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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}- 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 62–73), the cystine noose, or determinant loop (residues 87–94), and the carboxy-terminal loop (residues 94–104). An additional interesting feature of the hFSH structure is an extensive hydrophobic patch in the area formed by loops {alpha}L1, {alpha}L3, and ßL2. Glycosylation at {alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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 {alpha}- 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 {alpha}Asn78 appears to be important for thermal stability (16). Deglycosylation of hFSH and hCG at {alpha}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 {alpha}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 {alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of Recombinant hFSH-ßT26A
Amino acid sequencing of purified, Hi5 insect cell-expressed, hFSH-ßT26A revealed N termini of {alpha}-APDVQDCPEC and ß-CELTNITIAI, indicating that the {alpha}-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. 1AGo). As expected, the biological activity of hFSH-ßT26A was indistinguishable from wild-type hFSH, showing dose-response-related stimulation of progesterone production (Fig. 1BGo) and cAMP production (Fig. 1CGo) in Y1 cells stably transfected with hFSHR.



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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. 2Go). 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 {alpha}5 to {alpha}90 and ß3 to ß109 in hFSH molecule 1 (hFSH1) and residues {alpha}5 to {alpha}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 2Go shows the initial experimental electron density maps of a representative loop, residues ß87–94. The atomic coordinates and structure factors have been deposited with the Protein Data Bank, RCSB, entry number 1FL7.



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Figure 2. Electron Density of a Representative Loop in hFSH

The model for residues ß87–94 is shown in black sticks with the disulfide between ß87-ß94 in a thicker line, and the 3.5 Å experimental electron density map is displayed at a 1.2 {varsigma}-level. The refined average temperature factors for the residues included in the figure were 70Å for the main chain atoms and 85Å for the side chain atoms. Figures 2Go and 4Go were created using the program Setor (46 ).

 
Overall Structure
As expected, hFSH (Fig. 3Go) belongs to the family of cystine-knot growth factors (20, 21) and the overall fold is identical to that of hCG. Both the {alpha}- and ß- subunits have similar topologies, in which the cystine knot is the central motif. In this motif, a disulfide bond between Cys {alpha}10 (ß3) and {alpha}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 {alpha}-subunit ({alpha}L2) includes the only helical segment in the molecule, a 1.5-turn {alpha}-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, {alpha}7–31, {alpha}10–60, {alpha}28–82, {alpha}32–84, {alpha}59–87 and ß3–51, ß17–66, ß20–104, ß28–82, ß32–84, and ß87–94, 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, {alpha}L1, and {alpha}L3 form one end of the elongated, curved heterodimer and {alpha}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 ß84–104, wraps around ß-strands 2 and 3 of the {alpha}-subunit, resulting in the {alpha}-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).



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Figure 3. Cartoon Illustrating the Overall Fold of hFSH

The fold is typical of the cystine-knot growth factors in which both the {alpha}-subunit (light gray ribbon) and the ß-subunit (dark gray ribbon) have similar topologies. The carbohydrate and the disulfide bonds in both subunits are represented by ball-and-stick models. The N and C termini of each subunit, the N-glycosylated Asn residues (ß7 = Asn7 from the ßsubunit), and the loops discussed in the text are labeled. Figures 3Go, 6Go, 7Go, and 8Go were drawn with Molscript (47 ).

 
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 {alpha}52 and ß7 and for two GlcNAcs and one Man at site {alpha}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. 4Go). Residues in loops {alpha}L2, {alpha}L3, ßL2, and the carboxy terminus of the {alpha}- 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.



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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 {alpha}-carbon atoms (Fig. 5AGo). 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 {alpha}-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. 6AGo). The C-terminal end of the {alpha}-helix in loop 2 of the {alpha}-subunit is less helical in hFSH2, resulting in a shift in the position of the {alpha}-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 {alpha}-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 {alpha}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.



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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 {alpha}-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 {alpha}-subunit, both superpositions show that the largest deviations are seen around residue 72 (loop {alpha}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.

 


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Figure 6. Comparison of the Molecular Conformations of hFSH and hCG

A, Superposition of hFSH2 ({alpha}, green; ß, light blue) and hFSH1 ({alpha}, yellow; ß, dark blue) shown in two perpendicular views and produced by least-squares fitting of the {alpha}-carbon atoms. B, Similar figure of the superposition of hFSH2 ({alpha}, green; ß, light blue) and hCG ({alpha}, 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{alpha} 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. 5BGo and 6BGo). As expected, the {alpha}-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. 5BGo).

Loops ßL1, ßL2, andßL3.
ßL1 and ßL3, together with {alpha}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. 3Go and 6Go). 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. 7aGo). 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. 5CGo) 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 {alpha}L2 and the C terminus of the ß-subunit, which are discussed below.



