HEMATOPOIESIS1,2,3,4

SCF is a glycosylated, small-protein growth factor found in both membrane-

bound and soluble forms. The membrane-bound form is expressed on the surface
of bone marrow stromal cells and acts synergistically with other circulating
hematopoietic growth factors (M-CSF, GM-CSF, G-CSF, IL-3, erythropoietin,
etc.) to stimulate the proliferation of hematopoietic stem cells (HSCs) which
lead to a wide variety of cell lineages including the erythrocytic,
granulocytic and megakaryocytic lineages. Thus, SCF is a crucial component
of the hematopoietic microenvironment. On the stromal cell surface, SCF also
functions as a chemoattractant and adhesion structure for early hematopoietic
stem cells. The function of soluble SCF is unknown.

MAST CELL BIOLOGY5,6,7 & OTHER FUNCTIONS8,9

SCF also plays an important role in mast cell biology and thus also goes by
the name of mast-cell growth factor (MGF). Similar to its hematopoietic
functions, SCF acts to stimulate the proliferation and maturation of mast
cells in the peripheral tissues. In addition, SCF has been shown to
stimulate mast cell degranulation to release mediators such as serotonin and
histamine. SCF also plays a role in early germ cell development in which it
both enhances the proliferation and maintains the survival of primordial germ
cells (PGCs). SCF is also involved in melanocyte biology (possibly as a
chemoattractant) and in this way influences skin color.

DISCOVERY of SCF1,2
SCF was discovered through a classic combination of genetics and
biochemistry. It had been known for some time that two distinct genetic loci
(SI and W) were responsible for a similar set of mutant phenotypes in mice.
Both of these mutant phenotypes involved a typical triad of hematopoietic
defects, coat color alterations and defective gonadal development. Since the
W gene encodes the c-kit tyrosine kinase receptor, the search was on for the
ligand for this receptor. A protein that bound to the c-kit product was
isolated and purified which was also shown to map to the SI locus. Thus the
fact that SI encoded SCF and W encodes the c-kit product provided a cogent
biochemical explanation for the genetics.

CLINICAL ASPECTS10,11
Pathologically, SCF acts as an autocrine factor in some leukemias and some
small-cell lung cancers. Some anemias arising from bone-marrow failure may
also involve defects in the SCF/c-kit system. Therapeutically, SCF
antagonists may be useful agents in combating cancers that involve SCF as an
autocrine factor. SCF also has potential use in ameliorating various
acquired anemias (such as those resulting from chemotherapy) as well as
facilitating stem-cell mediated gene transfer & gene therapy.

REFERENCES
1 Martin FH, et al., (1990), Cell, 63:203.
2 Zsebo KB, et al., (1990), Cell, 63:195.
3 Migliaccio G, et al., (1991), J Cell Physiol., 148:503.
4 Andrews RG, et al., (1991), Blood, 78:1975.
5 Anderson DM, et al., (1990), Cell, 63:235.
6 Meininger CJ, et al., (1992), Blood, 79:958.
7 Tsuji K, Zsebo M, Ogawa M, (1991), J Cell Physio., 148:362.
8 Dolci S, et al., (1991), Nature, 352:809.
9 Matsui Y, et al., (1991), Nature, 353:750.
10 Cassel A, et al., (1993), Exp Hematol., 21:585.
11 Rygaard K, et al., (1993), Br J Cancer, 67:37.
HEMATOPOIESIS

MAST CELL BIOLOGY

DISCOVERY of SCF
SEQUENCE ANALYSIS1,12
Full-length SCF is composed of 248 amino acids and includes extracellular,
transmembrane and cytoplasmic domains. Soluble SCF is 164/165 amino acids
long and shares ~16% identity (~32% similarity) with another hematopoietic
growth factor M-CSF. M-CSF, in turn, has been found to be a member of a
growing list of growth factors (such as IL-4, GM-CSF, Growth Hormone, etc.)
which share a 4 -helix bundle folding motif.

