solubilization,
and purification of integral MPs, X-ray and NMR methods are
hampered with inherent technical difficulties. Diffraction quality
crystals of integral MPs are very difficult to obtain because solu-
bilized protein-detergent complexes do not usually form ordered
crystal lattices. Furthermore, the mobility of TM helices leads to
strong broadening of the signals in NMR spectra and presents
problems with spectra quality, resonance assignment, and detec-
tion of long-range interactions, all of which are necessary to
calculate the structure of a protein by NMR. Spin label-based
paramagnetic relaxation enhancement (PRE) approaches have
been used to address the paucity of long-distance constraints
associated with α-helical integral MPs (17, 18). However, the cost
of isotope labeling by in vivo heterologous expression in cells of
both prokaryotic and eukaryotic origins is often prohibitive for
NMR structural studies of even well-expressed MPs.
We addressed the aforementioned technical difficulties, devel-
oping a strategy for a cost-efficient structure determination of
MPs that has allowed us to quickly solve the backbone structures
of three HKR membrane domains by NMR. The chosen struc-
tures represent the three classes of this widespread bacterial
TM signaling system and belong to the abundant group of 2–4
TM crossers, which is poorly represented in the Protein Data
Bank (PDB). By studying the domains in isolation, we present
the structural insights into the signal-transducing membrane do-
mains of these receptors. The results demonstrate the efficiency
of the combinatorial dual-labeling (CDL) strategy. Thus, based
on synergy of fast cell-free (CF) expression and rapid NMR
analysis, CDL opens up possibilities for accelerated structural
analysis of MPs.
Results
Structures of MPs Produced in CF System are Comparable to Those
from Escherichia coli. To synthesize membrane domains of selected
histidine kinases we used the precipitating CF (p-CF) expression
mode (19), in which a protein is produced as a precipitate that is
subsequently solubilized by a nondenaturing detergent (a com-
prehensive description is given in SI Materials and Methods).
The p-CF mode is extremely useful because it allows NMR
studies of MPs without purification (Fig. S1). Because all of
the CF reaction components are soluble, they remain in a super-
natant and are easily removable after reaction by pellet wash. As
a result, the target MP can be expressed without affinity tags that
might affect its structure or stability. Although reminiscent of
bacterial inclusion bodies, the MP precipitate can be solubilized
with a mild lipid-like detergent (19) unlike true inclusion bodies,
which require strong denaturing conditions (20). Therefore, it is
likely that the CF precipitate has at least a partially folded
structure.
To verify this, we investigated the underlying structure of the
precipitates by magic angle spinning solid-state NMR (MAS SS-
NMR). Uniformly 13 C-labeled ArcB(1-115) and KdpD(397-502)
precipitates were measured directly after their production in the
p-CF reaction. By measuring the deviation of the 13 C chemical
shifts away from their typical values for a random coil peptide,
we could differentiate between helical, beta, and random coil sec-
ondary structures (21) in the precipitates. In these two samples,
all of the observed 13 Cα -13 Cβ cross-peaks for alanine and valine
residues fell within the regions of the 13 C-13 C correlation spectra
typical for α-helices (21) (Fig. 2A and Fig. S2A). The 13 Cα -13 Cβ
cross-peaks for valine and alanine residues that reside in the loop
regions are probably broadened beyond the detectability limits
in the MAS SS-NMR spectra. Moreover, the spectrum of the
ArcB(1-115) precipitate is very similar to the spectrum of the
ArcB(1-115) sample lyophilized after solubilization with a deter-
gent (Fig. 2A, red contours). The presence of secondary structure
in the precipitate was confirmed by solution NMR measurements
of proton-deuterium (H-D) exchange following detergent solubi-
lization. The results show that, in ArcB(1-115), 11 out of 16 va-
lines and 5 out of 7 alanine are located in protected and therefore
likely TM helical regions. Thus MAS SS-NMR analysis of chemi-
cal shifts together with solution NMR data on the exchange of
the labile backbone protons in the precipitate (Figs. S3 and
S4) have unambiguous interpretation: The TM helices of ArcB
(1-115) and KdpD(397-502) existed as secondary structure
elements before precipitation in a CF reaction.
To further validate the p-CF expression system, we compared it
with the standard E. coli expression in terms of sample quality and
structure, as well as time and cost efficiency (Fig. 3 and Table S1).
ArcB(1-115) was expressed and purified using both approaches.
The E. coli system required five consecutive days, beginning with
the transformation and growth of bacteria in minimal media and
then extraction and purification of the protein from the cell mem-
brane, in order to start the first NMR measurement. In contrast,
using the p-CF synthesis, the first NMR measurement was

