Three-Dimensional Structure of Human Follicle-Stimulating Hormone
Kristin M. Fox,
James A. Dias and
Patrick Van Roey
Department of Chemistry (K.M.F.) Union College Schenectady,
New York 12308
Division of Molecular Medicine (K.M.F.,
J.A.D., P.V.R.) Wadsworth Center Albany, New York
12201-0509
 |
ABSTRACT
|
---|
The crystal structure of a ßThr26Ala mutant of
human follicle-stimulating hormone (hFSH) has been determined to 3.0 Å
resolution. The hFSH mutant was expressed in baculovirus-infected Hi5
insect cells and purified by affinity chromatography, using a
ßhFSH-specific monoclonal antibody. The ßThr26Ala mutation
results in elimination of the ßAsn24 glycosylation site, yielding
protein more suitable for crystallization without affecting the
receptor binding and signal transduction activity of the glycohormone.
The crystal structure has two independent hFSH molecules in the
asymmetric unit and a solvent content of about 80%. The
- and
ßsubunits of hFSH have similar folds, consisting of central
cystine-knot motifs from which three ß-hairpins extend. The two
subunits associate very tightly in a head-to-tail arrangement, forming
an elongated, slightly curved structure, similar to that of human
chorionic gonadotropin (hCG). The hFSH heterodimers differ only in the
conformations of the amino and carboxy termini and the second loop of
the ß-subunit (L2ß). Detailed comparison of the structures of hFSH
and hCG reveals several differences in the ß-subunits that may be
important with respect to receptor binding specificity or signal
transduction. These differences include conformational changes and/or
differential distributions of polar or charged residues in loops L3ß
(hFSH residues 6273), the cystine noose, or determinant loop
(residues 8794), and the carboxy-terminal loop (residues 94104). An
additional interesting feature of the hFSH structure is an extensive
hydrophobic patch in the area formed by loops
L1,
L3, and ßL2.
Glycosylation at
Asn52 is well known to be required for full signal
transduction activity and heterodimer stability. The structure reveals
an intersubunit hydrogen bonding interaction between this carbohydrate
and ßTyr58, an indication of a mechanism by which the carbohydrate
may stabilize the heterodimer.
 |
INTRODUCTION
|
---|
FSH is a member of the family of pituitary glycoprotein hormones
(GPH) that play key roles in human fertility. The GPH, which also
include CG, LH, and TSH, are heterodimers, each consisting of a common
-subunit (92 amino acids) and a unique ß-subunit (111 amino acids
in FSH) (1, 2, 3, 4). FSH acts by binding to G protein-coupled receptors that
signal, in part, through the protein kinase A pathway (5, 6). FSH
enables ovarian folliculogenesis to the antral follicle stage and is
essential for Sertoli cell proliferation and maintenance of sperm
quality in the testis. The amino acids in GPH that have been identified
as critical for receptor binding are striking in similarity, yet they
are not the residues essential for signal transduction (7).
Identification of GPH residues that are key to signal transduction
could provide for the development of molecular mimetics or antagonists
of GPH action, with clinical applications in fertility management (8)
and treatment of thyroid disorders (9).
Glycosylation of the GPH has been shown to be important in circulatory
persistence and clearance, and in bioactivity (10, 11, 12, 13, 14). Each subunit
contains two glycosylation sites: at Asn52 and Asn78 in the
-
subunit and at conserved sites in the ß-subunit, Asn7 and Asn24
in human FSH (hFSH). ß-Subunit glycosylation has been reported to
affect disulfide bond formation and rate of secretion, with site 2
having a greater effect than site 1, especially on secretion (15).
Glycosylation at
Asn78 appears to be important for thermal stability
(16). Deglycosylation of hFSH and hCG at
Asn52 has long been
accepted to impair signal transduction while allowing full binding
activity, suggesting that receptor binding and signal transduction are
two separate functions involving different residues and that the
carbohydrate is key to signal transduction. Recent evidence suggests
that deglycosylation at
Asn52 causes hCG to be metastable, and
dissociation of the subunits occurs at 37 C (17). In that study,
disulfide bonds engineered between the subunits could overcome the
effect of deglycosylation on signal transduction. Although such studies
have not been performed for hFSH, they obscure previous results with
deglycosylated GPH and demonstrate that the carbohydrate at
Asn52 is
not essential for full signal transduction if the subunit association
is otherwise stabilized. It remains unclear whether the carbohydrate,
or the lack of it, affects the structure of all GPH, or whether the
formation of intersubunit disulfide bonds stabilizes hCG in a
conformation that is signal transduction competent.
Previous structural studies of GPH heteroolimers are limited to two
independent reports of the crystal structure of human CG (hCG),
partially deglycosylated by hydrogen fluoride treatment (3, 4),
and a recent report of a low resolution structure of the ternary
complex of fully glycosylated hCG with two Fv fragments (18). With the
goal of determining the structure of fully active, glycosylated hFSH,
we achieved high-level expression of hFSH in Hi5 insect cells and
established a method for purification that produces biologically active
hFSH. As part of studies of the glycosylation of hFSH, it was observed
that glycosylation at Asn24 of the ß-subunit is detectable in only
about half of the molecules (7). To reduce glycoform heterogeneity, in
anticipation that this would facilitate crystallization, glycosylation
at ßAsn24 was eliminated by site- directed mutagenesis,
converting Thr26 to Ala. This isoform of hFSH was fully active and
yielded crystals suitable for x-ray diffraction. Here, we report the
structure of ßT26A-hFSH and compare it to that of hCG.
 |
RESULTS AND DISCUSSION
|
---|
Characterization of Recombinant hFSH-ßT26A
Amino acid sequencing of purified, Hi5 insect cell-expressed,
hFSH-ßT26A revealed N termini of
-APDVQDCPEC and ß-CELTNITIAI,
indicating that the
-subunit signal peptide was cleaved as in
mammalian cells but that the ß-subunit lacked the two amino-terminal
residues (Asn, Ser). Purified hFSH-ßT26A was similar in activity to
hFSH expressed in insect cells, was stable during a 16-h incubation
period at room temperature, and bound receptor, effectively competing
with labeled pituitary hFSH for binding to hFSH receptors expressed in
Chinese hamster ovary (CHO) cells (Fig. 1A
). As expected, the biological activity
of hFSH-ßT26A was indistinguishable from wild-type hFSH, showing
dose-response-related stimulation of progesterone production (Fig. 1B
)
and cAMP production (Fig. 1C
) in Y1 cells stably transfected with
hFSHR.

