MINIREVIEW Hepatocyte growth factor activator (HGFA): molecular structure and interactions with HGFA inhibitor-1 (HAI-1) Charles Eigenbrot 1,2 , Rajkumar Ganesan 3 and Daniel Kirchhofer 3 1 Department of Structural Biology, Genentech, Inc., South San Francisco, CA, USA 2 Department of Antibody Engineering, Genentech, Inc., South San Francisco, CA, USA 3 Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Introduction Hepatocyte growth factor activator (HGFA) is a trypsin-like serine protease belonging to Clan PA, Family S1 (MEROPS data base, http://merops.sanger. ac.uk/). Full-length HGFA (96 kDa) has the same domain architecture as coagulation factor XII: an N-terminal fibronectin type II domain, an epidermal growth factor (EGF)-like domain, a fibronectin type I domain, a second EGF-like domain, a kringle domain, and a C-terminal protease domain (Fig. 1A). The HGFA protease domain amino acid sequence has the Keywords catalysis; hepatocyte growth factor; Kunitz domain; serine protease; structure Correspondence D. Kirchhofer, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA Fax: +1 (650) 225-3734 Tel: +1 (650) 225-2134 E-mail: dak@gene.com (Received 13 November 2009, revised 19 January 2010, accepted 8 February 2010) doi:10.1111/j.1742-4658.2010.07638.x The trypsin-like serine protease hepatocyte growth factor activator (HGFA) undergoes proteolytic activation during blood coagulation, result- ing in a 34 kDa ‘short form’, consisting mainly of the protease domain. The crystal structures of the recombinantly expressed HGFA ‘short form’ discussed herein have provided molecular insights into its interaction with inhibitors and substrates, as well as the regulation of catalytic activity. The HGFA structures revealed enzymatically competent and noncompetent forms associated with the conformational states of two substrate specific- ity-determining loops, the 220-loop and 99-loop. The implied dynamic behavior of these loops, which are intimately involved in substrate interac- tion, has precedents in other members of the S1 family of serine proteases, and may be associated with specific mechanisms of enzyme regulation. Furthermore, HGFA activity is strongly inhibited by HGFA inhibitor-1, a membrane-spanning multidomain inhibitor containing two Kunitz domains, of which only the N-terminal Kunitz domain-1 (KD1) inhibits enzymatic activity. In the structure of the KD1–HGFA complex, the inhibitor interacts with the active site region by making contacts with all substrate specificity-determining loops and by occupying subsites S1, S2 and S4 in a substrate-like manner. In fact, the side chains of KD1 residues occupying these sites are virtually superimposable on the P1, P2 and P4 residues of the pro-hepatocyte growth factor-derived substrate mimic Lys-Gln-Leu-Arg chloromethyl ketone bound to HGFA. These structures also allow us to rationalize the apparently narrow substrate specificity of HGFA, which is limited to the two known macromolecular substrates pro-hepatocyte growth factor and pro-macrophage-stimulating protein. Abbreviations EGF, epidermal growth factor; HAI, hepatocyte growth factor activator inhibitor; HGFA, hepatocyte growth factor activator; KD1, Kunitz domain-1; KD2, Kunitz domain-2; LDL, low-density lipoprotein; PDB, Protein Data Bank; pro-HGF, pro-hepatocyte growth factor; pro-MSP, pro-macrophage-stimulating protein; uPA, urokinase-type plasminogen activator. FEBS Journal 277 (2010) 2215–2222 ª 2010 The Authors Journal compilation ª 2010 FEBS 2215 highest sequence identity with that of factor XII (47%), tissue-type plasminogen activator (40%), uroki- nase-type plasminogen activator (uPA) (39%), and prostasin (38%). There is also a strong similarity at the structural level, as indicated by the rmsd values being lower than 1.0 A ˚ in a pairwise comparison of the protease domain canonical crystal structures (excluding loops): HGFA protease domain [Protein Data Bank (PDB) 1YC0] versus tissue-type plasmino- gen activator (PDB 1RTF, rmsd of 0.59 A ˚ ), versus uPA (PDB 1C5Y, rmsd of 0.58 A ˚ ), and versus prosta- sin (PDB 3DFL, rmsd of 0.67 A ˚ ) (the structure of the factor XII protease domain is not known). To date, there are only two known macromolecular substrates of HGFA, pro-hepatocyte growth factor (pro-HGF) [1] and pro-macrophage-stimulating protein (pro-MSP) [2], suggesting that HGFA has very limited substrate specificity. However, this may be an underestimation of the full complement of substrates, as no systematic substrate profiling has been performed yet. The AB CD Fig. 1. Conformational states of the HGFA active site region. (A) Cartoon of HGFA and HAI-1 domain architectures. HGFA contains a heavy chain (A-chain) disulfide-linked to the protease domain (B-chain). The subdomains of the A-chain are: fibronectin type I and type II (FNI and FNII), epidermal growth factor (EGF)-like and Kringle (Kr). Cleavage by thrombin (T) and plasma kallikrein (K) produces the serum form (‘short form’) of HGFA, comprising the protease domain and a disulfide-linked 35 amino acid peptide (Val373–Arg407) from the A-chain, which was used for crystallographic studies. HAI-1 is composed of a MANSC domain [25] followed by a structurally undefined region connecting to KD1, an LDL receptor (LDLR)-like domain, KD2, a transmembrane (TM) domain, and cytoplasmic domain (Cyt). The splice variant HAI-1B has an extra 16 amino acid stretch inserted (I) between KD1 and the LDLR-like domain. (B) The HGFA protease domain (beige, PDB 1YC0) with colored substrate ⁄ inhibitor specificity-determining loops (chymotrypsinogen numbering, i.e. ‘38-loop’, and the corresponding Perona and Craik [6] nomenclature, i.e. ‘Loop-A’) and substrate subsites (S1–S4). The catalytic triad Asp102–His57–Ser195 is indicated. (C) Conforma- tional states of the 220-loop in HGFA (left panel) as compared with prostasin (right panel). Left panel: the ‘open’ (standard conformation; PDB 1YC0) and ‘closed’ (nonstandard conformation; PDB 1YBW) HGFA forms are superimposed, with the two different 220-loop conforma- tions shown in cyan and magenta, respectively. Side chains of the catalytic triad residues (Asp102, His57, and Ser195) are indicated (yellow for ‘open’; magenta for ‘closed’), as is that of the 220-loop residues 215 and 216. The P1 Arg from the KQLR-cmk substrate mimic (see Fig. 3A) is also added to indicate the steric clash with the ‘closed’ form 220-loop. Right panel: the ‘open’ (PDB 3DFL) and ‘closed’ (PDB 3DFJ) prostasin forms are superimposed, showing the two different 220-loop conformations. The color codes are the same as for HGFA. The side chain of Asp217, which, in the ‘closed’ conformation, obstructs S1 access, is also indicated. (D) Conformational states of the HGFA 99-loop. As compared with the competent (or standard) conformation (slate blue), the 99-loop of the Fab40-inhibited HGFA (brick red: noncompetent) has shifted towards the substrate-binding cleft. The deleterious effects on catalysis derive from the repositioning of P99a and S99, both of which shape the S2 subsite. Molecular images were produced using PYMOL [38]. Molecular interactions of HGFA with inhibitor ⁄ substrate C. Eigenbrot et al. 2216 FEBS Journal 277 (2010) 2215–2222 ª 2010 The Authors Journal compilation ª 2010 FEBS cleavage site of pro-HGF is KQLR-VVNG(491–498) (P4–P4¢) and that of pro-MSP is SKLR-VVNG(487– 480) (P4–P4¢), indicating a preference for a P1 Arg. The potential roles of these substrates in mediating the proposed functions of HGFA in tissue regeneration and tumor promotion are discussed in other sections of this minireview series [3]. HGFA is secreted as a single-chain zymogen precur- sor, and is activated by cleavage at the Arg407-Ile408 bond (e.g. by thrombin during blood coagulation), resulting in the disulfide-linked two-chain form. Additional cleavage by plasma kallikrein at the Arg372-Val373 bond releases the 34 kDa HGFA ‘short form’ present in serum, containing a 35-residue peptide