COMPUTATIONAL BIOLOGY
BIOPHYSICS AND

Fig. 2. Verification of fold of ArcB(1-115), produced by CF system. (A) Overlay of 13 C DARR-NMR spectra (213.765 MHz) of uniformly 13 C-labeled ArcB(1-115)
expressed in p-CF reaction. Black contours correspond to the spectra of the washed precipitant of the p-CF reaction. Red contours correspond to the spectra of
the ArcB(1-115) sample lyophilized after the precipitant was solubilized in 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) . The green and
cyan lines correspond to random coil 13 Cα , 13 Cβ chemical shifts for valine (Val) and alanine (Ala), respectively; arrows show regions of chemical shifts corre-
sponding to the α-helical conformation. (B and C) ½1 H-15 N
-TROSY-HSQC spectra of 15 N-labeled ArcB(1-115) expressed (B) by p-CF synthesis and in (C) E coli.
(B) The precipitated protein was washed and solubilized in 5% LMPG. (C) The protein was extracted and purified from cell membrane with the FC-12 detergent
and the detergent was exchanged to LMPG. Cross-peaks denoted by red arrows correspond to the tag linker residues in the E. coli-expressed protein.

Maslennikov et al. PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 ∣ 10903
 and improved later by the high selectivity of isotope labeling in
CF synthesis and the efficiency of modern NMR heteronuclear
techniques (23). Several approaches were built upon this method
to perform site-selective screening (24) and to accelerate the re-
sonance assignment by using dual combinatorial labeling (25–27).
Simplified approach utilizing combinatorial selective 15 N labeling
was proposed for determination of the amino acid types for
1 HN , 15 NH resonances (28). Combinatorial selective labeling ap-

proaches (reviewed in refs. 29 and 30) use a different subset of

Fig. 3. Comparison of performance of the CF system with the standard
E. coli system. SDS-PAGE shows marker (M); CF reaction mixture (RM) before
reaction at 0 h (1); CF RM after ArcB(1-115) expression at 15 h (2); precipitate
after ArcB(1-115) p-CF (3); E. coli (EC) expressed ArcB(1-115) after extraction,
purification, Tag cleavage, size-exclusion chromatography (SEC), detergent
exchange on Q-sepharose, and concentration (4); arrow indicates ArcB(1-115).
possible in under 24 h, following an overnight expression and so-
lubilization of the protein. The comparison of ½1 H-15 N
-TROSY-
HSQC spectra obtained from protein produced by both the CF
(Fig. 2B) and the E. coli expression systems (Fig. 2C) shows that
the positions of all the backbone cross-peaks are nearly identical.
The difference in the number of the cross-peaks is a result of the
slight difference in the constructs used (the E. coli expression re-
quires an affinity tag). The backbone NOE data and 13 Cα chemi-
cal shifts (Fig. S5) are also very similar for both samples. Taken
together, these results lead us to conclude that structures of ArcB
(1-115) prepared using the CF and the E. coli expression systems
are the same.
Sequence-Optimized Isotope Labeling Scheme for Rapid Resonance
Assignments. Structure determination by NMR requires the se-
quential assignment of backbone resonances, a process that is
particularly laborious for α-helical MPs (discussed in ref. 15).
An alternative method of backbone resonance assignment utiliz-
ing dual selective 15 N and 13 C labeling and heteronuclear 13 C-15 N
spin coupling was originally proposed by Kainosho and Tsuji (22)
labeled amino acids in each sample in order to reduce the number
of samples required to achieve the assignment as compared to
one-by-one selective labeling. Most of the existing combinatorial
approaches use labeling schemes that are sequence blind and op-
timized for globular proteins (25, 26, 28). These work for proteins
that give good signal dispersion and good overall spectra quality,
but are impractical for MPs.
Expanding on the method originally proposed by Trbovic (27)
to resolve ambiguities in the NMR assignment of CF-expressed
MPs, we created a CDL strategy that is applicable to MPs (details
are described in Materials and Methods). At the core of the CDL
strategy is the individual optimization of the labeling scheme for
each protein sequence and each selected set of amino acids for
isotopic labeling. To derive these sequence-dependent schemes,
we have developed the software MCCL (Monte Carlo Combina-
torial Labeling, available at http://sbl.salk.edu/combipro), which
calculates the optimal labeling combination for a given protein
sequence with a defined number of samples using the Monte Car-
lo approach. As a result, the CDL method ensures that a minimal
number of samples are needed to assign the type of the first and
the second amino acid in every pair in a sequence, meanwhile
minimizing the spectral complexity of each sample.
The CDL strategy was used for the design of ½15 N-13 C
-labeling
schemes for both KdpD(397-502) (Fig. 4B) and QseC(1-185)
(Table S2), consisting of six and seven labeled samples, respec-
tively. Within 1 day after spectra collection, 27% and 22% of
1
H-15 N cross-peaks for KdpD(397-502) and QseC(1-185), respec-
tively, were unambiguously assigned. Additionally, the amino
acid type was defined for 100% and 74% of the 1 H-15 N cross-
peaks for KdpD(397-502) and QseC(1-185), respectively. The in-
complete rapid assignment provides evenly distributed multiple
starting points for traditional sequential assignment throughout