View larger version (21K):
[in this window]
[in a new window]
|
Figure 1. Characterization of the in Vitro
Bioactivity of ßT26A hFSH and Wild-Type (Wt) hFSH
The data are representative of experiments repeated at least twice.
Error bars represent sample error. A, Competition of
recombinant wild-type hFSH (iFSH) and ßT26A hFSH with
125I-hFSH (pituitary) for hFSH receptors expressed in CHO
cells. B, Progesterone production induced by wild-type hFSH or ßT26A
hFSH, measured in an in vitro bioassay. C, cAMP
production induced by treatment with either wild-type hFSH or ßT26A
hFSH.
|
|
Crystallization
The first hFSH crystals were obtained for glycohormone that was
extracted from human pituitary glands, purified as previously described
(19) and desialylated by neuraminidase treatment. These crystals grew
from 1.4 M
(NH4)2SO4
in phosphate buffer at pH 7.4. They were very large, up to 0.5 mm in
all three dimensions, but required about 6 months to grow to full size
and diffracted inconsistently, with some crystals diffracting to 3.8 Å
resolution but most diffracting to less than 5.0 Å resolution. Next,
crystals of insect cell-expressed wild-type hFSH were grown, initially
starting by cross-seeding with crystals of the pituitary hFSH. These
crystals were more consistent in quality, all diffracting to about 4.0
Å resolution, but still required several months to grow. The crystals
of the ßT26A mutant were grown by macroseeding methods under similar
conditions, and initial crystals were obtained by cross-seeding with
the recombinant wild-type hFSH. The final crystallization conditions
were similar to those of the initial pituitary protein conditions,
except that the crystals grew most consistently at pH 9.0. Crystals
grew to full size in less than 1 month and all were of similar
diffraction quality.
Structure Determination
The crystals belong to space group
P41212 with cell
parameters, a = 128.3 Å, c = 155.2 Å. Structure
determination revealed the presence of two hFSH molecules in the
asymmetric unit, with a solvent content of about 80%. The structure
was determined by multiple isomorphous replacement with anomalous
scattering, using four heavy atom derivatives, followed by solvent
flattening. Continuous electron density for more than 95% of the main
chain was observed in the initial 3.5Å electron density map (Fig. 2
). The model was refined to an R value
of 0.259 and an Rfree of 0.294 (2,584
reflections, 9.7% of the total data set) for all data from 30 to 3.0
Å resolution. The final model contains residues
5 to
90 and ß3
to ß109 in hFSH molecule 1 (hFSH1) and residues
5 to
90 and
ß3 to ß108 in hFSH molecule 2 (hFSH2) in addition to 14 sugar
residues and 2 sulfate ions. No attempt was made to include other
solvent molecules because of the relatively low resolution of the
structure and the uncertainty of the location of the disordered protein
and carbohydrate moieties. Thermal parameter refinement was performed
in a blocked mode, with main chain atoms and side chain atoms of each
residue represented as separate blocks. The thermal parameters are
high, due to a combination of the high solvent content and the high
flexibility of the molecule, with an average for all protein atoms of
58 Å2 and ranging from 28
Å2 for residues in the core of the molecule to
100 Å2 in some loops. Figure 2
shows the initial
experimental electron density maps of a representative loop, residues
ß8794. The atomic coordinates and structure factors have been
deposited with the Protein Data Bank, RCSB, entry number 1FL7.
Overall Structure
As expected, hFSH (Fig. 3
) belongs
to the family of cystine-knot growth factors (20, 21) and the overall
fold is identical to that of hCG. Both the
- and ß- subunits
have similar topologies, in which the cystine knot is the central
motif. In this motif, a disulfide bond between Cys
10 (ß3) and
60 (ß51) passes through a ring defined by disulfide bonds from Cys
28 to 82 and from Cys 32 to 84. Three ß-hairpins extend from the
cystine knot, two of which end in tight ß-turns at one end of the
molecule (loops L1 and L3), while the other one forms a longer, more
open loop (L2) at the opposite end. The L2 loop of the
-subunit
(
L2) includes the only helical segment in the molecule, a 1.5-turn
-helix that runs nearly perpendicular to the ß-strands. The
ß-hairpins are stabilized by, and associate through, disulfide
bridges. The disulfide bond pairings of hFSH,
731,
1060,
2882,
3284,
5987 and ß351, ß1766, ß20104,
ß2882, ß3284, and ß8794, are identical to those of hCG and
contradict earlier biochemical assignments (22, 23). The two subunits
are aligned head to tail and are slightly wound around each other so
that ßL2,
L1, and
L3 form one end of the elongated, curved
heterodimer and
L2, ßL1, and ßL3 form the other. The two
subunits are intimately associated via intermolecular contacts that
bury 32% of the total solvent-accessible surface of the monomers. A
loop at the C terminus of the ß-subunit, residues ß84104, wraps
around ß-strands 2 and 3 of the
-subunit, resulting in the
-subunit being surrounded on both sides by the ß-subunit.
Consequently, this loop has been referred to as the "seat belt"
loop in hCG (3).
Insect cell-expressed glycoproteins are glycosylated by short
high-mannose type, N-linked oligosaccharides, typically consisting of
the core (Man)4-(GlcNAc)2.
In both ßT26A hFSH molecules, interpretable electron density is
observed for the two GlcNAcs at site
52 and ß7 and for two GlcNAcs
and one Man at site
78. Although some additional density is observed
at all three sites in both hFSH molecules, disorder prevents modeling
of the remaining oligosaccharide.