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Figure 7. Overlap of Selected Areas of hFSH2 ({alpha}, green; ß, blue) and hCG ({alpha}, 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 {alpha}L1, {alpha}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. 6BGo), but the two hFSH molecules differ at this locus as much in conformation from each other as they differ from hCG (Fig. 6AGo). 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. 5CGo), 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 {alpha}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. 7bGo). This shift is part of the global conformational change at this end of the molecule, also including ßL1, ßL3, and {alpha}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. 7bGo). 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. 5CGo) 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 95–108, adopts a different conformation than in hCG, with maximum distances between equivalent residues of 7.0 Å (7.0 Å) (Figs. 5BGo and 6BGo) at hFSH residue ß99. Specifically, the loop spanning residues 95–103 in hFSH makes a tighter turn than the equivalent loop from 101–109 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. 5CGo). 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 95–100 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 {alpha}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 {alpha}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, {alpha}Phe17, {alpha}Phe74, and ßTyr39, together with {alpha}Pro16, {alpha}Phe18, and {alpha}Met71, forms a solvent-exposed hydrophobic patch at the end of the hFSH heterodimer composed of loops {alpha}L1, {alpha}L3, and ßL2 (Fig. 7cGo). This region includes only three charged residues ({alpha}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 {alpha}L1 loop in hFSH (residues 14–27) has not been evaluated by mutagenesis studies, epitope mapping with monoclonal antibodies revealed a discontinuous immunoneutralizing epitope comprised of residues {alpha}11–27 and {alpha}61–92 (29, 30). Mutational analysis of {alpha}Phe74 revealed only a modest decrease in hFSH binding to receptor. In contrast to the importance of {alpha}L1 and {alpha}L3, alanine scanning mutagenesis has shown that ßL2 (residues 33–53) is not essential for hFSH binding to receptor (31). Rather, ßL2 appears to be important in stabilizing the heterodimer association (32).

{alpha}-Subunit Carboxy Terminus.
The {alpha}-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, {alpha}91–92. 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 {alpha}Arg95 from the cystine noose of hCG with the carbonyl oxygen of {alpha}89, constraining the conformational flexibility of hCG. This bond is not possible in the hFSH structure, because the hFSH {alpha}-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 {alpha}-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 {alpha}L1, {alpha}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. 8Go). 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. 8Go), 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.



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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 {alpha}L2, Ser85, Thr86, Tyr88, and Tyr89 of the carboxy terminus of the {alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha} virus] and flasks were rotated for an additional 4–5 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 {alpha}-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 0.0–0.2 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 0.9–1.2 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 1Go). Initial MIRAS phases were calculated using SOLVE (41) (Table 1Go). 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.


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Table 1. Crystallographic Data