Fig. 4. The CDL strategy for the “point-directed assignment” of NMR spectra. (A) The ½1 H-15 N
cross-peak in HSQC will appear only if the second residue in a
pair is 15 N labeled (second box tagged 1). The ½1 H-15 N
cross-peak in the HSQC spectrum and the ½1 H-15 N-13 C
cross-peak in the HNCO spectrum will both appear
only if the peptide group is double ½13 C-15 N
-labeled (third box tagged 2). (B) Dual 15 N∕13 C combinatorial selective labeling scheme designed specifically for
backbone assignment of KdpD(397-502) (see details in SI Text). (C) Assignment of 1 H-15 N cross-peaks of KdpD(397-502) using a combinatorial scheme of se-
lective 13 C, 15 N labeling, presented in panel B. The overlays of ½1 H-15 N
-TROSY-HSQC (red contours) and ½1 H-15 N
projection of TROSY-HNCO (blue contours)
spectra are shown for each sample (I–VI). Absence of a cross-peak (tag “0”), a cross-peak present in TROSY only (tag “1”), and cross-peaks present in both the
TROSY and the HNCO spectra (tag “2”) in each combinatorially labeled sample determine the code (sequence of the tags) for every cross-peak A, B, and C in a
uniformly labeled sample. The scheme-designed code which is identical with the code determined from the recorded spectra defines a pair of amino acids for
the 1 HN , 15 NH , and 13 CO resonances.

10904 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001656107 Maslennikov et al.

the sequence. Starting from and building upon the results of the
CDL-derived assignment, the standard sequential assignment
procedure (31, 32) was tremendously accelerated, yielding an as-
signment for 100% of KdpD(397-502) and 76% of QseC(1-185)
backbone resonances in 2–3 weeks. ArcB(1-115) resonances
(96% of the backbone, 88% of Cβ , and most of Hα and Hβ ) were
assigned using the standard sequential assignment protocol. The
rapid assignment of backbone resonances enabled us to analyze
the secondary structure based on the chemical shift index and the
hydrogen exchange data (summarized in Fig. S4) and to proceed
with de novo NMR structure determination using the structural
constraints collected for assigned backbone atoms.

