Effector Sites in the Three-Dimensional Structure of ... - Cell Press

Structure, Vol. 8, 1179–1188, November, 2000, 2000 Elsevier Science Ltd. All rights reserved.

PII S0969-2126(00)00523-2

Effector Sites in the Three-Dimensional Structure of Mammalian Sperm ␤-Acrosin Rebecca Tranter,* Jon A. Read,* Roy Jones,† and R. Leo Brady*‡ * Department of Biochemistry University of Bristol Bristol BS8 1TD United Kingdom † Gamete Signalling Laboratory The Babraham Institute Cambridge CB2 4AT United Kingdom

Mammalian spermatozoa have evolved specialized structures and molecules to enable them to bind to and penetrate the zona pellucida, the extracellular matrix that surrounds all ovulated eggs. One of these specialized organelles is the acrosome, a membrane-bound vesicle that overlies the anterior sperm head. Upon receiving the correct stimulus from the egg, the acrosome undergoes exocytosis and releases its contents onto the surface of the zona pellucida, a process known as the acrosome reaction (reviewed in [1]). A major component of

the acrosome is proacrosin, the zymogen form of the serine protease ␤-acrosin (E.C.3.4.21.10), which is produced soon after release of the acrosomal contents. Although the precise role of ␤-acrosin in the fertilization process remains unclear, its location and unusually high cell specificity makes it a potential target for developing novel antifertility agents. Proacrosin is expressed postmeiotically in the testis, and its primary sequence has been elucidated in at least six species (boar, bull, rat, guinea pig, mouse, and human; [2, 3]). There is a high degree (70%–80%) of sequence identity between them and to other serine proteases such as trypsin (35%), chymotrypsin (33%), elastase (29%), and kallikrein (27%), especially in the catalytic domain containing the His, Asp, Ser triad. Most serine proteases are activated from their precursors by a single cleavage event at either the N or C terminus and, in this respect, proacrosin is unusual as its activation involves processing at both ends of the molecule. The conversion of boar proacrosin to ␤-acrosin is illustrative. Initially, there is a site-specific cleavage between Arg-22 and the adjacent Valine (Val-16 in the numbering scheme in Figure 1) to create a 22 residue “light” chain and a 376 residue “heavy” chain, which remain cross-linked by two disulphide bonds to form a heterodimer [4]. At this stage the protein (known as ␣-acrosin) acquires protease activity. Further activation is brought about by three sequential endoproteolytic cleavages at the C-terminal end to remove a total of 70 residues, including a characteristic polyproline motif [5], that yields highly active ␤-acrosin with a molecular weight of 36–38 kDa by SDS-PAGE. The enzyme is potentially N-glycosylated at two sites, Asn-2 and Asn-169. Although ␤-acrosin has been described as trypsin-like, it has more proline and arginine residues and a higher polarity index (41%) than pancreatic trypsin, which may reflect membraneassociating domains of the protein [2]. It also shares features with other trypsin-like blood coagulation proteases such as Factor Xa (FXa), tissue plasminogen activator (tPA), and the urokinase plasminogen activator (uPA). All of these enzymes are heterodimers with heavy and light chains joined by disulphide bonds. There are still conflicting views on the role of ␤-acrosin during fertilization. Traditionally, it has been thought to help spermatozoa penetrate through the zona pellucida (i.e., to act as a lysin; [6, 7]) although doubts about this have been expressed on numerous occasions, notably as a result of recent “knockout” experiments which have shown that mouse spermatozoa lacking ␤-acrosin are still capable of penetrating the zona [8, 9]. It has also been suggested that ␤-acrosin enhances dispersal of the acrosomal contents following the acrosome reaction [10], and there is evidence that it has a role as a secondary binding protein between spermatozoa and the zona pellucida [11–14]. The initial recognition and binding interactions that take place between gametes are highly specific and follow a sequential series of well-ordered steps (see [15]). Initially, primary ligand molecules on the sperm plasma membrane overlying the acrosome bind to primary receptors on a specific zona pellucida glycoprotein (ZP3), thereby tethering the motile spermatozoon to the egg. Binding is thought to cause the primary ligand

‡ To whom correspondence should be addressed (e-mail: l.brady@ bris.ac.uk).

Key words: ␤-acrosin structure; spermatozoa; zona pellucida; serine protease; fertilization

Summary Background: Proacrosin is a serine protease found specifically within the acrosomal vesicle of all mammalian spermatozoa. During fertilization proacrosin autoactivates to form ␤-acrosin, in which there is a “light” chain cross-linked to a “heavy” chain by two disulphide bonds. ␤-acrosin is thought to be multifunctional with roles in acrosomal exocytosis, as a receptor for zona pellucida proteins, and as a protease to facilitate penetration of spermatozoa into the egg. Results: The crystal structures of both ram and boar ␤-acrosins have been solved in complex with p-aminobenzamidine to 2.1 A˚ and 2.9 A˚ resolution, respectively. Both enzymes comprise a heavy chain with structural homology to trypsin, and a light chain covalently associated in a similar manner to blood coagulation enzymes. In crystals of boar ␤-acrosin, the carboxyl terminus of the heavy chain is inserted into the active site of the neighboring molecule. In both enzyme structures, there are distinctive positively charged surface “patches” close to the active site, which associate with carbohydrate from adjacent molecules and also bind sulfate ions. Conclusions: From the three-dimensional structure of ␤-acrosin, two separate effector sites are evident. First, proteolytic activity, believed to be important at various stages during fertilization, arises from the trypsin-like active site. Activity of this site may be autoregulated through intermolecular associations. Second, positively charged regions on the surface adjacent to the active site may act as receptors for binding zona pellucida glycoproteins. The spatial proximity of these two effector sites suggests there may be synergy between them. Introduction