disulfide-linked to the protease domain. All structural studies discussed herein were performed with the ‘short form’ of HGFA recombinantly expressed in insect cells. The seven available crystal structures of the HGFA protease domain, either as apoenzyme or in complex with an inhibitor, provide a basis for understanding the known biochemical functions of this enzyme. Our discussion is focused on the regulation of its catalytic machinery, its interactions with HGFA inhibitor (HAI)-1, and its substrate specificity. For mention of specific amino acid positions, we use the chymotrypsin- ogen numbering scheme to allow easy reference to the large number of related proteins (for conversion between native HGFA and chymotrypsinogen residue numbers, see [4]), and we employ the nomenclature of Schechter and Berger [5] in describing specific sites of protease–substrate (or inhibitor) interactions. The loops in and around the active site are named accord- ing to their chymotrypsinogen numbering (for conver- sion into the Perona and Craik [6] loop nomenclature, see Fig. 1B). The catalytically competent (standard) active site conformation The determined HGFA structures reveal three different conformational states of the active site region: a cata- lytically competent (standard) conformation, and two nonstandard conformations. Herein, we use the con- formation seen in the complex of HGFA with Kunitz domain-1 (KD1) as the representative of the standard conformation, which was also observed in complexes with two antibody fragments (Fab58 and Fab75) [7]. The standard, i.e. the conventional, form of HGFA (PDB 1YC0) displays features typical of the S1 family of serine proteases, such as the double b-barrel arrange- ment of the peptidase domain, a His57–Asp102–Ser195 catalytic triad, and distinct surface loops that determine substrate and inhibitor specificities (Fig. 1B). Among S1 family members, these loops display variable lengths, with HGFA falling comfortably within the ranges among close homologs. Additionally, some of these loops (the 140-, 180-, and 220-loop) undergo con- formational rearrangements during the zymogen to enzyme transition, and, together with the N-terminal peptide, are referred to as the ‘activation domain’ [8]. A zymogen form of ‘short HGFA’ has not been crystal- lized, but we presume that HGFA undergoes analogous changes during activation. A rare free Cys at posi- tion 187 is not found in any close homolog, but seems to have no special function [4]. Other key attributes of the catalytically competent structure are the ‘oxyanion hole’ formed by the amide nitrogens of Ser195 and Gly193, and substrate-binding subsites (S1, S2, S3, and S4), which interact with the corresponding P1–P4 resi- dues of the substrate (Lys-Gln-Leu-Arg for pro-HGF) (Fig. 1B). The principal determinant of substrate pref- erence is the substrate-binding pocket, S1 (Fig. 1B). As in trypsin, the Asp189 at the bottom of S1 confers a strong preference for substrates with an Arg or Lys as their P1 residue. In agreement with this, the two known macromolecular substrates, pro-HGF and pro-MSP [2], as well as synthetic substrates of HGFA, have a P1 Arg residue [7,9]. Nonstandard active site conformations The apo structure of HGFA (without inhibitor pres- ent) reveals an active site in which key elements of the substrate-binding site are changed in a way that is incompatible with substrate binding and catalytic activity. It shows a significant displacement of the Ser214–Asp217 segment (part of the 220-loop) as com- pared with the competent conformation with an ‘open’ active site. In apo-HGFA, the Ca atom of Trp215 is shifted by 2.8 A ˚ and that of Gly216 by 5.5 A ˚ (Fig. 1C). As a consequence of this difference, the entry of the substrate P1 residue into S1 is blocked, and the active site is ‘closed’. Figure 1C shows that this ‘closed’ conformation would cause a steric clash with the P1 Arg, thus precluding a productive interac- tion of a substrate with the catalytic