Crystal Packing
The two molecules in the asymmetric unit sit close together with
their concave surfaces associated in clasped hands-like fashion (Fig. 4
). Residues in loops
L2,
L3,
ßL2, and the carboxy terminus of the
- subunit of hFSH2
contact the carboxy-termini of both hFSH1 subunits as well as the hFSH1
seatbelt. In total, the interactions render approximately 10% of the
surface of the heterodimer solvent inaccessible. Intermolecular
contacts with symmetry-related molecules are limited to the loops at
the ends of the molecules. This packing arrangement results in an open
crystal lattice with channels of about 70 Å in diameter. All
carbohydrate chains extend into this open channel, as would be expected
because the crystals are ultimately derived from the initial crystals
produced from the more extensively glycosylated pituitary protein.
The average temperature factor of hFSH2, 49 Å2,
is significantly lower than that of hFSH1, 59Å2,
possibly due to the slightly larger number of crystal contacts in which
hFSH2 is involved.

View larger version (46K):
[in this window]
[in a new window]
|
Figure 4. Stereodiagram Showing the Crystal Packing of hFSH
Shown are all 16 hFSH heterodimers that have their center of mass
within the unit cell. The two dimers in the asymmetric unit associate
tightly with their concave surfaces overlapping, while contacts with
symmetry-related molecules are mostly limited to the ends of the
molecules.
|
|
Comparison of the Two Molecules in the Asymmetric Unit
The conformations of the two hFSH heterodimers in the asymmetric
unit are similar, with a root mean square deviation (r.m.s.d.) of 0.9
Å for all 180
-carbon atoms (Fig. 5A
). Although the subunits associate in
identical fashion in both heterodimers, there is a small difference in
the angle between the subunits, which results in an r.m.s.d. of 1.6 Å
between the ß-subunits when the
-subunits are superimposed. This
suggests that there is some flexibility in the association of subunits.
As expected, the core region of the protein, the cystine knot motifs,
and the surrounding ß-strands have the same conformation in both
molecules, and all of the major differences occur in the loops and at
the amino and carboxy termini (Fig. 6A
).
The C-terminal end of the
-helix in loop 2 of the
-subunit is
less helical in hFSH2, resulting in a shift in the position of the
-carbon of residue 48 by 2.4 Å. ßL2 differs most, with its
carboxy-terminal end shifted by about 5.5 Å at residue 43. This shift
appears to be correlated with a major difference in the conformation of
the carboxy terminus of the
-subunit, which lies in the middle of
loop ßL2 in hFSH2 but is positioned away from the molecule in hFSH1.
The final large difference is in
L3, which differs by about 3.7 Å
in the position of residue 72. While these conformational differences
between the two independent observations of the hFSH structure are
indicative of the flexibility of the hFSH heterodimer, they are small
compared with the differences between hFSH and hCG in many of the same
loops.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 5. Difference Mapping of the hFSH and hCG
Structures
A, Diagram showing the variation in temperature factor with residue
number for the two hFSH molecules in the asymmetric unit: hFSH1
(thin lines) and hFSH2 (thick lines). The
average temperature factor for hFSH1 is higher than that for hFSH2. B,
Diagram showing the -carbon atom r.m.s.d. for the least-squares
superposition of the two hFSH molecules (thin lines) and
of hFSH2 with hCG (thick lines). In the -subunit,
both superpositions show that the largest deviations are seen around
residue 72 (loop L3). The largest deviations between hFSH and hCG
are observed in the ß-subunit loops ßL1 around residue 25, ßL2
around residue 50, ßL3 around residue 80, and at the ß-carboxy
terminus. Large deviations are also observed in loop ßL2 in the
comparison of hFSH1 and hFSH2. This suggests that ßL2 is quite
flexible and can adopt a variety of conformations, while the changes in
the other three regions of the protein may be important for receptor
discrimination. C, Alignment of the sequences of the ß-subunits of
hFSH (top) and hCG (bottom) with the
important loops labeled. The cystine residues involved in the cystine
knot are highlighted with filled diamonds of different
shades according to their disulfide pairings. The N-glycosylated
asparagines are identified by an italicized
N. Major sequence differences can be found in ßL2, ßL3,
the cystine noose, and at the ß-carboxy terminus. The carboxy
terminus of hCG sequence is truncated at residue 111 for clarity. The
hCG sequence numbering is indicated in parentheses.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Figure 6. Comparison of the Molecular Conformations of hFSH
and hCG
A, Superposition of hFSH2 ( , green; ß, light
blue) and hFSH1 ( , yellow; ß, dark
blue) shown in two perpendicular views and produced by
least-squares fitting of the -carbon atoms. B, Similar figure of the
superposition of hFSH2 ( , green; ß, light
blue) and hCG ( , orange; ß,
magenta).
|
|
Comparison with hCG
For the comparisons of the conformations of hFSH and hCG, the hCG
coordinates with PDB code 1HCN (4) have been used because this
structure was determined at somewhat higher resolution than the 1HRP
structure (3). However, the two structures were of the same crystal
form and have an r.m.s.d. for all C
atoms of 0.7 Å, with maximum
main chain differences of approximately 1 Å in some of the loops. In
the absence of any systematic differences between the two structures,
the conformational analysis presented here is relevant to both
structures.
hFSH and hCG have identical folds, but significant differences occur in
the amino and carboxy termini and several loops (Figs. 5B
and 6B
). As
expected, the
-subunits, with r.m.s.d. values of 1.1 Å and 0.9 Å
for the least-squares fit of hCG with hFSH1 and hFSH2, respectively,
are more similar than the ß-subunits, with r.m.s.d. values of 1.5 Å
and 1.6 Å. The largest differences in the ß-subunits occur in the
three loops ßL1, ßL2, and ßL3, the cystine noose, and the
ß-subunit carboxy-terminal loop (Fig. 5B
).
Loops ßL1, ßL2, andßL3.