GLYCOSYLATION13,14
SCF undergoes rather extensive and heterogeneous N-linked & O-linked
glycosylation. However, the absence of glycosylation at normally
glycosylated sites does not significantly affect receptor binding or
biological activity. It is interesting to note that in human SCF one of the
glycosylation consensus sequences (at Asn120) is always glycosylated while
another such consensus sequence (at Asn72) is never glycosylated. O-linked
glycosylation occurs at residues 142,143 & 155 which are believed to be in
the flexible spacer domain just proximal to the transmembrane domain.

CONSERVED CYSTEINES & DISULFIDE BONDS14

Among the various species variants of SCF, four cysteines are absolutely
conserved. These have been shown to form two disulfide bonds at Cys 4-Cys89
and Cys43-Cys138.

SECONDARY STRUCTURE13
Circular dichroism studies show significant -helical secondary structure
ranging between 38% - 47% depending on the type of theoretical analysis used
to interpret the spectroscopic data. According to the CD results, about 33%
-sheet structure is also present.

DIMERIZATION13,15,16
Sedimentation equilibrium and gel filtration analyses indicate that SCF forms
non-covalently linked homo-dimers. Activation of the c-kit receptor has been
shown to involve receptor dimerization. Dimerization of SCF (the c-kit
ligand) may facilitate this signal transduction process.

REFERENCES
1 Martin FH, et al., (1990), Cell, 63:203.
12 Bazan, JF, (1991), Cell, 65:9.
13 Arakawa, T, et al., (1991), J Biol Chem., 266:18942.
14 Lu, HS, et al., (1991), J Biol Chem., 266:8102.
15 Arakawa T, et al., (1992), Anal Biochem., 203:53.
16 Lev S., et al., (1992), J Biol Chem., 267: 15970.
SEQUENCE ANALYSIS

N-Linked Glycosylation

Asn120 Glycosylated

Asn65 Some glycosylation

(accounts for heterogeneity)

Asn72 Not glycosylated

SECONDARY STRUCTURE13
l
RESTRICTED CONDITIONS
Initial crystallization trials of the 165 amino acid-long soluble form of SCF
were unsuccessful. The high solubility of this protein as well as the
postulated flexibility of the spacer domain proximal to the transmembrane
domain may account for these difficulties with crystallization. We
undertook, therefore, crystallization efforts with a 141 amino acid-long
truncated form of SCF which still retains receptor binding capability and
biological activity. In this case, crystallization trials were successful
but the conditions were highly restricted with even small changes in
precipitant concentration, pH and temperature having adverse effects. For
example, crystals grew in our warm room at about 22-23C but did not grow in
the 20C incubators. The optimum conditions for crystallization are shown in
the panel on the right. Until all of these conditions could be precisely
determined, reproducibility was a frustrating problem.

MULTIPLE ORTHORHOMBIC FORMS

Several closely related orthorhombic forms were grown sometimes within the
same droplet. One of these (form B) had relatively small unit cell
dimensions and diffracted very well. Unfortunately, this crystal was
impossible to reproduce with A form (with much poorer characteristics)
dominating. Stabilization protocols (see below) made it possible to observe
suitable diffraction from some of these A form crystals.

CALCIUM REQUIREMENT
Crystal growth was absolutely dependent on calcium (0.25M). Interestingly,
crystals did not grow with other divalent cations such as Mg, Zn or various
lanthanides; use of these cations resulted in heavy precipitation. However,
crystals treated with 10 mM EDTA were not disrupted suggesting that calcium
was not essential for crystal packing.

STABILIZATION
Stabilization of the crystals proved to be essential in improving diffraction
as well crystal lifetime. Unstabilized crystals diffracted rarely beyond
3.8Å spacings and typically had an x-ray lifetime of less than 18 hours.
Stabilization with slightly higher precipitant concentrations and a lower pH
(pH 7.2 as opposed to pH 7.5 during crystal growth) greatly improved
diffraction to beyond 3.0Å and increased the crystal lifetime to beyond 100
hours.