machinery of HGFA. The unconventional 220-loop arrangement is supported by new hydrogen bonds and hydrophobic interactions involving Trp215, also including some interactions from a crystal packing contact. This nonstandard 220-loop arrangement is not limited to HGFA, but has precedents in the apo forms of several other trypsin-like serine proteases, such as Na + -free thrombin [10], a1-tryptase [11], tonin [12], bacterial DegS [13], horse prostate kallikrein [14], and prostasin [15,16]. In addition, on the basis of its C. Eigenbrot et al. Molecular interactions of HGFA with inhibitor ⁄ substrate FEBS Journal 277 (2010) 2215–2222 ª 2010 The Authors Journal compilation ª 2010 FEBS 2217 zymogen structure, Hink-Schauer et al. [17] postulated that granzyme K may also have an S1 pocket obstructed by a distorted 220-loop. All of these struc- tures display significant displacement of residues 215– 220 and (usually) a few subsequent residues, although there is not a strong consensus for a single ‘alterna- tive ⁄ incompetent’ position for residues 215–220, per- haps partly because of the influence of crystal packing in some of these structures. Other features seen among incompetent active sites are not observed for HGFA, e.g. the loss of the oxyanion hole seen in Na + -free thrombin. Among these proteases, the closest homolog to HGFA is prostasin, also known as PRSS8 or chan- nel-activating protease-1. In apo-prostasin, the 220- loop is rearranged in a very similar manner as in apo- HGFA, and obstructs substrate access to S1 (Fig. 1C) [15,16]. As compared with the competent form of pro- stasin, the 220-loop Asp217 is shifted by 5.4 A ˚ towards S1 (Fig. 1C), and this new conformation is stabilized by a network of hydrogen bonds to a network of water molecules close to the catalytic Ser [15]. Moreover, the HGFA 99-loop can also adopt an unconventional conformation, which is incompatible with optimal enzyme activity. The 99-loop is important for substrate–inhibitor interactions, as it contributes to the formation of S2 and S4. The unconventional con- formation affects the proper interaction of the sub- strate with S2, owing to a rearrangement of the 99-loop residues Pro99a and Ser99, both of which shape the relatively hydrophobic S2 (Fig. 1D) [9]. Due to the 99-loop movement, S2 is now smaller, and interaction with the Leu P2 residue of substrates is significantly impaired. This particular 99-loop con- formation was observed in the structure of the inhibi- tory Fab40 bound to HGFA, reflecting the mechanism by which catalytic activity is inhibited by Fab40 [9]. Fab40 binds to a region outside of the substrate-bind- ing cleft located at the ‘back side’ of the 99-loop, and is a competitive allosteric inhibitor of HGFA [9]. It is possible that the 99-loop ‘switch’ reflects a natural reg- ulatory mechanism, as part of the Fab40-binding site corresponds to thrombin exosite II, which is a known effector-binding site regulating thrombin enzymatic activity [18]. The apparent 99-loop conformational flexibility is not restricted to HGFA, but has also been observed or implied to occur in other S1 family mem- bers, such as the close structural homolog prostasin, where the 99-loop can adopt three different conforma- tional states [16]. Members of the kallikrein family, such as horse prostate kallikrein, have relatively long 99-loops, and in some structures with no substrate mimic bound, the loop extends over the catalytic triad and would restrict access by substrate [14]. In coagula- tion factor IXa, the side chain of Tyr99 occludes S2 in the absence of a substrate mimic [19]. In addition to the 99-loop, other substrate specific- ity-determining loops, such as the 38-loop, 60-loop, and 170-loop, can adopt different conformations in various S1 family members [13,20,21], suggesting remarkable plasticity of the serine protease active site region. Thus, it is reasonable to assume that the unconventional 99-loop and 220-loop conformations of HGFA are part