ßL1 and ßL3, together with
L2 and the C terminus of the
ß-subunit, form one end of the heterodimer, extending beyond the area
where the two subunits interact (Figs. 3
and 6
). Comparison of the
structures reveals large differences in the conformations of hFSH and
hCG in this area, but no differences in the two independent molecules
of hFSH. The amino acid sequences of hFSH and hCG differ greatly in
ßL3, with only 4 identical residues in the 13-residue span between
hFSH residues ß64 and ß76. In addition the loop includes three
prolines in hCG but only one in hFSH. Consequently, the conformation of
ßL3 of hFSH is a more curved ß-hairpin than hCG, resulting in a
shift toward the concave surface of the heterodimer and nearer to the
area of the ß- subunit carboxy terminus (Fig. 7a
). The distances between corresponding
residues in the loop are as large as 6.4 Å (6.8 Å) for hFSH1 (hFSH2).
The amino acid sequences in ßL1 for hFSH and hCG are quite similar
(Fig. 5C
) and the conformation of the loop itself is also similar.
However, this loop moves by as much as 3.6 Å (3.9 Å) toward the
concave side of the molecule in hFSH in concert with ßL3, because the
two loops are tied together by the disulfide bridge, ßCys17-ßCys66.
These conformational changes are also correlated with conformational
changes in
L2 and the C terminus of the ß-subunit, which are
discussed below.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 7. Overlap of Selected Areas of hFSH2 ( ,
green; ß, blue) and hCG ( ,
orange; ß, magenta)
a, Loops ßL1 and ßL3. The conformation adopted by ßL3 is
different in the two hormones due to significant sequence differences.
Although the overall sequence and conformation of ßL1 is similar in
hFSH and hCG, the tethering of ßL1 to ßL3 by a disulfide bridge
results in a concerted movement of the two loops. b, Cystine noose.
Sequence variation with respect to charged residues in this area
results in a negatively charged patch on one side of the cystine noose
in hFSH and several positively charged residues in hCG. This loop may
be important for receptor discrimination. c, The solvent-exposed
hydrophobic patch in the area of L1, L3, and ßL2. The size and shape of the patch differs in hFSH and hCG. The
unusually large hydrophobic surface area suggests importance for
stabilizing intersubunit interaction and possibly for receptor
binding.
|
|
The conformation of hCG loop ßL2 is quite different from either one
of the conformations seen for hFSH (Fig. 6B
), but the two hFSH
molecules differ at this locus as much in conformation from each other
as they differ from hCG (Fig. 6A
). While the difference between hFSH
and hCG in part must result from differences in the sequences in this
loop, including a variation in the presence of proline residues (Fig. 5C
), overall this result suggests that the loop is highly flexible in
the GPH.
Cystine Noose.
The cystine noose, or determinant loop, is a short loop between two
disulfide-linked cysteines, ß87 and ß94 in hFSH (ß93-ß100 in
hCG) (24) located toward the center of the concave surface of the
molecule, adjacent to
L2. Residues in the cystine noose play a role
in determining the specificity of hCG receptor (25) and FSH receptor
(26) binding. The main chain conformation of the loop is very similar
in hFSH and hCG, although the entire loop is shifted by about 3 Å at
its tip (Fig. 7b
). This shift is part of the global conformational
change at this end of the molecule, also including ßL1, ßL3, and
L2. More important than the conformational differences between the
cystine nooses are their strikingly different surface charge
characteristics. There are three negatively charged residues, Asp 88,
Asp 90, and Asp 93, and no positively charged residues in hFSH, while
hCG has two positively charged residues (Arg 94 and Arg 95) and one
negatively charged residue (Asp 99). The conserved aspartic acid
residues near the C terminus of the cystine noose, Asp 93 in hFSH and
Asp 99 in hCG, have similar conformations in the two hormones. The
structure is consistent with the biochemical data since this residue
has been shown to be essential for both hFSH activity (7, 27) and
hCG activity. The three aspartic acid residues in hFSH create a
negatively charged patch on one side of the cystine noose. In contrast,
the positively charged arginines in hCG are not arranged to form a
charged patch; rather the side chains are directed to opposite sides of
the loop (Fig. 7b
). These observations are consistent with biochemical
data, showing that while neither hFSH Asp 88 nor Asp 90 is
essential for hFSH binding to receptor (27), both play a role in
discriminating between the hFSH and hCG/LH receptors (26). Sequence
comparison (Fig. 5C
) shows that the cystine noose and ßL3 are the
only two areas where there is a significant charge differential between
hFSH and hCG. The only other possibly significant area is in ßL2, but
the change in this case is from a generally charged surface in hFSH
to a generally uncharged surface in hCG.
ß-Carboxy-Terminal Loop.
The carboxy terminus of the hFSH ß-subunit, residues 95108, adopts
a different conformation than in hCG, with maximum distances between
equivalent residues of 7.0 Å (7.0 Å) (Figs. 5B
and 6B
) at hFSH
residue ß99. Specifically, the loop spanning residues 95103 in hFSH
makes a tighter turn than the equivalent loop from 101109 in hCG.
Both hFSH molecules have identical conformations in this area, despite
the fact that this loop in hFSH1 is involved in more intermolecular
contacts than in hFSH2. Therefore, differences between hFSH and hCG are
caused by divergent sequences in this area (Fig. 5C
). Biochemical data
indicate that the ß-subunit carboxy terminus plays a role in hFSH
receptor binding, because alanine substitution of the three residues
Arg97-Gly98-Leu99 (27), as well as replacement of the hFSH residues
95100 with the corresponding residues of human LH (hLH) (26),
diminishes hFSH receptor binding. However, swapping hLH residues for
hFSH residues at this locus does not allow for LH receptor binding by
the hFSH-LH chimera (26). The biochemical data are consistent with the
structure, in that these residues clearly adopt a different
conformation, allowing for differential recognition by the appropriate
receptor. However, the changes in this region alone are not sufficient
to change the receptor specificity of the hormone.
Glycosylation.
Comparison of the structures of hFSH and hCG indicate that
glycosylation has no global effect on the glycohormone conformations.
Although the conformation of the insect cell-expressed, and fully
active, hFSH differs significantly from that of more extensively
deglycosylated, HF-treated hCG in several loops, none of these
differences can be directly correlated with differences in the
glycosylation. The structure of hFSH reveals one likely mechanism by
which the oligosaccharide contributes to heterodimer stability (17).