MISCELLANEOUS ISSUES
Addition of the crystallization conditions with -octyl glucoside was
unproductive. In addition crystallization with MOPS or TRIS at the
equivalent or slightly different pH ranges resulted in poorly formed
crystals. HEPES was the only effective buffer at the crystallization pH of
7.5. The crystals were moderately biaxially birefringent. For diffraction
purposes, the best crystals measured 0.35  0.25  0.25 mm.
DIFFRACTION PARAMETERS
0-level precession photographs were used to determine unit cell dimensions as
well as Laue group symmetry. As mentioned before, several orthorhombic
crystal forms were obtained many of which had very similar unit cell
dimensions and varying diffraction quality. Inspection of the morphology did
not lend any clues as to which form a particular crystal belonged to. This
of course complicated our initial analysis of the diffraction. The best
crystals had unit cell dimensions of a=88.1Å, b=83.4Å, c=72.7Å. Based on
these results, four molecules per asymetric unit gave the most plausible
solvent content (46%). These results were also confirmed using a biochemical
analysis based on amino acid composition (see the panel on PACKING MODEL).
Initial diffraction patterns showed 90C reciprocal lattice angles, mm
symmetry each of the 0-level nets, and systematic absences of odd reflections
along all three axes h, k, and l. The space group of these protein crystals
is thus P212121.

SCF hk TRANSFORM17,18,13
Unusual diffraction patterns can sometimes indicate large-scale structural
features in the crystal structure. This fact was used by Watson & Crick to
lead them to the double-helical structure of DNA. A similar situation may be
possible with SCF although a closer analogy would be the crystallographic
studies of bacteriorhodopsin or more recently a leucine zipper. In the case
of bacteriorhodopsin strong reflections are observed at 10Å and 5Å spacings.
These patterns, termed the 10Å band & 5Å band are mutually perpendicular to
each other and reflect (no pun intended) the interhelical spacings and
helical-rise spacings respectively of this predominantely -helical membrane
protein. The SCF hk transform also shows a stronger ring of diffraction at
about 9.25Å spacings along the k-axis. The presence of this pattern may
indicate an approximately side-by-side packing of the -helices in SCF along
the k(c) axis. This data is consistent with the largely -helical content of
SCF as determined by CD & sequence analysis.

SCF hl TRANSFORM
Both a 10Å band (10.5Å) along the l-axis and a 5Å band (5.2Å) along the h-
axis are observed in the SCF hl 0-level net. This is suggestive of the -
helices lining up along the h(a) axis and packing side-by-side along the l
axis. Of course, for this to be possible all four molecules in the asymetric
unit must be oriented in approximately the same direction. Such a packing
model is consistent with Patterson self-rotation searches we have computed
with native data (see the SELF-ROTATION panels).

REFERENCES
13 Arakawa, T, et al., (1991), J Biol Chem., 266:18942.
17 Blaurock, A, J Mol Biol.
18 Rasmussen, R., et al., (1991), Proc Natl Acad Sci USA, 88:561.

DIFFRACTION PARAMETERS

Unit cell dimensions a=88.1Å, b=83.4Å, c=72.7Å.

Za 4

Solvent content 46%

Space Group P212121

BACTERIORHODOPSIN TRANSFORM

SCF hk TRANSFORM

HELIX PARAMETERS
SCF hl TRANSFORM
lDATA COLLECTION STRATEGY

The most important factor in data collection was the stabilization and
careful handling of the SCF crystals. Even with such care, data collected
was often poor. For example, data collection from one crystal gave an Rsym
of 0.087 out to 3.8Å but rapidly decayed in quality further out. Another
crystal gave reliable and complete data out to 3.0Å and some data out to
2.8Å. Data was collected on a Hamlin area detector using a home x-ray source
at 50kV and 100mA. Our best native data set was collected from a single
crystal for a total irradiation time of 42 hours. Three orientations were
used to meet the competing demands of crystal decay, data completeness and
adquate overlap for merging statistics. Attempts to collect data beyond 2.8Å
never succeeded although there is hope that using a synchrontron source may
substantially improve the observable diffraction.