of an ensemble of conformational states, and that the substrate is simply sampling the conventional conformation, in effect shifting the equi- librium towards the competent state. It is common for structural studies of proteases to include an inhibitor to limit autolysis and stabilize the protein during crystallization. The characterization of incompetent active sites among the relatively small number of uninhibited S1 family X-ray structures sug- gests that such conformational plasticity is widespread and forms part of the biological regulation of enzyme activity. Examples of regulation also include Na + effects on thrombin [10], the PDZ domain of DegS [13], and interactions between the 220-loop and 99-loop of horse prostate kallikrein [14], and Ca 2+ effects on prostasin [16]. For systems without factors beyond the protease domain playing a role, the free energy requirement for moving between incompetent and competent conformations is probably quite low, well within the energy provided by substrate interac- tions, and consistent with the notion of ‘induced fit’. This is probably the case for HGFA, which, despite adopting a catalytically incompetent conformation, is enzymatically fully active. This suggests that HGFA can easily undergo transition between the two active site conformations. A contrary example is found for the catalytically inactive a1-tryptase, which adopts an incompetent 220-loop stabilized by a unique sequence [11]. Inhibition of HGFA by HAI-1 The activity of HGFA is inhibited by naturally occur- ring protein inhibitors belonging to different classes, such as the Kunitz domain inhibitors HAI-1 and HAI- 2, and the serpin protein C inhibitor (SerpinA5) [22] (refer to the review by Suzuki [23]). The first identified inhibitor was HAI-1 [24], which is composed of an N-terminal MANSC domain [25], a structurally unas- signed region, KD1, low-density lipoprotein (LDL) receptor-like domain, Kunitz domain-2 (KD2), a trans- membrane domain, and a cytoplasmic domain (Fig. 1A). A human splice variant containing an extra 16 amino acids inserted between KD1 and the LDL Molecular interactions of HGFA with inhibitor ⁄ substrate C. Eigenbrot et al. 2218 FEBS Journal 277 (2010) 2215–2222 ª 2010 The Authors Journal compilation ª 2010 FEBS receptor-like domain was identified, and was found to have identical tissue expression and inhibitory specific- ity to those of HAI-1 [26] (Fig. 2A). HAI-1 deficiency is embryo lethal, owing to defective placental tissue architecture caused by dysregulated enzymatic activity [27–29]. KD1 inhibits the enzymatic activity of HGFA, matriptase, hepsin, and prostasin, whereas the C-termi- nal KD2 does not [26,30–33]. Domain characterization studies suggest a complex interplay between various HAI-1 domains in regulating the activity of KD1 towards the examined proteases, HGFA and matrip- tase [30,32]. In the case of matriptase, the reported K i values range from 647 pm for the entire HAI-1 extra- cellular domain to 1.6 pm for a smaller KD1-contain- ing HAI-1 fragment [32]. The different binding affinities of the full-length and truncated HAI-1 ver- sions may be of physiological relevance, as several sol- uble HAI-1 forms were found in the cell culture medium [24] and in association with matriptase in human milk [34]. HAI-1 inhibits HGFA by forming a tight associa- tion as a pseudosubstrate between its KD1 and the enzyme active site [4]. KD1 makes contacts (hydrogen bonding and hydrophobic) to all substrate–inhibitor specificity-determining loops of HGFA (Fig. 2A,B). The conformations of these loops are essentially the same as found in all examples of catalytically compe- tent HGFA structures. There is a close correspondence between the KD1 interactions with the substrate-bind- ing cleft and those seen for the substrate mimic KQLR-cmk. KD1 places the side chains of residues Arg260, Cys259-Cys283, and Arg258 in the S1, S2 and S4 subsites in a way almost identical to the way that the substrate mimic