The nitrogen atom of the acetamido group of the Asn-proximal GlcNAc at
Asn52 forms a hydrogen bond with the hydroxyl group of ßTyr58,
thereby adding an additional intersubunit contact. In hCG, the
equivalent residue is Phe64, incapable of forming a similar hydrogen
bond, but able to make a hydrophobic interaction with the hydrophobic
side of the sugar ring. Indeed, Lapthorn et al. (3) report
contacts between the
Asn52 carbohydrate and residues ßTyr59,
ßVal62, ßPhe64, ßAla83, and ßThr97. While the single additional
hydrogen bond observed in the hFSH structure may not be adequate to
explain the higher stability of the glycosylated heterodimer, it is
quite possible that sugar groups not fully defined in the electron
density map make additional contacts.
Hydrophobic Patch.
A propeller-shaped triad of aromatic residues,
Phe17,
Phe74, and ßTyr39, together with
Pro16,
Phe18, and
Met71, forms a solvent-exposed hydrophobic patch at the end of the
hFSH heterodimer composed of loops
L1,
L3, and ßL2 (Fig. 7c
).
This region includes only three charged residues (
Lys75, ßLys40,
and ßAsp41), all of which are directed away from the hydrophobic
patch. In hCG, ßLeu45 replaces hFSH ßTyr 39 and the patch is
smaller because of a difference in the conformation of the ßL2 loop.
This area may play a major role in receptor binding. Although the
relevance of the
L1 loop in hFSH (residues 1427) has not been
evaluated by mutagenesis studies, epitope mapping with monoclonal
antibodies revealed a discontinuous immunoneutralizing epitope
comprised of residues
1127 and
6192 (29, 30). Mutational
analysis of
Phe74 revealed only a modest decrease in hFSH binding to
receptor. In contrast to the importance of
L1 and
L3, alanine
scanning mutagenesis has shown that ßL2 (residues 3353) is not
essential for hFSH binding to receptor (31). Rather, ßL2 appears to
be important in stabilizing the heterodimer association (32).
-Subunit Carboxy Terminus.
The
-subunit carboxy terminus has been implicated in receptor
binding in hCG (33, 34, 35), hFSH (36), and TSH (37). Clearly, this region
is very flexible in hFSH because it adopts different conformations in
hFSH1 and hFSH2 and electron density is lacking for the last two
residues,
9192. This makes it difficult to draw conclusions about
how this region is involved in receptor binding. The only clear
difference between hFSH and hCG is the formation of a hydrogen bond
between the side chain NH of
Arg95 from the cystine noose of hCG
with the carbonyl oxygen of
89, constraining the conformational
flexibility of hCG. This bond is not possible in the hFSH structure,
because the hFSH
-carboxy terminus is not as close to the cystine
noose. This difference in conformation is interesting in the context of
receptor interaction, as the residues in the carboxy terminus of the
-subunit are essential for hFSH and hCG binding (34, 35, 36).
Conclusion
The overall structures of hFSH and hCG are similar, but several
intriguing differences are observed in specific loops, especially in
the ß-subunit. The largest difference is at one end of the molecule
where ßL1 and ßL3 move together toward the concave side of the
molecule in hFSH compared with hCG. In addition, the ßL3 loop has a
very different conformation as a result of major differences in the
amino acid sequence. Currently, no biological data are available
regarding the importance of these loops in receptor binding or signal
transduction. The three other areas with different conformations, the
ß carboxy-terminal loop, the cystine noose, and the hydrophobic patch
area between loops
L1,
L3, and ßL2 also have different surface
characteristics. In addition, several residues in these areas have been
shown, by scanning alanine mutagenesis or epitope mapping, to play a
role in receptor binding (Fig. 8
).
Interestingly, the ß-carboxy-terminal loop, the cystine noose, and
the ßL1 and ßL3 loops are located on the concave side of the
heterodimer (Fig. 8
), resulting in a face that is very different in the
two gonadotropins. These differences are therefore very likely to be
important for discrimination between hFSH and hCG by their respective
receptors.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 8. Ribbon Image of the Structure of hFSH Highlighting
Areas of Interest
Magenta, areas with known biological importance and
structural differences with hCG; blue, areas of
biological significance but no defined conformational difference with
hCG; and, yellow: areas of conformational differences
between hFSH and hCG, without known biological relevance. Residues
known to be important for hFSH-receptor binding are shown: Lys51 of
L2, Ser85, Thr86, Tyr88, and Tyr89 of the carboxy terminus of the
-subunit (blue) and Asp93 (red) on the
ß-carboxy terminal loop. The residues forming the exposed hydrophobic
patch are shown in green. All areas of interest, except
the hydrophobic patch, are located at the concave surface of the
molecule.
|
|
 |
MATERIALS AND METHODS
|
---|
Preparation of hFSH Isoform ßT26A
Site-directed mutagenesis experiments, aimed at elimination of
hFSHß glycosylation site 2, were performed as previously described
for other hFSH mutants (36). The oligonucleotide used to create the
mutation was 5'-A-AGC-ATC-AAC-ACC- GCT-TGG-TGT-GCT-GGC-3'.
Production of Recombinant hFSH
Production of recombinant hFSH in insect cells was carried out
using procedures as previously described (26, 31). Each production run
consisted of about 30 roller bottles, seeded with 1 x
108 Hi5 cells and kept at 27 C for 48 h. At
that time, virus was added directly to each flask [1.0 MOI
(multiplicity of infection) of hFSHßT26A virus and 3.5 MOI of hFSH
virus] and flasks were rotated for an additional 45 days. Next,
media were collected by centrifugation in 1-liter bottles (2,500
x g, 10 min). The clarified media were pooled and made 1.0
mM with phenylmethylsulfonyl fluoride, and 0.1%
with sodium azide. Typically, recombinant hFSH-ßT26A was expressed at
levels of 2.7 mg/liter (n = 9) as determined by enzyme-linked
immunosorbent assay (ELISA) (32). Media (typically 4 liters) were then
concentrated at 4 C utilizing an Amicon (Waltham, MA) radial flow
cartridge (10,000 mwc), to a volume of about 200 ml, and then frozen
until processed. The concentrate was thawed, clarified by
centrifugation (16,000 x g, 30 min) and then applied
to the affinity column without further processing.