HKR TM Domain Structures. Having verified that the p-CF system

yields a folded protein and completed the NMR resonance
assignments, we collected the NOE and PRE data required to
calculate the structures of ArcB(1-115), QseC(1-185), and
KdpD(397-502) (see Materials and Methods). The folds depicted
in Figs. 1B and 5 show the helical nature of the TM domains from
these three proteins whose calculated structures have a crystallo-
graphic equivalent resolution (33) in the range of 2.7–3.2 Å
(Table 1).
The structures of ArcB(1-115) and QseC(1-185) (Figs. 1B
and 5 A and B) are both two-helical motifs whose helices have
the appropriate length (30–32 Å) to cross the lipid bilayer. The
large periplasmic signaling domain of QseC was not included
in the structure calculations and so its TM domain is simply
composed of two antiparallel weakly interacting α-helices with
a crossing angle of 157  4°. Similarly, the connecting loop in
ArcB(1-115) is not well defined and its TM domain comprises
two antiparallel α-helices with a crossing angle of 142  6.5°
and a distance of 11.1 Å at the closest point between the helices
(all helical packing parameters are summarized in Table S3). In
comparison, the crossing angle between two helices of HTR-II
Transducer in complex with sensory Rhodopsin (34) is 169° and
the distance between the helices is 10 Å, whereas for two tightly
packed helices in dimeric human glycophorin A, the distance is just
6.4 Å (35). Prolines at position 67 of ArcB and positions 166 and
173 in QseC disrupt the helical hydrogen bond patterns (see the1 H
exchange data for ArcB(1-115) in Fig. S4) and create kinks of
22  2° [ArcB(1-115)], 22  5° and 24  4° [QseC(1-185)] in the
second helix, adding local flexibility to the helices and increasing
the interhelical distances near the periplasmic side of the mem-
brane, thus additionally weakening the helix–helix interactions.
The TM domain of KdpD comprises a four-helix bundle
(Figs. 1B and 5C, and Table S3), in which the second and third

COMPUTATIONAL BIOLOGY
helices are relatively short (23–24 Å) and loosely packed with the

BIOPHYSICS AND
crossing angle of −165  6° and the interhelical distance of
∼9.4 Å. The second helix interacts with the first and third helices
only. The first and the fourth helix show the crossing angle of
−157  4°. These two helices weakly interact only near their cy-
toplasmic ends and this is the only consistent interaction involving
the first helix, which causes the whole bundle to be packed rather
loosely. It is possible that the loose helical packing observed in all
three structures could be an artifact of the detergent solubiliza-
tion or particular choice of HKR truncation. Nonetheless, the Fig. 5. Twenty superimposed structures of the TM domains of (A) ArcB(1-115),
TM structures lead us to surmise that the loose helical packing (B) QseC(1-185), and (C) KdpD(397-502). Backbones are shown for the stable
provides an inherent flexibility in the TM domains and that this is regions: ArcB(1-115), residues 20-83; QseC(1-185), residues 10-38; and 156-185
perhaps essential to the mechanism of signal transduction across and KdpD(397-502), residues 397-502. Consecutive TM helices are colored in
the membrane. The idea needs further verification including the following order: green, blue, orange, and coral. Structures on the right
computational dynamics simulation studies. are rotated 90° vertically.

Discussion the impetus is usually to remove offending domains (those that

We have taken a deconstructionist approach to tackling the
problems of MP structure determination. Because the size and
mobility of MPs are limiting factors in NMR analysis, the study
of individual domains has obvious advantages. Although domain-
wise structure determination is nothing new to crystallography,
inhibit crystallization), which in the case of MPs is most often
the membrane domain itself, or conversely engineering artificial
soluble protein domains into MPs in order to obtain diffraction
quality MP crystals (36). NMR techniques, on the other hand,
are more amenable to the structural study of flexible membrane