Structure 1180

Figure 1. Sequence of ␤-Acrosin (a) Nucleotide and corresponding amino acid sequence for ram acrosin. (b) A comparison of the amino acid sequence of ram ␤-acrosin with other known ␤-acrosin sequences. Residues forming the light chain, which extends from residues labeled 1–22, in pink, are aligned with the pink bar. The heavy chain begins immediately after this region, with the N-terminal residue numbered 16. The C termini of the heavy chain, as observed in the crystal structure, correspond to Thr-254 in ram acrosin and Arg-257 in boar acrosin (both marked in red). The two N-linked glycosylation sites are shown in green, and the catalytic triad residues (His, Asp, Ser) in blue. Amino acid numbering follows the convention for ␣-chymotrypsin. Residues forming the positively charged exosites are highlighted in yellow.

molecules to cluster together and induce the acrosome reaction. To retain these acrosome-reacted spermatozoa on the zona surface, secondary ligand molecules exposed on the acrosomal matrix or inner acrosmal membrane interact with secondary receptors on a different zona glycoprotein (ZP2). The sperm head then begins to penetrate through the zona pellucida largely as a consequence of the thrust forces generated by the motile flagellum [16]. Finally, tertiary ligands on the equatorial segment of the spermatozoon bind it to integrinlike receptors on the oolemma [17]. Evidence from several laboratories is strongly supportive of a role for ␤-acrosin as a secondary ligand molecule. It has been shown to bind nonenzymically to zona glycoproteins through interaction of basic residues on the surface of ␤-acrosin, forming strong ionic bonds with polysulphate groups on the carbo-

hydrate moiety of the zona glycoprotein [13, 18, 19]. The nature of the “backbone” structure carrying the polysulphate groups is relatively unimportant (polyvinylsulphate, fucoidan, and dextran sulfate are all effective inhibitors of zona glycoprotein binding to ␤-acrosin) except insofar as it presents the sulfate groups in the correct stereochemical alignment for interacting with basic residues on ␤-acrosin. Synthetic peptides of polylysine or polyarginine have little or no affinity for zona glycoproteins [18], indicating that the tertiary structure of ␤-acrosin is equally important for the formation of the polysulphate “docking” site in a manner analogous to the heparin binding site on antithrombin III [20]. This is enforced by the high level of selectivity of binding between zona glycoproteins and ␤-acrosin; other serine proteases (e.g., trypsin and elastase) or proteins with a similarly high pI (e.g., myelin basic protein) show

Structure of Sperm ␤-Acrosin 1181

approximately 10-fold less uptake of zona glycoproteins [21]. Furthermore, suramin, a polysulphonated drug, binds strongly to ␤-acrosin and inhibits sperm-zona binding in in vitro fertilization assays [15]. Suramin is thought to adopt the conformation of a shallow ladle [22] with three sulphonate groups at each of its extremities, suggesting that there is an optimal spatial configuration that is compatible with the docking site on ␤-acrosin. It is known from a limited number of site-directed mutagenesis experiments that some of the important residues within the docking site are His-37.D, Arg-37.G, and Arg-38 and Arg-221.A, Lys-223, and Arg 224 [18, 23] (numbering as in Figure 1). However, the stoichiometry of suramin or zona glycoprotein binding to ␤-acrosin is not known and other residues may be involved. In total, the above evidence suggests that the three-dimensional structure of ␤-acrosin is crucial for its functionality, both as a protease and as a secondary binding molecule at fertilization. In this paper we describe the crystal structures of ␤-acrosin from two species, boar and ram. Both enzymes have been isolated from spermatozoa and hence contain the correct posttranslational modifications. The proteins crystallize in different crystal lattices, and hence the associations observed within each crystal environment provide an opportunity to examine the surface interactions of ␤-acrosin. Results and Discussion Sequence of Ram ␤-Acrosin Figure 1 shows the nucleotide sequence and corresponding deduced amino acid sequence of ram ␤-acrosin aligned by homology with other known mammalian acrosin sequences. Both ram and boar ␤-acrosin sequences have been numbered by convention using the chymotrypsin numbering system. The heavy chain of ram ␤-acrosin is therefore numbered from 16 to 254, and boar ␤-acrosin is numbered from 16 to 257. Ram ␤-acrosin is most closely related to bovine ␤-acrosin in sequence, sharing 93% sequence identity. There is also considerable homology with the human, boar, mouse, and rat ␤-acrosins, especially in the catalytic domains. The proteins vary most at the carboxyl termini of the heavy chain, a region rich in prolines and previously suggested as a site of interaction or anchoring with acrosomal membranes. Overall Structure ␤-acrosin from both species comprises two polypeptide chains, here referred to as H (heavy chain) and L (light chain). These chains are joined covalently by two disulphides between residues L5 and H114 and L9 and H122. The heavy chain, for which there are 260 residues visible in the electron density in ram ␤-acrosin and 263 in boar ␤-acrosin, adopts a very similar fold to that observed for trypsin and the trypsin-like serine proteases such as FXa, tPA, and uPA (Figure 2). This wellstudied fold comprises a bilobal structure, with each subdomain formed almost exclusively from antiparallel ␤ barrel structure. The subdomains are related by a pseudo-2-fold axis, with the catalytic triad (His-57, Asp-102, and Ser-195) located close to the bilobal interface. All of the core features of the trypsin fold are also present in ␤-acrosin, with significant changes being restricted to surface loops. Many of the inserted loop regions in ␤-acrosin are similar to those observed in uPA; these include insertions in loop regions between residues 37 and 38, 59 and 62, and 201 and 203 and the extended surface helical region 170 and172. Two features which appear unique to ␤-acro-