Preparation of the Affinity Support
An affinity column was prepared using monoclonal antibody (mAb)
46.3H6.B7. This antibody binds to both the
monomeric ß-subunit of native hFSH, and the heterodimeric hormone,
but has no measurable cross-reactivity with the
-subunit or with hLH
(19, 38).
The cell line producing mAb 46.3H6.B7 was expanded as ascites tumors in
mice (Animal Welfare Committee Approval was obtained for these
studies). Approximately 38 ml of 46.3H6.B7 ascitic fluid were diluted
1:3 with PBS and subjected to ammonium sulfate precipitation as
follows. Saturated ammonium sulfate (pH 7.2) was added to the diluted
ascitic fluid in a ratio of 4.5 volumes of ammonium sulfate solution to
5.5 volumes of antibody. The mixture was stirred (4 C, 30 min), and the
antibody precipitate was collected by centrifugation (5, 858 x
g, 15 min). Each precipitate was dissolved in PBS and
reprecipitated twice as before. Wet pellets were dissolved and dialyzed
against 0.01 M potassium phosphate, pH 7.2.
Dialyzed samples were clarified by centrifugation, the pH and
conductivity were adjusted to the buffer values, and the sample was
applied to a 1.9 x 18 cm diethylaminoethyl (DEAE) Sephacel
column. Antibody was eluted with a gradient of 0.00.2
M NaCl. The procedure used for coupling of the
DEAE Sephacel-purified antibody (100 mg) to CNBr-activated Sepharose
was exactly as described by the manufacturer (Pharmacia Biotech, Piscataway, NJ).
Purification of hFSH ßT26A
Concentrates of conditioned media, collected from cells
producing hFSH ßT26A, were applied directly to the affinity support.
Typically a flow rate of 0.6 ml/min was used. The mAb 46.3H6.B7 column
dimensions were 0.9 x 10 cm. The sample buffer and column buffer
were 0.1 M potassium phosphate, made 0.3 M with
NaCl, pH 7.0. The absorbance (275 nm) of the fractions was determined
during the procedure. After a decrease of absorbance to baseline,
elution buffer (0.1 M sodium acetate, pH 2.0, 0.5
M NaCl) was pumped through the column. Fractions were
collected into tubes containing 2.0 M Tris base and mixed
to neutralize each fraction.
Biological Characterization of hFSH-ßT26A Heterodimer
Recombinant hFSH-ßT26A was compared with wild-type hFSH in a
RRA using CHO cells, stably expressing hFSH receptors (36, 39). Signal
transduction induced by hFSH-ßT26A was determined by measuring
progesterone (36) or cAMP (38) production by Y1 cells that stably
express hFSH receptors (39).
Protein Crystallization
Crystals of ßT26A hFSH were grown by macroseeding into drops
containing 3 µl protein (9 mg/ml in 10 mM Tris, pH 7) and
1 µl reservoir solution. The reservoir solution was 100
mM glycine, pH 9.0, and 0.91.2 M ammonium
sulfate.
Data Collection and Structure Determination
The crystals were transferred to a solution consisting of the
crystallization buffer enriched with 25% wt/vol sucrose before flash
cooling in liquid nitrogen. The crystals were highly sensitive to the
addition of heavy atom compounds. Crystals tolerated soaking in heavy
atom solutions for no more than about 10 h, with some heavy atom
compounds, especially platinum and mercury compounds resulting in
significant degradation of the diffraction quality after as little as
2 h. Data were collected at Stanford Synchrotron Radiation
Laboratory (SSRL) Beamline B9.1, processed with MOSFLM (40), and then
scaled and merged with SCALA (40) (Table 1
). Initial MIRAS phases were calculated
using SOLVE (41) (Table 1
). The correct space group and
enantiomer were determining by examining the figure-of-merit
(FOM) for both space groups with each hand. This clearly indicated that
P41212 was the correct
space group, FOM 0.57 vs. 0.43, and that the positive
hand was correct. All four derivatives had relatively weak phasing
power due to low occupancy, and the sites of the platinum and osmium
derivatives were similar. However, solvent flattening [CCP4 program DM
(40)] greatly improved the quality of the phases because of the very
high solvent content of the crystals (
80%). MIRAS phasing in
program SOLVE resulted in an overall FOM of 0.57. After solvent
flattening, the electron density clearly showed two molecules in the
asymmetric unit.
Structure Refinement
An initial model was built into the
MIRAS/solvent-flattened electron density map. Rounds of simulated
annealing refinement using the torsion angle dynamics option in the CNS
(42) were followed by phase combination in SIGMAA (40) before
rebuilding in O (43) during early stages of refinement. Once the
majority of the sequence had been placed in the model, composite omit
maps in CNS were used for rebuilding in an effort to reduce model bias.
The quality of the model was monitored throughout with PROCHECK (44).
Grouped temperature factor refinement, where the main chain atoms of
each residue formed one group and the side chain atoms formed a second
group, was used in later stages of refinement. Thermal parameters that
exceeded 100 Å2 were reset to this value during
the refinement. Experiments were done using NCS constraints and
restraints to tether the two molecules in the asymmetric unit, but in
all cases this caused Rfree to increase. The
final hFSH structure has an R value of 25.9% and an
Rfree of 29.4% for all data from 50.0 to 3.0 Å
resolution. The model contains 2,992 protein atoms, 14 carbohydrate
residues, and 2 sulfate ions. The geometry is good, with 74.3% of
residues in the most favored regions of the Ramachandran plot, and only
three residues (0.9%) from the loops in disallowed regions. The
r.m.s.d. values from ideality for the protein are as follows: bonds,
0.008 Å; angles, 1.5°; dihedrals, 24.6°; impropers, 1.0°.