KQLR places its P1, P2 and P4 side chains (Fig. 3). In addition, KD1 makes two main chain to main chain hydrogen bonds with Ser214 and Gly216 that are also formed by the substrate mimic KQLR-cmk (Fig. 3A,B). KD1 is one of the ‘standard mechanism’ or ‘Laskowski mechanism’ inhibitors, which tightly bind to the enzyme in a substrate-like manner but undergo cleavage at an extremely low rate. Indeed, the structure shows KD1 presenting its intact P1–P1¢ (Arg-Gly) peptide bond for nucleophilic attack by Ser195, the P1 backbone carbonyl being stabilized by the main chain amide nitrogen atoms of Gly193 and Ser195 (Fig. 3B). On the basis of biochemical and structural studies on a related Kunitz domain–enzyme pair, the bovine AB Fig. 2. Interaction of HGFA protease domain with HAI-1-derived KD1 (PDB 1YC0). (A) KD1 (magenta) interacts with HGFA (beige) in a sub- strate-like manner by occupying subsites S4, S2 and S1 (in orange) with Arg258, Cys259–Cys283, and Arg260 (P1 residue), respectively (side chains in blue). The binding region is delineated by the dotted line, and corresponds to the green surface in (B). (B) Open book representa- tion of the HGFA–KD1 interaction. Residues on HGFA (green) and KD1 (blue) with an atom within 4.0 A ˚ of the other protein (= binding region) are indicated. KD1 makes contact with all substrate ⁄ inhibitor specificity-determining loops on HGFA (compare with Fig. 1B), and uses the protruding P1 Arg260 for insertion into the deep S1 pocket. The catalytic His57 and Ser195 are also within 4.0 A ˚ of KD1 and are in yellow. C. Eigenbrot et al. Molecular interactions of HGFA with inhibitor ⁄ substrate FEBS Journal 277 (2010) 2215–2222 ª 2010 The Authors Journal compilation ª 2010 FEBS 2219 pancreatic trypsin inhibitor–trypsin complex, it was proposed that cleavage at the inhibitor P1–P1¢ peptide bond can readily occur. However, owing to the tight association of the cleavage product, the peptide bond is more rapidly resynthesized, so that the intact form of the Kunitz domain inhibitor predominates in the crystal structure [35]. According to this model, the intact KD1 peptide bond in the KD1–HGFA structure thus reflects the capture of the dominant form, owing to the more favorable rate of peptide bond resynthesis during crystallization. Enzymatic kinetic experiments showed that the enzyme specificity of HAI-1 is completely determined by KD1 alone, and does not require additional interac- tions [4], although other HAI-1 domains may nega- tively regulate the affinity of binding between KD1 and HGFA [30,32]. Therefore, the specificity must arise from structural features of each inhibited enzyme (e.g. HGFA, matriptase, hepsin, prostasin, and tryp- sin) [4,8,15,36,37] around the inhibitor binding site. The use of the KD1–HGFA complex to rationalize the structural basis of enzyme specificity has obvious limi- tations, as other KD1–enzyme structures are not avail- able. Also, structural adjustments made by the enzyme can be significant and difficult to predict, as in the case of the related aprotinin–prostasin complex, in which the 99-loop moves away from the substrate-binding cleft to accommodate the Kunitz-type inhibitor aproti- nin [15]. In some cases, however, good structural argu- ments can be made, such as the complete lack of inhibition by KD1 of the closely related uPA. A likely reason is the conformation of the uPA 99-loop. Although it is only one amino acid longer than the 99- loop in HGFA, its conformation is quite different and, in a hypothetical complex, it would extend well into the location where Arg258 is found in HGFA S4, causing a steric conflict with Leu97b of uPA (PDB 1LMW). For a more detailed structure-based analysis, see a previously published study by Shia et al. [4]. Substrate interaction and specificity The structures of the substrate mimic KQLR-cmk bound to HGFA and the KD1–HGFA complex pro- vide insights into salient features determining substrate interactions and specificity. The KQLR peptide