Maslennikov et al. PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 ∣ 10905
Table 1. Summary of NMR data and statistics for the calculated sets tion process. The CDL strategy, founded in the synergy between
of 20 lowest energy structures of ArcB(1-115), QseC(1-185), and the CF and NMR methods, opens up new possibilities for fast
KdpD(397-502) determination of backbone structures of MPs, especially those
ArcB(1-115) QseC(1-185) KdpD(397-502)
recalcitrant to crystallization. It is also complementary with
the recently proposed approach by Raman et al. (42) for fast de-
NMR distance constraints termination of backbone structure of large globular proteins.
NOE* 72 — — Backbone structures determined quickly by the CDL strategy
PRE 291 295 845
would provide excellent starting points for high-throughput
Hydrogen bonds 31 28 56
NMR dihedral angle modeling of a large number of classes of integral MPs and further
constraints structure-function prediction.
Phi 37 42 72
Psi 37 42 72 Materials and Methods
Structures statistics† Protein Expression, Characterization, and NMR Sample Preparation. ArcB(1-115)
Violations (mean and SD) was expressed with a thrombin-cleavable N-terminal His9 tag in E. coli BL21
Distance constraints, Å 0.80 ± 0.01 1.18 ± 0.12 0.72 ± 0.02 DE3 cells (Invitrogen) and purified as described in SI Materials and Methods.
Dihedral angle 10.3 ± 1.70 10.3 ± 0.40 5.70 ± 0.51 ArcB(1-115), QseC(1-185), and KdpD(397-502) were expressed as p-CF in the
constraints, ° absence of detergents (19). Washed precipitate was solubilized in 300 μL
Max. distance 0.17 ± 0.01 0.27 ± 0.03 0.22 ± 0.02 5% ðwt∕volÞ 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]
violation, Å (Avanti Polar Lipids; Anatrace), 20 mM Mes-BisTris pH 5.5 [ArcB(1-115)] or
Max. dihedral angle 1.74 ± 0.01 2.12 ± 0.40 0.81 ± 0.01 pH 6.0 [QseC(1-185) and KdpD(397-502)]. The proteins were characterized
violation, ° by SDS-PAGE, surface-enhanced laser desorption/ionization MS analysis,
Backbone rms deviation, Å and light scattering coupled with size-exclusion chromatography and refract-
Average pairwise 1.45 ± 0.45 2.35 ± 0.85 1.61 ± 0.48 ing index measurements (Fig. S2). Preparation of the samples for H-D ex-
in the set change experiments, solid-state NMR, and PRE measurements is described
To the mean structure 1.41 ± 0.46 2.18 ± 0.86 1.56 ± 0.49 in SI Materials and Methods.
Equivalent resolution‡ (Å) 2.9 3.2 2.7
NMR Experiments. Solid-state NMR, 2D 13 C-DARR, experiments (43) were
*Interresidue sequential (ji − jj ¼ 1) constraints only.
† performed on Bruker AVANCE 850 spectrometer (Centre for Biomolecular
Applied in TM helical regions.
‡ Magnetic Resonance). High-resolution NMR spectra of ArcB(1-115) expressed
Calculated by PROCHECK program (33).
in E. coli were recorded at 45 °C on a Bruker AVANCE 900 MHz spectrometer
(Korea Basic Science Institute). NMR spectra of TM domains of ArcB, QseC,
domains. However, this does not mean that such domains neces- and KdpD expressed in the CF system were recorded at 45° and 37 °C on a
sarily retain their native states, and the interpretation of the struc- Bruker AVANCE 700 MHz spectrometer (Salk). Details of NMR experiments
tures needs to be made with this caveat in mind. are described in SI Materials and Methods.
The hallmark feature of these three TM domain structures
appears to be their loose helical packing. The packing of TM CDL Strategy. The CDL strategy comprises four steps: (i) designing the com-
binatorial labeling scheme with the MCCL program; (ii) parallel expression of
α-helices is related to protein function and can be rigid, as in
6–7 CDL samples using the p-CF expression system (0.5–1.0 mL of reaction
the case of channel pores like KcsA (37), ionotropic receptors mixture for each CDL sample) and solubilization in the same buffer to elim-
like nAChR (38), the glutamate receptor channel (39), and tightly inate any differences in cross-peak positions; (iii) short measurement of
packed multihelical proteins like membrane respiratory enzymes ½1 H-15 N
