Hepatocyte growth factor activator HGFA: molecularstructure and interactions with HGFA inhibitor-1 HAI-1 Charles Eigenbrot1,2, Rajkumar Ganesan3and Daniel Kirchhofer3 1 Department of Str
Trang 1Hepatocyte growth factor activator (HGFA): molecular
structure and interactions with HGFA inhibitor-1 (HAI-1) Charles Eigenbrot1,2, Rajkumar Ganesan3and Daniel Kirchhofer3
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’, consistresult-ing 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.
Trang 2highest 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
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].
Trang 3cleavage 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 numberaccord-ing (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
Trang 4zymogen 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 Ca2+ 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 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
Trang 5receptor-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 matripmatrip-tase, the reported Ki
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
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 interacrepresenta-tion 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.
Trang 6pancreatic 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
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.
Trang 7small 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
In conclusion, the intriguing structural features of
HGFA interactions with a substrate mimic and the
pseudosubstrate KD1 suggest that HGFA has unique
substrate preferences This may be helpful in
identify-ing additional macromolecular substrates
Addition-ally, the noncanonical conformations that have been
seen among HGFA protease structures may be useful
in discovering highly specific peptidic and nonpeptidic
inhibitors of HGFA
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