consti- tutes the P4–P1 sequence of the natural substrate pro- HGF, and thus should serve as a good approximation of natural substrate interactions with the HGFA active site. The KQLR-cmk peptide, covalently linked to the catalytic Ser195 and His57, inserts into the active site groove in a manner that is typical for substrate inter- actions with trypsin-like serine proteases. It adopts a twisted antiparallel conformation, forming the inter- main chain hydrogen bonds between P1 Arg and Ser214 and between P3 Gln and Gly216 (Fig. 3A). S1 is filled with the P1 Arg, which engages in standard salt bridge interactions with HGFA Asp189, located at the bottom of S1. The P2 Leu tightly packs into the ABC Fig. 3. Substrate interaction with HGFA. (A) The crystal structure of HGFA (surface representation, beige, PDB 2WUC) in complex with Ac-KQLR-cmk (stick representation, green). The KQLR inhibitor is covalently bonded to Ser195 and His57, and it is stabilized by two inter- main chain (P1 ArgÆSer214 and P3 GlnÆGly216) hydrogen bonds (red dotted lines). Additional hydrogen bonds with side chains include P1 ArgÆGly193, P2 LeuÆGln192, and P4 LysÆSer99. The hydrophobic S2 pocket is formed by His57, Ser99, Pro99a and Trp215 (orange). (B) The structure of HGFA (surface representation, beige, PDB 1YC0) in complex with KD1 (stick representation, magenta). The Arg260 is bound in the deep S1 pocket, and forms a salt bridge with Asp189 in a similar manner to the P1 Arg of KQLR. The carbonyl oxygen of Arg260 is hydrogen bonded to the amide nitrogens of the oxyanion hole (Gly193 and Ser195). The hydrophobic S2 pocket (formed by His57, Ser99, Pro99a, and Trp215) (blue) is occupied by thiols of disulfide-bonded Cys259–Cys283. Apart from forming a hydrogen bond with Ser99, Arg258 of KD1 interacts with Trp215 via a p-stacking interaction. (C) Superposition of KQLR with the KD1 residues Arg258-Cys259 ⁄ Cys283- Arg260 indicates an overlap of main chains P1–P3 and excellent correspondence of the side chains occupying subsites S1, S2, and S4. Molecular interactions of HGFA with inhibitor ⁄ substrate C. Eigenbrot et al. 2220 FEBS Journal 277 (2010) 2215–2222 ª 2010 The Authors Journal compilation ª 2010 FEBS small hydrophobic S2 pocket formed by Pro99a, Ser99, Trp215, and His57, suggesting a strong prefer- ence for a Leu at P2. The P3 Gln points outward towards the solvent-exposed region of the active site, suggesting poor specificity at this position, as in most S1 family proteases. Some degree of specificity for S4 is suggested by the hydrogen bond formation between P4 Lys and Ser99. Most intriguingly, the occupancy of S1, S2 and S4 is reprised by the KD1 inhibitor, using its Arg260 side chain, the thiol groups from the disul- fide bonded Cys259–Cy283, and the Arg258 side chain, respectively (Fig. 3B,C). This remarkable correspon- dence may indicate that HGFA has preference for a P1 Arg and a basic P4 (Lys ⁄ Arg) residue. Molecular modeling studies indicate that a P4 Arg of the hypo- thetical RQLR peptide would be an excellent fit, as it may compensate for the negative electrostatic potential created in S4 by Asp217 and Ser99 (data not shown). Our structural arguments about the S4 specificity need to be tempered by the facts that, for most S1 prote- ases, the degree of specificity generally diminishes beyond S2, and that our analysis is based on only two crystal structures. In addition, the presence of a P2 Leu in both macromolecular and synthetic substrates of HGFA is consistent with the structural features of S2, suggesting a strong preference for Leu as a P2 resi- due. The P1¢ residue for HAI-1 is a Gly, whereas it is a Val for both known macromolecular substrates. This may indicate a preference for small hydrophobic residues at this position. 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Abbreviations EGF, epidermal growth factor; HAI, hepatocyte growth factor activator inhibitor; HGFA, hepatocyte growth factor activator; KD1, Kunitz domain-1;