-TROSY-HSQC and 2D-HNCO spectra (about 0.5–1.5 h per spectrum)
(40), or flexible, as observed in the case of many metabotropic for each CDL sample, all the samples for the combinatorial assignment of a
membrane receptors like G protein-coupled receptors (36) and particular protein were measured in only 1–2 days, depending on the
kinase receptors. The majority of the solved structures of integral concentration of the protein; (iv) analysis of the spectra and assignment
MPs (>97%) represents proteins that actively or passively trans- of 1 H-15 N cross-peaks using the CARA program (44).
port something (a molecule, ion, proton, or electron) across the
Structure Calculation and Analysis. The 13 Cα chemical shift deviations from
biological membrane (channels and transporters) and proteins
random coil values were used to define backbone torsion angle restraints
that tightly bind another molecule to catalyze a reaction (oxi- (45). Sequential distance constraints were derived from the integral intensi-
dases, ATPases, intramembrane proteases, etc.). The metabotro- ties of NOE cross-peaks measured in 3D 15 N-resolved TROSY-½1 H;1 H
-NOESY
pic membrane receptors are still a much underrepresented family (mixing time 120 ms). The long-range distance constraints were derived by
in the PDB. Their primary role in a cell is to transmit signals analysis of the PRE effect as described (18). An interactive procedure, which
through the membrane. Therefore, they do not require a well- included structure calculation by the CYANA program (46) followed by the
defined conformational state of the TM domain that would be distance constraints refinement, was used to calculate the backbone spatial
needed, for example, for coordinating transported ions or mole- structures of ArcB(1-115), QseC(1-185), and KdpD(397-502). The summary of
the constraints used in the calculation of the structures is presented in Table 1.
cules. On the contrary, in order to transmit a signal, the TM
The 20 conformers with the lowest target function of the last CYANA calcu-
domain must act as a conformational switch, an action which lation cycle were energy-minimized using the CNS program (47). The helical
can be supported by its intrinsic mobility (41). packing parameters, such as interhelical crossing angles, interhelical dis-
Furthermore, although it is known that HKRs act as dimers in tances, and helical kinks, were subsequently analyzed with the Helix Packing
the membrane, the fact that all of the structures are monomers Pair program (48).
can be explained by the absence of the cytoplasmic dimerization
domain in our constructs. The dimerization of HKRs is unlikely Point-Directed Assignment. The point-directed assignment is based on selec-
mediated by TM domains as reflected by their loose helical pack- tive dual-isotope (15 N and 13 C) labeling of two types of amino acids, located
ing. Further studies need to be done to address the significance consecutively in a protein sequence. For every pair of residues in which the
first amino acid is labeled with 13 CO and the second amino acid is labeled with
of the loose helical packing of the transmembrane signaling 15 NH , cross-peaks in both the ½15 N-1 H
-HSQC and the HNCO spectra arise (tag
mechanism of HKRs. “2” in Fig. 4A). If the second amino acid in a pair is labeled with 15 NH and the
first amino acid is not labeled with 13 CO , there will only be a cross-peak in the
Concluding Remarks HSQC spectrum (tag “1” in Fig. 4A). For pairs in which the second amino acid
The three structures presented in this study offer a glimpse into is not labeled with 15 N, there will be no cross-peaks in either spectrum (tag
the abundant class of 2-4 TM crossers, underrepresented in the “0” in Fig. 4A). Thus, by analyzing the presence and absence of cross-peaks in
PDB, and provide an important inroad toward understanding the the ½15 N-1 H
-HSQC and the HNCO spectra recorded from a selectively labeled
mechanistic aspects of the conformation-driven signal transduc- sample, one can define the type of amino acids for backbone resonances