sin are an insertion of five residues in the 74–79 loop and an additional short helix at the carboxyl terminus formed by residues 242–252. The root mean square deviation (rmsd) of 210 equivalent C␣ positions between ram ␤-acrosin and bovine trypsin is 1.2 A˚, and 1.9 A˚ for the same 210 equivalent atoms of boar ␤-acrosin to trypsin. The overall structures of the heavy chains for ram and boar ␤-acrosins are very similar (rmsd 0.51 A˚ for 273 equivalent C␣ positions). One notable area of difference between the two ␤-acrosin structures is seen in the carboxy-terminal residues, where the polypeptide chain is visible in the structure of boar ␤-acrosin for five additional residues beyond the terminal helix. These residues are stabilized in this conformation by their interaction with the active site of a neighboring molecule (see below). The hydrophobic residues of the amino terminus of both heavy chains insert between the helices immediately behind the active site (Figure 2), consistent with conformational changes associated with autocleavage at this site leading to activation of the enzyme. Some measure of evolutionary similarity within a protein family can be ascertained from the placement and conservation of disulphide bonds. ␤-acrosin has six disulphide bonds: four intrachain bonds within the heavy chain and two interchain links between the heavy and light chains. The four intrachain disulphides in the heavy chain of ␤-acrosin (42–58, 136–201, 168–182, and 191–220) are all shared by trypsin, FXa, uPA, and tPA. ␤-acrosin does not have the link from position 22 as seen in trypsin (22–157) and FXa (22–27). Additionally, ␤-acrosin lacks the 50–111 disulphide characteristic of uPA and tPA. The heterodimeric trypsin-like enzymes FXa, tPA, and uPA all link their light chains through an interchain disulphide arising from position 122 of the heavy chain. ␤-acrosin also shares a link at this site but is unique in having two sites of attachment for the light chain: L5-H114 and L9-H122. These disulphides are conserved in all known sequences of ␤-acrosin. These observations suggest that although these enzymes undoubtedly share a common evolution, ␤-acrosin forms a distinct subgroup of this family. The light chain in both structures is only partially ordered in the electron density, with residues L3-L16 visible in the ram structure and residues L3-L15 in the boar structure. This chain is located on the opposite face of the heavy chain from the active site, similar to the accessory chains seen in the blood coagulation enzymes such as FXa and uPA. However, the composition and conformation of the light chains in all of these serine proteases differs significantly (Figure 3). In both forms of ␤-acrosin, the six residue segment between the two cysteine residues where the polypeptide chain is anchored to the heavy chain adopts a similar, extended conformation, but lacks any secondary structure. The light chain is formed by specific cleavage between Arg-L22 and Ile/Val-H16 (the first residue of the heavy chain), but only residues in the light chain up to AsnL16 are visible in the electron density. It is not clear whether the terminal six residues of the light chain are present within the crystals; the inclusion of an arginine at position L17 of ram acrosin may result in further cleavage and hence trimming of the chain. However, from the relative locations of the light chain carboxyl terminus and the amino terminus of the heavy chain, it is apparent that considerable distortion would be necessary to form the proenzyme in an intact, single polypeptide chain. This distortion would undoubtedly extend to the nearby active site residues, explaining the inactivity of the proenzyme. Apart from the disulphide linkages, the light chain makes limited contacts with its partner heavy chain, with 1483 A˚2 of solvent-

Structure 1182

Figure 2. Structure of ␤-Acrosin Stereo diagrams showing C␣ traces of the structures of ram ␤-acrosin (a), with the light chain in pale green and heavy chain in red. Every 20th residue is numbered and the amino and carboxyl termini of each chain are labeled N and C, respectively. The active site is located to the left on this figure, where the upper of the two bound benzamidine molecules is shown in black. The carbohydrate visible in the electron density is shown at the top of the figure, with Asn-169 also in black. (b) In the same orientation, the structure of bovine trypsin (blue) overlaid on the structure of ram ␤-acrosin (red and green; color scheme as in [a]), and (c) the structure of boar ␤-acrosin (heavy chain in blue, light chain in orange, carbohydrate in pink) overlaid on the structure of ram ␤-acrosin (colored as in [a]), again in the same orientation. The four sulphate ions bound to boar ␤-acrosin are shown in orange.