DATA REDUCTION

The data was reduced using the CCP4 crystallographic computing package
(ROTAVATA & AGROVATA). The data totaled 21,028 measurements with 5934 of
these independent. The cumulative Rsym was 0.039 out to 2.8Å although by
this point the data was rapidly falling off in quality since the last shell
(2.87-2.8Å) had an Rsym of 0.377. The fact that the data looks good at high
intensities points favorably to the possibility that data collection at a
synchrontron may prove useful. Note also that the average intensity tends to
rise between 5.5Å and 4.5Å which is a relatively common phenomenon but in
this case may also be indicative of the diffraction arising from the helical
rise repeats (see DIFFRACTION panel).

PSEUDO-CENTERING

As a possible confirmation of potential packing models, a rough search for

the presence of pseudo-centering was conducted. Ratio's of even to odd
reflections (k+l,h+l,h+k) were computed and nothing indicative of psuedo-
centering was observed either at low or high resolution.
AGVROATA RESULTS
SELF-ROTATION FUNCTIONS
We know (see BIOCHEMISTRY panel) that SCF naturally forms homo-dimers. In
addition, since apparently there are four molecules per asymmetric unit we
would like to characterize the non-crystallographic symmetry of these
crystals. Patterson self-rotation searches can be used to find such non-
crystallographic symmetry elements. The basic idea is to compute a native
Patterson map, systematically rotate this map by a specified angle and then
compare it to the original, unrotated map. A high correlation between the
rotated and unrotated map is indicative (although not necessarily so) of
crystallographic or non-crystallographic symmetry. In the latter case may be
masked by noise or other such uncertainties. In any case, the search for a
dimer axis should reveal strong peaks when searching at  = 180. Several
programs are used to calculate these self-rotation functions and the three we
have used are MERLOT, GLRF and XPLOR. These results are described below.

MERLOT
The MERLOT plot ( = 180) is shown on the right side of this panel. Data
used for the calculation range from 35 to 10Å and a 30Å Patterson radius
cutoff. Many such plots were produced with varying data limits and cutoff
radii. The most common peaks were at =90 and =12, =78; the two peaks
are symmetrically related. The peaks look curiously elongated in the
direction of the a*-axis. This suggests that each of these peaks is actually
a summation of two unresolved peaks. An analysis of peak heights (from the
GLRF program discussed below) shows that the height of these fused peaks
(being about 1/2 as large as teh origin peak) is to large for it to be a
single peak. In addition, since there are actually two dimers in the
asymmetric unit, we should likewise see two dimer axes. Given the fact that
the two SCF dimers are equivalent to each other, it would be highly
improbable that only one of these axes would be apparent. Our conclusion is
that the two dimer axes are on either side of the fused peak. The two dimer
axes are approximately:

=81 =78

=99 =78

The other possibility is that one axis is along the c* axis and the other
two-fold is just flanking it.

GLRF18
Prior knowledge about non-crystallographic symmetry can be used in GLRF to
enhance self-rotation function peak heights. In our calculations we
presupposed the existence of a two-fold axis so that the GLRF program would
rotate the Patterson by 180 for each calculated comparison. A plot as well
as quantitative data is shown on the right side of this panel. Note the
fused peaks (1 & 2) and the flanking peaks (3 & 4).
REFERENCES
18 Tong L, and Rossmann M, (1990), Acta Cryst., A46:783.

MERLOT

GLRF
THE "DIMER-DIMER" AXIS
After locating the two-folds relating each of the two dimers we need now to
find the non-crystallographic symmetry element relating the two dimers to
each other. We refer to this as the "dimer-dimer" axis. Because this axis
relates all four molecules to each other we would expect the corresponding
Patterson self-rotation function peak height to be rather large. Because the
search is throughout the complete angle space, we have used XPLOR to
calculate the self-rotation functions. The results are given below.