10906 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1001656107 Maslennikov et al.

participating in these cross-peaks and bind them to a particular amino acid
pair. If a pair is unique in the sequence, an exact assignment of the 1 HN , 15 NH ,
and 13 CO resonances to the residues that comprise that pair is instantly made.
Numerically, each pair of residues in a given sample is specified by a tag
depending on its labeling combination, as explained in Figure 4A. Therefore,
in a series of combinatorially labeled samples a pair of residues is defined by a
series of tags, that is, a code which is directly related to the presence or ab-
sence of cross-peaks in both spectra for each sample. The scheme-designed
code which is identical with the code derived from the recorded spectra
defines a pair of amino acids for the 1 HN , 15 NH , and 13 CO resonances. If
the code is unique, i.e., there is only one pair in the sequence it corresponds
to, the assignment of the 1 HN and 15 NH resonances to the second residue, as
well as the assignment of the 13 CO resonance to the first residue of the pair, is
made. This simple analysis provides an unambiguous assignment for
∼30–40% of the backbone 1 HN , 15 NH , and13 CO resonances and defines the
amino acid type for the rest of the backbone 1 HN , 15 NH , and 13 CO resonances,
limiting the number of their possible positions in a sequence to as few as 2–4.
1. Wolanin PM, Thomason PA, Stock JB (2002) Histidine protein kinases: Key signal
transducers outside the animal kingdom. Genome Biol 3(10):REVIEWS3013.
2. Etzkorn M, et al. (2008) Plasticity of the PAS domain and a potential role for signal
transduction in the histidine kinase DcuS. Nat Struct Mol Biol 15:1031–1039.
3. Rogov VV, et al. (2006) A new structural domain in the Escherichia coli RcsC hybrid
sensor kinase connects histidine kinase and phosphoreceiver domains. J Mol Biol
364:68–79.
4. Marina A, Mott C, Auyzenberg A, Hendrickson WA, Waldburger CD (2001) Structural
and mutational analysis of the PhoQ histidine kinase catalytic domain. Insight into the
reaction mechanism. J Biol Chem 276:41182–41190.
5. Tanaka T, et al. (1998) NMR structure of the histidine kinase domain of the E. coli
osmosensor EnvZ. Nature 396:88–92.
6. Tomomori C, et al. (1999) Solution structure of the homodimeric core domain of
Escherichia coli histidine kinase EnvZ. Nat Struct Biol 6:729–734.
7. Ikegami T, et al. (2001) Solution structure and dynamic character of the histidine-con-
taining phosphotransfer domain of anaerobic sensor kinase ArcB from Escherichia coli.
Biochemistry 40:375–386.
8. Kato M, Mizuno T, Shimizu T, Hakoshima T (1997) Insights into multistep phosphorelay
from the crystal structure of the C-terminal HPt domain of ArcB. Cell 88:717–723.
9. Rogov VV, Bernhard F, Lohr F, Dotsch V (2004) Solution structure of the Escherichia coli
YojN histidine-phosphotransferase domain and its interaction with cognate
phosphoryl receiver domains. J Mol Biol 343:1035–1048.
10. Pappalardo L, et al. (2003) The NMR structure of the sensory domain of the
membranous two-component fumarate sensor (histidine protein kinase) DcuS of
Escherichia coli. J Biol Chem 278(40):39185–39188.
11. Cheung J, Bingman CA, Reyngold M, Hendrickson WA, Waldburger CD (2008)
Crystal structure of a functional dimer of the PhoQ sensor domain. J Biol Chem
283:13762–13770.
12. Cheung J, Hendrickson WA (2009) Structural analysis of ligand stimulation of the
histidine kinase NarX. Structure 17:190–201.
13. Moore JO, Hendrickson WA (2009) Structural analysis of sensor domains from the
TMAO-responsive histidine kinase receptor TorS. Structure 17:1195–1204.
14. McLuskey K, Roszak AW, Zhu Y, Isaacs NW (2010) Crystal structures of all-alpha type
membrane proteins. Eur Biophys J 39(5):723–755.
15. Kim HK, Howell SC, Van Horn WD, Jeon YH, Sanders CR (2009) Recent advances
in the application of solution NMR spectroscopy to multi-span integral membrane
proteins. Prog Nucl Mag Res Sp 55:335–360.
16. White SH (2009) Biophysical dissection of membrane proteins. Nature 459(7245):
344–346.
17. Battiste JL, Wagner G (2000) Utilization of site-directed spin labeling and high-resolu-
tion heteronuclear nuclear magnetic resonance for global fold determination of large
proteins with limited nuclear overhauser effect data. Biochemistry 39:5355–5365.
18. Roosild TP, et al. (2005) NMR structure of Mistic, a membrane-integrating protein for
membrane protein expression. Science 307:1317–1321.
19. Klammt C, et al. (2004) High level cell-free expression and specific labeling of integral
membrane proteins. Eur J Biochem 271:568–580.
20. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia
coli. Nat Biotechnol 22:1399–1408.
21. Wishart DS, Sykes BD (1994) The 13C chemical-shift index: A simple method for the
identification of protein secondary structure using 13C chemical-shift data. J Biomol
NMR 4:171–180.
22. Kainosho M, Tsuji T (1982) Assignment of the three methionyl carbonyl carbon
resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15