accessible surface area buried between the residues from each chain that are ordered within these structures. Nonetheless, the unique arrangement and strict conservation of the two disulphides in all forms of ␤-acrosin (Figure 1) suggests that the light chain might play a functional role beyond activation from the proenzyme. There are discrepancies within the literature regarding the

authentic carboxyl terminus of the heavy chain, which is trimmed in the autocatalytic conversion of the proenzyme to ␤-acrosin. Sequence comparisons (Figure 1) show there are a number of arginines in the region beyond residue 250 that could form potential cleavage sites, but no absolutely conserved consensus site in all ␤-acrosin sequences. Polypeptide chain termini are frequently disordered within crystal lattices, compli-


Figure 3. Structure of the Light Chain of ␤-Acrosin Figure shows an overlay of the light chain conformations of ram ␤-acrosin (orange), boar ␤-acrosin (red), and the equivalent light chain conformations observed in Factor Xa (blue), urokinase plasminogen activator (dark green), and thrombin (light green). ␤-acrosin is unique in having two interchain disulphide points of attachment with its associated heavy chain, which tightly restricts the conformation of the light chain amino acids between these links. In all cases the extremities of the light chains are disordered in the respective crystal structures.

cating the determination of the true chain terminus. In the structure of boar ␤-acrosin, however, the heavy chain carboxyl terminus is bound within the active site of a neighboring molecule (see below). This arrangement shows clearly that the polypeptide chain terminates at Arg-257 within these crystals. However, the sequence in this region (PPRP), unique to boar acrosin, is similar to a consensus segment 280–283 further along the chain of the proenzyme, (V/I/A) (R/H)PP, which has previously been assigned as the ␤-acrosin carboxyl terminus. As crystals of both forms of ␤-acrosin grow over extended periods of time (4–6 months), it is possible that slow hydrolysis over this period leads to further trimming from the carboxyl terminus. The final residue visible in the ram ␤-acrosin structure is Thr-254, from a stretch of sequence completely devoid of positively charged amino acids. However, this residue is adjacent to an open, solvent channel in the crystals that may contain further, disordered amino acids extending from the carboxyl terminus. Effector Site I: The Catalytic Active Site Upon activation, ␤-acrosin acts as a trypsin-like protease during the acrosome reaction. From the high structural homology of ␤-acrosin with the trypsin-like family of proteases, it is logical to assign this behavior to the familiar active site of these enzymes. Both ram and boar ␤-acrosin have been cocrystallized in the presence of benzamidine, which is seen to bind in the S1 pocket (using the terminology of Schechter and Berger [24]) of the active site, in contact with the invariant Asp-189 (Figure

2). A second molecule of benzamidine is also found bound between adjacent copies of ram ␤-acrosin in the crystal lattice (Figure 2a). This molecule makes contacts with the Glu-85 side chain and the main chain carbonyl of Ser-180 and a watermediated hydrogen bond with Asp-164, but its binding is likely to be an artefact of the crystallization matrix. The identity and conformation of the active site residues in both forms of ␤-acrosin bears considerable homology to the equivalent region in trypsin and other trypsin-like enzymes. The S1 pocket, which regulates specificity for Arg and Lys substrates, is highly similar in both sequence and conformation in crystal structures of ␤-acrosin, trypsin, tPA, Fxa, and uPA. In all cases the pocket is bordered by the segments 213–220, and 225–229, with the conserved Trp-215 at the entrance to the pocket and Asp-189 and Ser-195 at its base. Autocatalysis within the ␤-acrosin polypeptide appears to occur predominantly at sites adjacent to Arg residues. It has previously been proposed that the preference of tPA for Arg over Lys substrates can be explained by its extended and largely nonpolar S1 pocket [25]. Despite its apparent similar specificity for Arg substrates, the S1 pocket in ␤-acrosin, however, does not share these features and is more similar to those seen in trypsin and FXa. The S2 and S4 pockets have been used to achieve inhibitor specificity for FXa, tPA, and uPA (e.g., see [26–28]). In both tPA and FXa, the S2 pocket is dominated by a tyrosine at position 99, leading to restricted accommodation for side chains in the P2 position and hence a preference for substrates with Gly in this location. uPA has an insertion after position 97, resulting in an extended loop that diminishes the size of both S2 and S4 pockets. Position 99 is occupied by Leu in trypsin, whereas in ␤-acrosin there is a conserved glutamine. The positioning of the alkyl chain of the glutamine side chain and variations in the surrounding main chain conformation lead to a relatively enlarged S2 pocket compatible with bulkier residues in the P2 position of substrates. It is noteworthy that many of the reported autocleavage sites at the carboxyl terminus of the heavy chain in ␤-acrosin have proline, leucine, or valine in the P2 position. Significant changes are also observed in the adjacent S4 pocket, the wall of which is formed by residues from the 172–177 strand. Here the main chains in trypsin, uPA and ␤-acrosin are similar and vary considerably from FXa and tPA. Within this cohort of proteinases, ␤-acrosin is unique in contributing two arginines into the pocket, a feature which could be exploited in the design of specific ␤-acrosin inhibitors. The arginine in position 174 of tPA has been successfully used to achieve specificity for this enzyme [26]. Although the active site of boar ␤-acrosin is, as expected, very similar to that of ram ␤-acrosin, the molecular packing in these crystals is very diffferent. Despite access to the active site being partially occluded by the presence of the benzamidine inhibitor, segments from a neighboring molecule within the crystal lattice are seen to dock in the active site. These regions are the heavy chain C terminus (residues 255 to 257) and the 201–205 loop (Figure 4). We note that there are several similarities in the mode of association between ␤-acrosin molecules and structures of inhibitory peptide complexes of trypsin. The ␤-acrosin C-terminal residue Arg-257 is prevented from fully entering the S1 pocket, which is occupied by benzamidine. Nevertheless, the arginine lies close to the entrance to S1, and only a small shift would be required to allow the arginine to enter the pocket on removal of inhibitor. Instead, the arginine side chain sits in the pocket normally occupied by P1⬘ residues, where it makes a salt bridge with Lys-59. The adjacent proline,