Structure Comparisons/Surface Area Calculations
Protein structures were compared using LSQMAN (45), and values
quoted in the text are the results of comparing regions of the
structure after the entire structure was superimposed by least-squares
fitting of the
-carbon atoms only. Solvent-accessible surface area
was calculated using CNS with a 1.4-Å probe (42).
 |
ACKNOWLEDGMENTS
|
---|
We thank Carrie Arnold, Smita D. Mahale, Maria Patrascu, Barbara
Sheppard, and Yiqiu Zhang for assistance with the hFSH and the ßT26A
hFSH expression, purification, and characterization; Xiaochun Ding for
assistance with the crystallization experiments, and Jeffrey A. Bell
and Christopher A. Waddling for assistance with the data collection at
SSRL. The authors gratefully acknowledge the support of the Wadsworth
Center Core Facilities, including the Molecular Genetics, Amino Acid
Analysis and Sequencing Facilities, as well as the Wadsworth Centers
Tissue Culture Facility.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Patrick Van Roey or James Dias, Wadsworth Center, P.O. Box 509, Albany, New York 12201-0509. E-mail: vanroey{at}wadsworth.org; james.dias@
This research was supported by NIH Grants HD-18407 (J.A.D.) and
GM-50431 (P.V.R.) and a grant from ARES Advanced Technologies/Serono.
The x-ray crystallography facilities of SSRL are funded by the
Department of Energy and the National Institutes of Health.
Received for publication June 30, 2000.
Revision received August 29, 2000.
Accepted for publication September 19, 2000.
 |
REFERENCES
|
---|
-
Pierce JG, Parsons TF 1981 Glycoprotein hormones:
structure and function. Annu Rev Biochem 50:465495[CrossRef][Medline]
-
Parsons TF, Strickland TW, Pierce JG 1985 Disassembly and
assembly of glycoprotein hormones. Methods Enzymol 109:736749[Medline]
-
Lapthorn AP, Harris DC, Littlejohn A, Lustbader JW, Canfield
RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human
chorionic gonadotropin. Nature 369:455461[CrossRef][Medline]
-
Wu H, Lustbader JW, Liu Y, Canfield RE, Hendrickson WA 1994 Structure of human chorionic gonadotropin at 2.6 A resolution from MAD
analysis of the selenomethionyl protein. Structure 2:545558[Medline]
-
Simoni M, Gromoll J, Nieschlag E 1997 The
follicle-stimulating hormone receptor: biochemistry, molecular biology,
physiology, and pathophysiology. Endocr Rev 18:739773[Abstract/Free Full Text]
-
Milgrom E, de Roux N, Ghinea N, Beau I, Loosfelt H, Vannier
B, Savouret JF, Misrahi M 1997 Gonadotrophin and thyrotrophin
receptors. Horm Res 48[Suppl 4]:3337
-
Dias JA, Lindau-Shepard B, Hauer C, Auger I 1998 Human
follicle-stimulating hormone structure-activity relationships. Biol
Reprod 58:13311336[Medline]
-
Chappel S, Buckler D, Kelton C, Tayar NE 1998 Follicle
stimulating hormone and its receptor: future perspectives. Hum Reprod
13[Suppl 3]:1835, discussion 4751
-
Grossmann M, Leitolf H, Weintraub BD, Szkudlinski MW 1998 A
rational design strategy for protein hormone superagonists. Nat
Biotechnol 16:871875[Medline]
-
Baenzinger JU 1994 Glycosylation and glycoprotein hormone
function. In: Lustbader JW, Puett D, Ruddon RW (eds)
Glycoprotein Hormones: Structure, Function and Clinical Implications.
Springer-Verlag, New York, pp 167174
-
Ulloa-Aguirre A, Midgley Jr AR, Beitins IZ, Padmanabhan V 1995 Follicle-stimulating isohormones: characterization and physiological
relevance. Endocr Rev 16:765787[Medline]
-
Ulloa-Aguirre A, Timossi C 1998 Structure-function
relationship of follicle-stimulating hormone and its receptor. Hum
Reprod Update 4:260283[Abstract/Free Full Text]
-
Szkudlinski MW, Thotakura NR, Tropea JE, Grossmann M,
Weintraub BD 1995 Asparagine-linked oligosaccharide structures
determine clearance and organ distribution of pituitary and recombinant
thyrotropin. Endocrinology 136:33253330[Abstract]
-
Ulloa-Aguirre A, Timossi C, Damian-Matsumura P, Dias JA 1999 Role of glycosylation in function of follicle-stimulating hormone.
Endocrine 11:205215[CrossRef][Medline]
-
Feng W, Matzuk MM, Mountjoy K, Bedows E, Ruddon RW, Boime I 1995 The asparagine-linked oligosaccharides of the human chorionic
gonadotropin ß subunit facilitate correct disulfide bond pairing.
J Biol Chem 270:1185111859[Abstract/Free Full Text]
-
van Zuylen CW, Kamerling JP, Vliegenthart JF 1997 Glycosylation beyond the Asn78-linked GlcNAc residue has a significant
enhancing effect on the stability of the
subunit of human chorionic
gonadotropin. Biochem Biophys Res Commun 232:117120[CrossRef][Medline]
-
Heikoop JC, van den Boogaart P, de Leeuw R, Mulders JW,
Grootenhuis PD 1998 Partially deglycosylated human choriogonadotropin,
stabilized by intersubunit disulfide bonds, shows full bioactivity. Eur
J Biochem 253:354356[Abstract]
-
Tegoni M, Spinelli S, Verhoeyen M, Davis P, Cambillau C 1999 Crystal structure of a ternary complex between human corionic
gonadotropin (hCG) and two Fv fragments specific for the
and
ß-subunits. J Mol Biol 289:13751385[CrossRef][Medline]
-
Weiner RS, Dias JA, Andersen TT 1991 Epitope mapping of human
follicle stimulating hormone-
using monoclonal antibody 3A
identifies a potential receptor binding sequence. Endocrinology 128:14851495[Abstract]
-
Isaacs NW 1995 Cystine knots. Curr Opin Struct Biol 5:391395[CrossRef][Medline]
-
Sun PD, Davies DR 1995 The cystine-knot growth-factor
superfamily. Annu Rev Biophys Biomol Struct 24:269291[CrossRef][Medline]
-
Rathnam P, Tolvo A, Saxena BB 1982 Elucidation of the
disulfide bond positions of the ß-subunit of human
follicle-stimulating hormone. Biochim Biophys Acta 708:160166[Medline]
-
Fujiki Y, Rathnam P, Saxena BB 1980 Studies on the disulfide
bonds in human pituitary follicle-stimulating hormone. Biochim Biophys
Acta 624:428435[Medline]
-
Lapthorn AJ, Janes RW, Isaacs NW, Wallace BA 1995 Cystine
nooses and protein specificity. Nat Struct Biol 2:266268[Medline]
-
Campbell RK, Dean-Emig DM, Moyle WR 1991 Conversion of human
choriogonadotropin into a follitropin by protein engineering. Proc Natl
Acad Sci USA 88:760764[Abstract]
-
Dias JA, Zhang Y, Liu X 1994 Receptor binding and functional
properties of chimeric human follitropin prepared by an exchange
between a small hydrophilic intercysteine loop of human follitropin and
human lutropin. J Biol Chem 269:2528925294[Abstract/Free Full Text]
-
Lindau-Shepard B, Roth KE, Dias JA 1994 Identification of
amino acids in the C-terminal region of human follicle-stimulating
hormone (FSH) ß-subunit involved in binding to human FSH receptor.