double-labeling technique. A new strategy for structural studies of proteins in
solution. Biochemistry 21:6273–6279.
Maslennikov et al.
CDL Assignment Process. The CDL assignment process is demonstrated
for three KdpD(397-502) cross-peaks in Figure 4C. Here, an 1 H-15 N cross-peak
for residue C is present in the TROSY spectra of samples I, III, IV, and V and
in the HN plane of the HNCO spectrum of sample IV, therefore, its code is
101210 (the digit place corresponds to sample number). This code is
unique and corresponds to the Phe481-Ala482 pair in the sequence, which
gives us an unambiguous assignment for Ala482. Cross-peak B has the
code 021102 and was assigned to three possible Ala-Val pairs in the
sequence (Val411/472/483). Cross-peak A has the code 011101 and was
assigned to nine possible pairs, with Val as the second amino acid in
every pair and Arg, Val, or Thr, which are not labeled by 13 C, as the first
amino acid.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health (GM074929), Incheon Free Economic Zone, and World-Class University
program (S.C.), and Korea Membrane Protein Initiative and 21C Frontier
Microbial Genomics program (Y.H.J.). C.K. was partly supported by Deutsche
Forschungs Gemeinschaft Research Fellowship.
23. Yabuki T, et al. (1998) Dual amino acid-selective and site-directed stable-isotope label-
ing of the human c-Ha-Ras protein by cell-free synthesis. J Biomol NMR 11:295–306.
24. Weigelt J, van Dongen M, Uppenberg J, Schultz J, Wikström M (2002) Site-selective
screening by NMR spectroscopy with labeled amino acid pairs. J Am Chem Soc
124:2446–2447.
25. Shi J, Pelton JG, Cho HS, Wemmer DE (2004) Protein signal assignments using specific
labeling and cell-free synthesis. J Biomol NMR 28:235–247.
26. Parker MJ, Aulton-Jones M, Hounslow AM, Craven CJ (2004) A combinatorial selective
labeling method for the assignment of backbone amide NMR resonances. J Am Chem
Soc 126:5020–5021.
27. Trbovic N, et al. (2005) Efficient strategy for the rapid backbone assignment of
membrane proteins. J Am Chem Soc 127:13504–13505.
28. Wu PS, et al. (2006) Amino-acid type identification in 15N-HSQC spectra by combina-
torial selective 15N-labelling. J Biomol NMR 34:13–21.
29. Ozawa K, Wu PS, Dixon NE, Otting G (2006) N-Labelled proteins by cell-free protein
synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand
complexes. FEBS J 273:4154–4159.
30. Sobhanifar S, et al. (2010) Cell-free expression and stable isotope labelling strategies
for membrane proteins. J Biomol NMR 46:33–43.
31. Wüthrich K (1986) NMR of Proteins and Nucleic Acids (Wiley, New York), pp 130–161.
32. Clore GM, Gronenborn AM (1994) Multidimensional heteronuclear nuclear magnetic
resonance of proteins. Methods Enzymol 239:349–363.
33. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: A program to
check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291.
34. Gordeliy VI, et al. (2002) Molecular basis of transmembrane signalling by sensory
rhodopsin II-transducer complex. Nature 419:484–487.
35. MacKenzie KR, Prestegard JH, Engelman DM (1997) A transmembrane helix dimer:
Structure and implications. Science 276:131–133.
36. Cherezov V, et al. (2007) High-resolution crystal structure of an engineered human
beta2-adrenergic G protein-coupled receptor. Science 318:1258–1265.
37. Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R (2001) Chemistry of ion
coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.
Nature 414:43–48.
38. Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A
resolution. J Mol Biol 346:967–989.
39. Sobolevsky AI, Rosconi MP, Gouaux E (2009) X-ray structure, symmetry and mechanism
COMPUTATIONAL BIOLOGY
of an AMPA-subtype glutamate receptor. Nature 462:745–756.
40. Wittig I, Schagger H (2009) Supramolecular organization of ATP synthase and respira-
BIOPHYSICS AND
tory chain in mitochondrial membranes. Biochim Biophys Acta 1787:672–680.
41. Hendrickson WA (2005) Transduction of biochemical signals across cell membranes.
Q Rev Biophys 38:321–330.
42. Raman S, et al. (2010) NMR structure determination for larger proteins using back-
bone-only data. Science 327:1014–1018.
43. Takegoshi K, Nakamura S, Terao T (2001) 13C-1H dipolar-assisted rotational resonance
in magic-angle spinning NMR. Chem Phys Lett 344:631–637.
44. Keller R (2004) The Computer Aided Resonance Assignment Tutorial (Cantina Verlag,
Goldau, Switzerland).
45. Luginbuhl P, Szyperski T, Wuthrich K (1995) Statistical basis for the use of 13Calpha
chemical shifts in protein structure determination. J Magn Reson Ser B 109:229–233.
46. Guntert P (2004) Automated NMR structure calculation with CYANA. Method Mol Biol
278:353–378.
47. Brunger AT, et al. (1998) Crystallography & NMR system: A new software suite for
macromolecular structure determination. Acta Crystallogr D 54:905–921.
48. Dalton JA, Michalopoulos I, Westhead DR (2003) Calculation of helix packing angles in
protein structures. Bioinformatics 19:1298–1299.
PNAS ∣ June 15, 2010 ∣ vol. 107 ∣ no. 24 ∣ 10907