Structure 1184

Figure 4. Active Site of Boar ␤-Acrosin (a) The boar ␤-acrosin heavy chain carboxyl terminus inserts into the active site of a neighboring molecule. The figure shows the placement of the heavy chain carboxyl terminus (residues Pro-255, Pro-256, and Arg-257) and adjacent loop residues Asp-201, Arg-202, and Ala-203 within the active site of another molecule (shown as a molecular surface). The location of the catalytic site serine (Ser-195) is marked by the pink surface, the p-aminobenzamidine inhibitor is shown in green, and the specificity pockets are labeled according to the convention of [24]. (b) Corresponding electron density (2|Fobs| ⫺ |Fcalc| coefficients contoured at 1 ␴) for the active site region shown in part (a). Density for the residues inserted into the active site are shown, along with the density corresponding to the active site serine (Ser-195) and the p-aminobenzamidine inhibitor.

Pro-256, forms a complimentary fit in the S2’ pocket. Note that this segment of the polypeptide chain is accommodated in the reverse direction (C→N rather than N→C) from bound substrates. The inserted fragment terminates where the scissile peptide bond is normally located in the activated complex, and the S2S4 pockets are occupied by amino acids from the 201–205 loop. The Arg-202 side chain is located in the S4 pocket of the enzyme, and the S2 pocket is partially occupied by Ala-203, with Glu-99 at its entrance forming a salt bridge with Asp201 from the inserted segment. The total solvent-accessible surface area buried in this interaction between two adjacent molecules of ␤-acrosin is 2478 A˚2. Although this binding may be completely fortuitous, the proline-rich regions of the various ␤-acrosins contain several examples of the (R/K)PP motif present at the carboxyl terminus of the bound fragment, and it is tempting to speculate that this may frequently lead to interactions similar to these observed in the crystal lattice. In the crystal, these intermolecular associations generate an extended helical rod, reminiscent of the polymers reported in the crystal structure of the serpin ␣1-antitrypsin [29]. Intermolecular assemblies of ␣1-antitrypsin are generated by insertion of the uncleaved serpin active site loop into adjacent molecules, and the resulting inactivation of the enzyme is associated with a variety of diseases. The ability of boar ␤-acrosin to form helical polymers suggests that assembly of this enzyme may provide an efficient means of autoinhibition. This interaction also illustrates surfaces of the ␤-acrosin active site that are accessible for the design of specific inhibitors. Effector Site II: ␤-Acrosin as a Receptor for Zona Pellucida Glycoproteins Both proacrosin and ␤-acrosin have been suggested to act as secondary binding molecules for receptors on zona pellucida glycoproteins, hence assisting attachment of acrosome-reacted sperm to the egg surface. The matrix of the zona pellucida is composed of a limited range of proteins (in most species only

3–4), all of which are heavily glycosylated and sulphated [30, 31]. Previous structural analyses of the serine protease thrombin have identified a highly charged patch on its surface termed the anion binding exosite-I, through which it binds to the thrombomodulin receptor [32]. This region, which is separate from the active site, is formed from a cluster of lysine and arginine residues primarily in the 35–40 and 75–80 loop regions. uPA also has a concentration of positive charges in a similar location [33]. A consistent feature in both ram and boar ␤-acrosin structures is the presence of two regions of concentrated positively charged amino acids on the periphery of the active site (Figure 5). These regions differ from those previously described for thrombin and are formed primarily by residues from the 35–40, 60–65, and 87–94 loop regions (exosite I) and the 145–150, 170–178, 186–188, and 220–225 loops (exosite II). Many of the positively charged residues in these regions are highly conserved throughout all known ␤-acrosin sequences (Figure 1). These two patches straddle the catalytic active site described above, forming a semicontinuous ring of positive surface charge at its periphery. There have been several reports demonstrating saturable, high-affinity (10⫺8 M) binding between ␤-acrosin and sulphated polymers [13, 14, 18, 21]. This activity is also seen in recombinant forms of ␤-acrosin expressed in bacterial systems, which, because of the high disulphide content of ␤-acrosin, are unlikely to have fully native folded conformations. This suggests a level of degeneracy in the sulfate binding ability of ␤-acrosin consistent with a multivalent surface patch of net negative charge. This affinity for anions is also maintained in truncated forms of ␤-acrosin, which has allowed predictions of the regions of the sequence likely to be involved in formation of the binding site. These have been reported to involve residues 60–65, 87–94, and 250–253 [18, 23, 34, 35]. The former two of these clusters correspond to the regions giving rise to exosite I. Two features of the ram and boar crystal structures demonstrate the ability of these two exosite regions to act as generic anion receptors. There are two conserved N-linked glycosyla-