XPLOR SELF-ROTATION FUNCTION CALCULATIONS

There are two possibilities for this "dimer-dimer" axis. As can be seen on
the right side of this panel, XPLOR shows similar results under a wide
variety of data limits & Patterson cutoff parameters. Two large peaks
invariably show up.

The =0, =7.5, =180 peak

This first peak is also visible in the MERLOT plot as an elongation of the
origin peak along the a*-axis where the axis points out approximately in the
same direction as the b*-axis. Unfortunately, because it is so close to the
origin peak it is difficult to properly evaluate; this peak, for example, is
not picked up by the GLRF package. In any case, if we consider the M-CSF
dimer structure as prototypical, a "dimer-dimer" axis in the b*-axis
direction would suggest that the helices are oriented approximately in the b-
axis direction. According to our general analysis of the diffraction (see
the DIFFRACTION panel) the helices are oriented in the a-axis direction.
Because of its proximity to the origin and its inconsistency with the other
diffraction results the next peak may be a more plausible candidate for the
"dimer-dimer" axis.

The =0, =90, =15 peak

Since in this case   180, this peak does not correspond to a perfect two-
fold. The axis in this case, is approximately oriented in the direction of
the a*-axis so that all the helices would now point in the direction of the
a-axis. This is consistent with the model discussed in the DIFFRACTION
panel. The strength and consistency of this peak using the XPLOR package is
also impressive and we regard this peak as the most plausible one
corresponding to the "dimer-dimer" axis.
XPLOR RESULTS: Rmax=8Å,Rmin=5Å,Patrad=20Å

XPLOR RESULTS: Rmax=35Å,Rmin=4Å,Patrad=15Å

Za calculation19

In order to more definitively determine the number of molecules in teh

asymmetric unit we used a quantitative amino acid analysis protocol. This
protocol involves carefully measuring the macroscopic volume of a specific
crystal and then submitting this crystal to an amino acid analysis in order
to determine the number of molecules in the crystal and thus the number of
molecules in the asymmetric unit. The results of this study are summarized
in the chart on the right side of this panel.

PACKING MODEL

Based on the previously described self-rotation functions, a packing model

for the asymmetric unit can be proposed. All four monomors have their a-
helices approximately disposed in the direction of the a-axis. The two dimer
two-folds do not overlap since the axis relating the two dimers involves a
rotation of 15°.

REFERENCES

19 Kwong PD, et al., (1990), Proc Natl Acad Sci USA, 87:6423.
PHILOSOPHY OF MOLECULAR REPLACEMENT
SCF shares a common 4 -helix folding motif along with several other growth
factors and hormones such as GM-CSF, G-CSF, M-CSF, IL-4, Growth hormone, IL-
2. More such examples will no doubt be discovered in the near future. In
an attempt to begin obtaining phasing information we have tried to generate
conventional heavy atom as well lanthanide derivatives but to no avail.
Because of the structural similarity of these proteins we have attempted a
molecular replacement solution to the phase problem. In addition, with the
ubiquity of this folding motif, this study will also establish the
feasibility of using molecular replacement as a possible strategy for quickly
solving growth factor structures in the future.

TRIAL STRUCTURE20 and INITIAL RESULTS

Our first trial structure is the hematopoietic growth factor GM-CSF which is
a prototypical 4 -helix bundle but nevertheless has little sequence
similarity with SCF. In order to use our knowledge of the dimer two-fold, we
have used the GLRF package with a locked two-fold (whose orientation is
determined by the self-rotation functions). Patterson cross-rotation
searches (in a manner analogous to the self-rotation procedures) were
performed using the GM-CSF monomer as a trial structure. Results with and
without the locked two-fold are shown on the right side of this panel. Note
that there are four to five relatively high peaks which could potentially
correspond to solutions for the four SCF monomers in the asymmetric unit.
The peak heights for these "solutions" are enhanced when the GLRF calculation
is done with the locked two-fold. In addition, noise peaks have become less
prominent.