Endocrinology 135:12351240[Abstract]
-
Chen F, Wang Y, Puett D 1991 Role of the invariant aspartic
acid 99 of human choriogonadotropin ß in receptor binding and
biological activity. J Biol Chem 266:1935719361[Abstract/Free Full Text]
-
Szkudlinski MW, Teh NG, Grossmann M, Tropea JE, Weintraub BD 1996 Engineering human glycoprotein hormone superactive analogs. Nat
Biotechnol 14:12571263[Medline]
-
Weiner RS, Dias JA 1992 Identification of assembled epitopes
on the
-subunit of human follicle stimulating hormone. Mol Cell
Endocrinol 85:4152[CrossRef][Medline]
-
Roth KE, Dias JA 1995 Scanning-alanine mutagenesis of long
loop residues 3353 in follicle stimulating hormone ß subunit. Mol
Cell Endocrinol 109:143149[CrossRef][Medline]
-
Roth KE, Dias JA 1996 Follitropin conformational stability
mediated by loop 2 ß effects follitropin-receptor interaction.
Biochemistry 35:79287935[CrossRef][Medline]
-
Chen F, Wang Y, Puett D 1992 The carboxy-terminal region of
the glycoprotein hormone
-subunit: contributions to receptor binding
and signaling in human chorionic gonadotropin. Mol Endocrinol 6:914919[Abstract]
-
Yoo J, Zeng H, Ji I, Murdoch WJ, Ji TH 1993 COOH-terminal
amino acids of the
subunit play common and different roles in human
choriogonadotropin and follitropin. J Biol Chem 268:1303413042[Abstract/Free Full Text]
-
Zeng H, Ji I, Ji TH 1995 Lys91 and His90 of the
-subunit
are crucial for receptor binding and hormone action of
follicle-stimulating hormone (FSH) and play hormone-specific roles in
FSH and human chorionic gonadotropin. Endocrinology 136:29482953[Abstract]
-
Arnold CJ, Liu C, Lindau-Shepard B, Losavio ML, Patrascu MT,
Dias JA 1998 The human follitropin
- subunit C terminus
collaborates with a ß-subunit cystine noose and an
-subunit loop
to assemble a receptor-binding domain competent for signal
transduction. Biochemistry 37:17621768[CrossRef][Medline]
-
Grossmann M, Szkudlinski MW, Zeng H, Kraiem Z, Ji I, Tropea
JE, Ji TH, Weintraub BD 1995 Role of the carboxy-terminal residues of
the
-subunit in the expression and bioactivity of human
thyroid-stimulating hormone. Mol Endocrinol 9:948958[Abstract]
-
Roth KE, Liu C, Shepard BA, Shaffer JB, Dias JA 1993 The
flanking amino acids of the human follitropin ß- subunit 3353
region are involved in assembly of the follitropin heterodimer.
Endocrinology 132:25712577[Abstract]
-
Kelton CA, Cheng SV, Nugent NP, Schweickhardt RL,
Rosenthal JL, Overton SA, Wands GD, Kuzeja JB, Luchette CA, Chappel SC 1992 The cloning of the human follicle stimulating hormone receptor and
its expression in COS-7, CHO, and Y-1 cells. Mol Cell Endocrinol 89:141151[CrossRef][Medline]
-
CCP4 1994 Collaborative Computing Project No. 4. The CCP4
suite: programs for protein crystallography. Acta Crystallogr D 50:760763[CrossRef]
-
Terwilliger TC, Berendzen J 1999 Automated MAD and MIR
structure solution. Acta Crystallogr D 55:849861[CrossRef][Medline]
-
Brunger A, Adams P, Clore G, DeLano W, Gros P,
Grosse-Kunstleve R, Jiang J, Kuszewski J, Nilges M, Pannu N, Read R,
Rice L, Simonson T, Warren G 1998 Crystallography & NMR system: a new
software suite for macromolecular structure determination. Acta
Crystallogr D 54:905921[CrossRef][Medline]
-
Jones TA, Zou JY, Cowan SW, Kjeldgaard M 1991 Improved methods
for the building of protein models in electron-density maps and the
location of errors in these maps. Acta Crystallogr A 47:110119[CrossRef][Medline]
-
Laskowski RA, McArthur MW, Moss DS, Thornton JM 1993 PROCHECK:
a program to check the stereochemical quality of protein structures.
J Appl Crystallogr 26:282291[CrossRef]
-
Kleywegt G 1999 Experimental assessment of
differences between related protein crystal structures. Acta
Crystallogr D 55:187884[CrossRef][Medline]
-
Evans SV 1993 SETOR: hardware-highlighted three-
dimensional solid model representation of macromolecules. J Mol
Graph 11:134138[CrossRef][Medline]
-
Kraulis PJ 1991 MOLSCRIPT: a program to produce both
detailed and schematic plots of protein structures. J Appl
Crystallogr 24:946950[CrossRef]