Figure 5. Surface of ␤-Acrosin GRASP [48] surface representation of ([a], left) ram ␤-acrosin and ([b], right) boar ␤-acrosin. In both cases the view is looking directly toward the active site face. The position of the active site serine (S195) is labeled, as are positively charged side chains contributing to exosite I (labeled ExoI) and exosite II (labeled ExoII). The conserved carbohydrate at Asn169 is labeled CHO.

tion sites in mammalian acrosin sequences (Figure 1): one close to the N terminus of the light chain at position Asn-L2 and the other in the heavy chain at Asn-169. As both forms of ␤-acrosin used in this study were isolated from spermatozoa, the native form of glycosylation is present within the crystals. Whereas glycoprotein carbohydrate structures are usually highly mobile within crystal lattices, much of the bound carbohydrate at the heavy chain site is clearly visible in the electron density map, especially in the ram ␤-acrosin structure. In the model for ram ␤-acrosin, it has been possible to trace five sugar residues with confidence: the first two N-acetyl glucosamines, a 1→6 branched fucose, and two 1→4 mannoses. These residues are located on the periphery of the face of the enzyme containing the active site. In the hexagonal crystals, crystallographic dimers are formed by face-to-face assembly of pairs of molecules (Figure 6). In the ram ␤-acrosin structure, the unusual level of order associated with the conserved Asn169 carbohydrate arises from a plethora of contacts made primarily between the hydroxyl groups of the sugar rings and the positively charged amino acid side chains from the 60–65 and 87–94 loops from neighboring molecules in the crystal lattice. These are the same regions which give rise to the anionic exosite I described above. The clear visibility of the carbohydrate groups in this region of the electron density map demonstrates the ability of this region of the ␤-acrosin surface to form stable complexes with charged groups, perhaps mimicking its interactions with the glycoproteins of the zona pellucida. We note that if similar associations are made with other proteins, this would present the catalytic site of ␤-acrosin in close proximity to the bound glycoprotein. These observations are supported by the molecular associations observed in the cubic crystals of boar ␤-acrosin, where the molecular packing is very different. Although the arrangement in these crystals is dominated by the head-to-tail interactions allowing the C terminus of each molecule to insert into the active site on the opposite molecular face of a neighboring molecule as described above, we note that the carbohydrate from the conserved Asn-169 site is now predominantly located adjacent to either residues 87–94 or 172–178 from neighboring molecules in the crystal lattice. These regions correspond to

exosites I and II, respectively. Although in this case the electron density is less well ordered and only allows us to trace four of the sugar rings with confidence, it is clear that low-energy, favorable interactions are formed between the sugars and adjacent protein surfaces. A second fortuitous outcome of the crystallization procedure is also encountered in the crystal structure of boar ␤-acrosin. These crystals were obtained in the presence of 200 mM ammonium sulfate buffer, and in the refined structure, four sulfate groups are particularly evident in the electron density. Three of these groups are seen to cluster in one region of the ␤-acrosin surface (Figure 2c), where they bind directly to the side chains of Arg-170.B, Arg-175, Arg-201.A, Arg-201.A, Arg-221.A, and Lys-223. All of these amino acids contribute to the surface region of high negative charge density, which we have described as exosite II. These observations provide a direct illustration of the ability of ␤-acrosin to bind to negatively charged sulfate groups and hence by analogy to polysulphated compounds such as the heavily glycosylated and sulphated proteins that are common in the matrix of the zona pellucida. In combination with the carbohydrate associations described above, this supports the notion of the two exosite regions forming a receptor-like face on ␤-acrosin that dominates its interactions with other molecules. This ability of ␤-acrosin to bind anions is likely to explain the tight association reported for the anticancer drug suramin [15]. This highly symmetrical compound contains two polysulphonated napthalene groups at its extremities. Binding of these groups to any of the cationic patches surrounding the active site face could feasibly obstruct access of substrates, hence explaining the inhibitory effect of suramin on the amidase activity of ␤-acrosin [36]. The proximity of these highly charged regions to the active site provides obvious opportunities for the design of specific inhibitors. A Combined Model for ␤-Acrosin Action The catalytic and receptor binding activities of ␤-acrosin have frequently been studied and described separately, and indeed up to this point we have also described the regions from which these activities are derived individually. However, one promi-

Structure 1186

Figure 6. Molecular Associations of ␤-Acrosin Figure shows the arrangement of ␤-acrosin molecules in crystals of (a) ram ␤-acrosin and (b) boar ␤-acrosin. In (a) residues forming exosite II (colored cyan) in the orange molecule contact the ordered carbohydrate (colored green and marked CHO) from the symmetryrelated red molecule. Additionally, residues forming exosite I (colored yellow) contact the carbohydrate from another symmetry-related molecule (colored magenta; carbohydrate only from this molecule shown). In crystals of boar ␤-acrosin (b), despite the very different crystal packing, the carbohydrate from one molecule (colored magenta) is ordered through binding to residues forming exosite II on the symmetry-related (orange) molecule. In both figures some of the positively charged residues that are involved in carbohydrate binding are labeled.