FURTHER STUDIES
The next step is to refine the dimer axes and re-do the cross-rotation
functions in the hope of enhancing the peaks of interest. The strategy then
would be to apply a translation function to search for the orientation and
position of one of the GM-CSF monomers. The refined non-crystallographic
symmetry elements would then be used to generate the second GM-CSF molecule
and a translation search would then be applied to this molecules. The third
and fourth molecules would be similarly generated and positioned. Variations
on this theme could include the use of the GM-CSF fold with a polyalanine
sequence or an averaging of several 4 -helix bundle proteins. Ideally, we
would like to use the M-CSF molecule since this is both more closely related
to SCF as well as shares its dimerization properties.

REFERENCES
20 Diederichs K, Boone T, and Karplus PA, (1991), Science, 254:1779.
GM-CSF TRIAL STRUCTURE

INITIAL GLRF RESULTS

THE M-CSF STRUCTURE21
The three-dimensional x-ray crystallographic structure of M-CSF has been
recently determined. As expected, it has a 4 -helix bundle folding motif
characteristic of other such growth factors. It shares about 16% sequence
identity with SCF and as with SCF it is a dimeric structure. However, unlike
SCF which is a non-covalently bound dimer, the M-CSF dimer is covalently
bound via a single intramolecular disulfide bond.

FEATURES OF THE SCF MODEL

Based on sequence comparisons and helical hydrophobicity moments a model
structure of SCF superimposed on a schematic rendition of M-CSF structure has
been proposed that corroborates some of the biochemical data. The conserved
cysteines, for example, easily fit into positions that allow the formation of
the two intramolecular disulfides. The first disulfide bond attaches the C-
terminus to the top of helix B while the second disulfide bond attaches the
N-terminus to the top of helix C. It has also been observed that not all of
the consensus sites for N-linked glycosylation are, in fact, glycosylated in
the native protein. In particular, Asn120 is always glycosylated, Asn69
sometimes glycosylated and Asn72 is never glycosylated. According the model
structure Asn120 is on the outer face of helix D while Asn72 is situated
towards the core of the molecule at the confluence of helices A and B.
Obviously glycosylation at this site would seriously disrupt the SCF
structure. Asn69 is situated on the outer edge of helix C. Other important
features of this model include the acidic surfaces of the helices, and the
conserved dimer interface.

ACIDIC RIDGES
The helices in SCF have rather marked hydrophobic moments: the interior
facing portions of the helices are quite hydrophobic while the exterior
surfaces are often charged. Helices B, C and D, in particular have a large
preponderance of negatively charged acidic aspartates and glutamates situated
on their surfaces. Helix B for example has an uninterrupted, continuous
ridge of five acidic residues on its surface. It is possible that the role
of divalent cations in both the precipitation and and crystallization of SCF
may in part be derived from the neutralization of these "acidic ridges" by
these cations.

CONSERVED DIMER INTERFACE

The region of the molecule distal to the C-terminus turns out to be
completely and strictly conserved among the SCFs from different species.
This region is also analogous to the dimer interface for M-CSF so it is very
likely that it too is the dimer interface for SCF. Knowledge of the dimer
site may help in designing SCF antagonists that may prove useful in cancer
chemotherapy, etc.

PROPOSED RECEPTOR BINDING SITE

Because the C-terminus leads towards the cell surface and the dimer interface
interacts with the other SCF monomer, it seems plausible to propose that the
receptor binding site is composed of the surfaces of helices A & C. A more
detailed knowledge of these surfaces in conjunction with mutant studies would
assist in the development of SCF agonists --potentially useful agents in
treating various clinical anemic states.
REFERENCES
21 Pandit J, et al., (1992), Science, 258:1358.
M-CSF STRUCTURE

SCF MODEL