 
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 {alpha}-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 Center’s 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Pierce JG, Parsons TF 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465–495[CrossRef][Medline]
  2. Parsons TF, Strickland TW, Pierce JG 1985 Disassembly and assembly of glycoprotein hormones. Methods Enzymol 109:736–749[Medline]
  3. Lapthorn AP, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human chorionic gonadotropin. Nature 369:455–461[CrossRef][Medline]
  4. 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 2:545–558[Medline]
  5. Simoni M, Gromoll J, Nieschlag E 1997 The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 18:739–773[Abstract/Free Full Text]
  6. 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]:33–37
  7. Dias JA, Lindau-Shepard B, Hauer C, Auger I 1998 Human follicle-stimulating hormone structure-activity relationships. Biol Reprod 58:1331–1336[Medline]
  8. Chappel S, Buckler D, Kelton C, Tayar NE 1998 Follicle stimulating hormone and its receptor: future perspectives. Hum Reprod 13[Suppl 3]:18–35, discussion 47–51
  9. Grossmann M, Leitolf H, Weintraub BD, Szkudlinski MW 1998 A rational design strategy for protein hormone superagonists. Nat Biotechnol 16:871–875[Medline]
  10. 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 167–174
  11. Ulloa-Aguirre A, Midgley Jr AR, Beitins IZ, Padmanabhan V 1995 Follicle-stimulating isohormones: characterization and physiological relevance. Endocr Rev 16:765–787[Medline]
  12. Ulloa-Aguirre A, Timossi C 1998 Structure-function relationship of follicle-stimulating hormone and its receptor. Hum Reprod Update 4:260–283[Abstract/Free Full Text]
  13. 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 136:3325–3330[Abstract]
  14. Ulloa-Aguirre A, Timossi C, Damian-Matsumura P, Dias JA 1999 Role of glycosylation in function of follicle-stimulating hormone. Endocrine 11:205–215[CrossRef][Medline]
  15. 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 270:11851–11859[Abstract/Free Full Text]
  16. 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 {alpha} subunit of human chorionic gonadotropin. Biochem Biophys Res Commun 232:117–120[CrossRef][Medline]
  17. 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 253:354–356[Abstract]
  18. 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 {alpha} and ß-subunits. J Mol Biol 289:1375–1385[CrossRef][Medline]
  19. Weiner RS, Dias JA, Andersen TT 1991 Epitope mapping of human follicle stimulating hormone-{alpha} using monoclonal antibody 3A identifies a potential receptor binding sequence. Endocrinology 128:1485–1495[Abstract]
  20. Isaacs NW 1995 Cystine knots. Curr Opin Struct Biol 5:391–395[CrossRef][Medline]
  21. Sun PD, Davies DR 1995 The cystine-knot growth-factor superfamily. Annu Rev Biophys Biomol Struct 24:269–291[CrossRef][Medline]
  22. 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 708:160–166[Medline]
  23. Fujiki Y, Rathnam P, Saxena BB 1980 Studies on the disulfide bonds in human pituitary follicle-stimulating hormone. Biochim Biophys Acta 624:428–435[Medline]
  24. Lapthorn AJ, Janes RW, Isaacs NW, Wallace BA 1995 Cystine nooses and protein specificity. Nat Struct Biol 2:266–268[Medline]
  25. Campbell RK, Dean-Emig DM, Moyle WR 1991 Conversion of human choriogonadotropin into a follitropin by protein engineering. Proc Natl Acad Sci USA 88:760–764[Abstract]
  26. 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 269:25289–25294[Abstract/Free Full Text]
  27. 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 135:1235–1240[Abstract]
  28. 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 266:19357–19361[Abstract/Free Full Text]
  29. Szkudlinski MW, Teh NG, Grossmann M, Tropea JE, Weintraub BD 1996 Engineering human glycoprotein hormone superactive analogs. Nat Biotechnol 14:1257–1263[Medline]
  30. Weiner RS, Dias JA 1992 Identification of assembled epitopes on the {alpha}-subunit of human follicle stimulating hormone. Mol Cell Endocrinol 85:41–52[CrossRef][Medline]
  31. Roth KE, Dias JA 1995 Scanning-alanine mutagenesis of long loop residues 33–53 in follicle stimulating hormone ß subunit. Mol Cell Endocrinol 109:143–149[CrossRef][Medline]
  32. Roth KE, Dias JA 1996 Follitropin conformational stability mediated by loop 2 ß effects follitropin-receptor interaction. Biochemistry 35:7928–7935[CrossRef][Medline]
  33. Chen F, Wang Y, Puett D 1992 The carboxy-terminal region of the glycoprotein hormone {alpha}-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin. Mol Endocrinol 6:914–919[Abstract]
  34. Yoo J, Zeng H, Ji I, Murdoch WJ, Ji TH 1993 COOH-terminal amino acids of the {alpha} subunit play common and different roles in human choriogonadotropin and follitropin. J Biol Chem 268:13034–13042[Abstract/Free Full Text]
  35. Zeng H, Ji I, Ji TH 1995 Lys91 and His90 of the {alpha}-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 136:2948–2953[Abstract]
  36. Arnold CJ, Liu C, Lindau-Shepard B, Losavio ML, Patrascu MT, Dias JA 1998 The human follitropin {alpha}- subunit C terminus collaborates with a ß-subunit cystine noose and an {alpha}-subunit loop to assemble a receptor-binding domain competent for signal transduction. Biochemistry 37:1762–1768[CrossRef][Medline]
  37. 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 {alpha}-subunit in the expression and bioactivity of human thyroid-stimulating hormone. Mol Endocrinol 9:948–958[Abstract]
  38. Roth KE, Liu C, Shepard BA, Shaffer JB, Dias JA 1993 The flanking amino acids of the human follitropin ß- subunit 33–53 region are involved in assembly of the follitropin heterodimer. Endocrinology 132:2571–2577[Abstract]
  39. 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 89:141–151[CrossRef][Medline]
  40. CCP4 1994 Collaborative Computing Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50:760–763[CrossRef]
  41. Terwilliger TC, Berendzen J 1999 Automated MAD and MIR structure solution. Acta Crystallogr D 55:849–861[CrossRef][Medline]
  42. 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 54:905–921[CrossRef][Medline]
  43. 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 47:110–119[CrossRef][Medline]
  44. Laskowski RA, McArthur MW, Moss DS, Thornton JM 1993 PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:282–291[CrossRef]
  45. Kleywegt G 1999 Experimental assessment of differences between related protein crystal structures. Acta Crystallogr D 55:1878–84[CrossRef][Medline]
  46. Evans SV 1993 SETOR: hardware-highlighted three- dimensional solid model representation of macromolecules. J Mol Graph 11:134–138[CrossRef][Medline]
  47. Kraulis PJ 1991 MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr 24:946–950[CrossRef]