nent feature in the surface representations in Figure 5 is the proximity of the exosite regions to the catalytic active site. It is tempting to speculate, therefore, that the role of the exosite regions is to target ␤-acrosin to particular substrates. As the active site cavity itself has a small overall negative charge (see Figure 5), proteins with excess surface negative charge are likely to be disfavored as substrates. Addition of the exosites at the periphery reverses this tendency. Although previous studies [18, 35, 37] have noted that the receptor binding activity of ␤-acrosin is maintained in the presence of trypsin-specific inhibitors, this division of functions is compatible with the separate physical identities of the two sites noted in these crystal structures. We anticipate that combined functioning of the two activities would result in significant enhancement in hydrolysis of proteins rich in posttranslational modifications that generate an overall negative net surface charge. These crystal structures are compatible with an enhanced hydrolytic role for ␤-acrosin within the zona pellucida matrix. Low concentrations of zona glycoproteins and sulphated polymers potentiate proacrosin activation but become inhibitory beyond a threshold molar ratio [36, 38]. This has been attributed to contact activation following adsorption onto a “soluble” surface (the glycoprotein or sulphated polymer) analogous to that observed for enhanced conversion of factor XII to factor XIIa in the presence of dextran sulfate or bound to glass [39]. The subsequent inhibition of ␤-acrosin activity may be due to masking of the active site following saturation

binding of zona glycoproteins. These interpretations are supported by the fact that active ␤-acrosin has only a limited proteolytic effect on the zona surface when incubated for several hours with intact eggs [40], in contrast to pronase, which causes complete degradation of all egg investments to small glycopeptides. It has been speculated that this limited proteolysis actually enhances binding between zona glycoproteins and ␤-acrosin, thereby causing enzyme inhibition [13].

Biological Implications ␤-acrosin is a sperm-specific serine protease released from the acrosomal vesicle at the time of fertilization. Historically, it has been regarded as a lysin to facilitate penetration of spermatozoa through the zona pellucida that surrounds all mammalian eggs. Current evidence, however, indicates that it is a multifunctional protein with additional roles in dispersal of the acrosomal matrix and as a secondary binding molecule to retain acrosome-reacted spermatozoa on the zona surface. To elucidate the structural requirements for these different roles, we have analyzed the crystal structures of native ␤-acrosin from ram and boar spermatozoa. Our results reveal that ␤-acrosin shares many features with other trypsin-like serine proteases, with a unique active site signature presenting new opportunities for specific inhibitor design. ␤-acrosin is a heterodimer formed by covalent linkage


Table 1. Summary of Data Collection and Refinement Statistics for the Crystal Structures of Ram and Boar ␤-Acrosin Ram ␤-Acrosin Boar ␤-Acrosin Resolution range (A˚) Rsym Redundancy Completeness (%) Number of unique reflections Number of reflections used in refinement Number of reflections in free R set Number of waters R factor R free Average Biso for protein atoms (A˚2) Rms deviation for bond length (A˚) Rms deviation for angle distances (⬚)

30–2.1 0.068 4.8 93.7 23,810 20,601 1,120 203 0.187 0.234 40.2 0.021 2.310

100–2.9 0.078 5.9 97.4 8,711 8,303 408 43 0.235 0.289 65.8 0.043 2.118

of a heavy and light chain similar to many of the blood coagulation enzymes such as Factor Xa, urokinase plasminogen activator, and tissue plasminogen activator. The 23 residue light chain is, unusually, anchored through two conserved interchain disulphide bonds, perhaps reflecting a required rigidity for effector function. Distinctive cationic surface patches are present near to the active site and contain residues previously demonstrated by site-directed mutagenesis to be involved in binding zona glycoproteins, sulphated polymers, and sulphonated drugs such as suramin. Molecular associations within the crystal lattices show that these patches bind sulfate groups and carbohydrate chains from neighboring molecules. The proximity of these regions to the active site suggests a correlation between the two independent functions previously ascribed to this family of enzymes. The structure is consistent with an “inch worm” mode of action for ␤-acrosin at fertilization: the exosites attract the enzyme and spermatozoa to sulphated zona glycoproteins, thereby promoting their cleavage. Since inhibition of both these activities leads to a decrease in fertility of spermatozoa, the information provides new opportunities for designing nonsteroidal contraceptive compounds. Experimental Procedures Purification, Crystallization, and Structure Analysis of ␤-Acrosin ␤-acrosin was purified from ejaculated ram and boar spermatozoa with slight modifications to established protocols [36, 41]. Briefly, the 35% ammonium sulfate precipitate enriched in proacrosin was redissolved in 1 mM HCl (pH 3.0) and dialyzed for 48 hr at 5⬚C against three changes of the same buffer. The protein solution was made 0.1 M Tris-HCl (pH 8.5) and proacrosin allowed to autoactivate to ␤-acrosin by incubation for 16 hr at 5⬚C or, in some cases, for 4 hr at 20⬚C. The pH was reduced to 3.0 with HCl and dialyzed exhaustively against 1mM HCl. Approximately 10 mg total protein was applied to a 2 ml column of Benzamidine-Sepharose 6B (Amersham Pharmacia) equilibrated in 0.1 M Triethanolamine/1.0 M NaCl (pH 7.8) at 5⬚C and unbound protein eluted with the same buffer. Protein concentrations were monitored by absorbance at 280 nm. Bound protein (mostly ␤-acrosin) was desorbed with 10 mM ammonium formate/formic acid (pH 3.0), and protein-containing fractions were pooled and rechromatographed by FPLC on a Superose-12 column equilibrated in 50 mM acetate buffer/0.2 M NaCl (pH 4.0) at 5⬚C. The highly purified ␤-acrosin obtained gave a single band at ⵑ38 kDa by SDS-PAGE/Coomassie blue staining and was judged to be ⬎95% pure. Amidase activity of the purified enzyme was measured spectrophotometrically at 405 nm using 0.375 mM L-BAPNA substrate in 0.1 M Tris-HCl/50 mM CaCl2 (pH 8.2) at 20⬚C [36, 42]. The total ␤-acrosin purified amounted to ⵑ12 mg for ram and ⵑ20 mg for boar. Crystals of both ram and boar ␤-acrosin were grown in the presence of 10 mM p-amino-benzamidine by vapor diffusion over extended periods of time (4–6 months). The precipitant for crystals of ram ␤-acrosin was 0.1 M sodium acetate (pH 4.6), 2 M sodium formate. These crystals are hexagonal

prisms with dimensions 0.1 ⫻ 0.1 ⫻ 0.2 mm and belong to space group P6522 with a ⫽ b ⫽ 105.3 A˚, c ⫽ 120.8 A˚, and ␥ ⫽ 120.0⬚. Crystals of boar ␤-acrosin grew from 0.1 M sodium cacodylate (pH 6.5), 30% PEG 8000, and 0.2 M ammonium sulfate precipitant and formed cubes with dimensions up to 0.1 ⫻ 0.1 ⫻ 0.1 mm. These crystals belong to the cubic space group P4332 with a ⫽ 130.6 A˚. Diffraction data from crystals of ram ␤-acrosin were collected on station PX7.2 (␭ ⫽ 1.488 A˚) and data from crystals of boar ␤-acrosin were collected on station PX14.1 (␭ ⫽ 1.244 A˚), both at the Daresbury SRS synchrotron. All data were processed with DENZO/SCALEPACK [43] and are summarized in Table 1. Both structures were solved by molecular replacement using AMORE [44]. The coordinates of bovine trypsin (PDB entry 1ce5) were used as a search model to initially solve the ram ␤-acrosin structure. Using data in the range of 10–3 A˚, the correct rotation and translation solution gave an initial R factor of 0.52 (next highest solution 0.58). The boar ␤-acrosin structure was solved using the refined structure of ram ␤-acrosin as a search model. Data in the range of 10–3 A˚ gave an initial solution with R factor of 0.47. Both structures were refined using iterative cycles of manual rebuilding using QUANTA (MSI) and maximum likelihood refinement with REFMAC [45] and CNS [46]. Water molecules were added automatically using ARPP [47], and checked manually to ensure sensible geometry. Statistics for the two final models are included in Table 1. There are no outliers in the Ramachandran plots of the two final models. Sequencing of Ram ␤-Acrosin The amino acid sequence of ram ␤-acrosin was deduced from the DNA sequence. Total RNA was isolated from ram testes using a Trizol Reagent kit (Life Technologies), and the acrosin sequence was amplified by RT-PCR. Two primers (5⬘-GATGGTCAGCCTCC-AGATCTT-3⬘ and 3⬘-GGAGAGTA CACGTTTCTGTC-5⬘) were designed from regions of known sequence conservation in other ␤-acrosin sequences and used to sequence the core residues 22–201. Residues at the C terminus were then derived from further RT-PCR using two further primers (5⬘-CTTGTGTAACTCGACCAGATGGTAC-3⬘ and 3⬘-GGTGGAACCAAGAAGGTTGT-5⬘). The N-terminal sequence, which includes all of the light chain, was obtained by rapid amplification of cDNA ends (RACE) using a further primer (5⬘-GAAGATCAGCCTCCAGTCGGTCA CTTT-3⬘). Acknowledgments We thank the BBSRC for financial support and the EPSRC for the award of a CASE studentship to one of us (R. T.). We are grateful to Peter James (The Babraham Institute) for his dedication during purification of large amounts of ␤-acrosin from ram and boar spermatozoa, Dr John Pascall (The Babraham Institute) for helpful discussions, John Coadwell (BI) for assistance with database searches, Dr Jan Frayne (University of Bristol) for assistance and advice with the sequencing studies, and the staff at the Daresbury SRS for provision of data collection facilities. Received: July 26, 2000 Revised: September 22, 2000 Accepted: October 3, 2000 References 1. Yanagimachi, R. (1994). Mammalian fertilization. In The Physiology of Reproduction, Second Edition, E. Knobil and J.D. Neill, eds. (New York: Raven Press), pp.189–317. 2. Urch, U.A. (1991). Biochemistry and function of acrosin. In Elements of Mammalian Fertilization, Volume 1, P.M. Wassarman, ed. (Boca Raton, FL: CRC Press), pp. 233–248. 3. Adham, I.M., et al., and Engel, W. (1996). The structures of the bovine and porcine proacrosin genes and their conservation among mammals. Biol. Chem. Hoppe Seyler. 377, 261–265. 4. Fock-Nuzel, R., Lottspeich, F., Henschen, A., and Muller-Esterl, W. (1984). Boar proacrosin is a two-chain molecule. Eur. J. Biochem. 141, 441–446. 5. Baba, T., et al., and Arai, Y. (1989). Activation and maturation mechanisms of boar acrosin zymogen based on the deduced primary structure. J. Biol. Chem. 264, 11920–11927. 6. Nakano, M., Tanaka, Y., Kimura, T., Hatanaka, Y., and Tobita, T. (1989).

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