Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.     

Strand 6B deformation and residues exposure towards N-terminal end of helix B during proteinase inhibition by Serpins

Serine Protease inhibitors (Serpins) like antithrombin, antitrypsin, neuroserpin, antichymotrypsin, protein C-inhibitor and plasminogen activator inhibitor is involved in important biological functions like blood coagulation, fibrinolysis, inflammation, cell migration and complement activation. Serpins native state is metastable, which undergoes transformation to a more stable state during the process of protease inhibition. Serpins are prone to conformation defects, however little is known about the factors and mechanisms which promote its conformational change and misfolding. Helix B region in serpins is with several point mutations which result in pathological conditions due to polymerization. Helix B analysis for residue burial and cavity was undertaken to understand its role in serpin structure function. A structural overlap and an accessible surface area analysis showed the deformation of strand 6B and exposure of helix B at N-terminal end in cleaved conformation but not in the native and latent conformation of various inhibitory serpins. A cleaved polymer like conformation of antitrypsin also showed deformation of s6B and helix B exposure. Cavity analysis showed that helix B residues were part of the largest cavity in most of the serpins in the native state which increase in size during the transformation to cleaved and latent states. These data for the first time show the importance of strand 6B deformation and exposure of helix B in smooth insertion of the reactive center loop during serpin inhibition and indicate that helix B exposure due to variants may increase its polymer propensity. ABBREVIATIONS: serpin -serine protease inhibitors RCL -reactive center loop ASA -accessible surface area.     

The structure of active serpin 1K from Manduca sexta.

BACKGROUND: The reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding. RESULTS: The 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin. CONCLUSIONS: Serpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.     

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.     

The length of the reactive center loop modulates the latency transition of plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1.     

Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering alpha(1)-antitrypsin to inhibit cathepsin proteases

The closely related serpins squamous cell carcinoma antigen-1 and -2 (SCCA-1 and -2, respectively) are capable of inhibiting cysteine proteases of the papain superfamily. To ascertain whether the ability to inhibit cysteine proteases is an intrinsic property of serpins in general, the reactive center loop (RCL) of the archetypal serine protease inhibitor alpha(1)-antitrypsin was replaced with that of SCCA-1. It was found that this simple substitution could convert alpha(1)-antitrypsin into a cysteine protease inhibitor, albeit an inefficient one. The RCL of SCCA-1 is three residues longer than that of alpha(1)-antitrypsin, and therefore, the effect of loop length on the cysteine protease inhibitory activity was investigated. Mutants in which the RCL was shortened by one, two, or three residues were effective inhibitors with second-order rate constants of 10(5)-10(7) M(-)(1) s(-)(1). In addition to loop length, the identity of the cysteine protease was of considerable importance, since the chimeric molecules inhibited cathepsins L, V, and K efficiently, but not papain or cathepsin B. By testing complexes between an RCL-mimicking peptide and the mutants, it was found that the formation of a stable serpin-cysteine protease complex and the inhibition of a cysteine protease were both critically dependent on RCL insertion. The results strongly indicate that the serpin body is intrinsically capable of supporting cysteine protease inhibition, and that the complex with a papain-like cysteine protease would be expected to be analogous to that seen with serine proteases.     

Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.     

Corticosteroid-binding globulin, a structural basis for steroid transport and proteinase-triggered release

Corticosteroid-binding globulin (CBG) is a serine proteinase inhibitor (serpin) family member that transports glucocorticoids in blood and regulates their access to target cells. The 1.9A crystal structure of rat CBG shows that its steroid-binding site resembles the thyroxin-binding site in the related serpin, thyroxin-binding globulin, and mutagenesis studies have confirmed the contributions of key residues that constitute the steroid-binding pocket. Unlike thyroxin-bound thyroxin-binding globulin, the cortisol-bound CBG displays an "active" serpin conformation with the proteinase-sensitive, reactive center loop (RCL) fully expelled from the regulatory beta-sheet A. Moreover, the CBG structure allows us to predict that complete insertion of the proteolytically cleaved RCL into the serpin fold occurs in concert with a displacement and unwinding of helix D that would disrupt the steroid-binding site. This allosteric coupling between RCL positioning and occupancy of the CBG steroid-binding site, which resembles the ligand (glycosamino-glycan)-dependent activation of the thrombin inhibitory serpins heparin cofactor II and anti-thrombin RCLs, ensures both optimal recognition of CBG by target proteinases and efficient release of steroid to sites of action.     

Distinct encounter complexes of PAI‐1 with plasminogen activators and vitronectin revealed by changes in the conformation and dynamics of the reactive center loop

Plasminogen activator inhibitor‐1 (PAI‐1) is a biologically important serine protease inhibitor (serpin) that, when overexpressed, is associated with a high risk for cardiovascular disease and cancer metastasis. Several of its ligands, including vitronectin, tissue‐type and urokinase‐type plasminogen activator (tPA, uPA), affect the fate of PAI‐1. Here, we measured changes in the solvent accessibility and dynamics of an important unresolved functional region, the reactive center loop (RCL), upon binding of these ligands. Binding of the catalytically inactive S195A variant of tPA to the RCL causes an increase in fluorescence, indicating greater solvent protection, at its C‐terminus, while mobility along the loop remains relatively unchanged. In contrast, a fluorescence increase and large decrease in mobility at the N‐terminal RCL is observed upon binding of S195A‐uPA to PAI‐1. At a site distant from the RCL, binding of vitronectin results in a modest decrease in fluorescence at its proximal end without restricting overall loop dynamics. These results provide the new evidence for ligand effects on RCL conformation and dynamics and differences in the Michaelis complex with plasminogen activators that can be used for the development of more specific inhibitors to PAI‐1. This study is also the first to use electron paramagnetic resonance (EPR) spectroscopy to investigate PAI‐1 dynamics. Significance : Balanced blood homeostasis and controlled cell migration requires coordination between serine proteases, serpins, and cofactors. These ligands form noncovalent complexes, which influence the outcome of protease inhibition and associated physiological processes. This study reveals differences in binding via changes in solvent accessibility and dynamics within these complexes that can be exploited to develop more specific drugs in the treatment of diseases associated with unbalanced serpin activity.     

Canonical inhibitor-like interactions explain reactivity of alpha1-proteinase inhibitor Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, alpha1-proteinase inhibitor (alpha1PI) Pittsburgh (P1 Met --> Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound alpha1PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2'. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with alpha1PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for alpha1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.     

Crystal structure of human maspin, a serpin with antitumor properties: reactive center loop of maspin is exposed but constrained

Maspin, a member of the serpin superfamily, has tumor suppressing activity against breast and prostate cancer. Maspin inhibits tumor growth by blocking cell invasion, and its reactive center loop (RCL) is thought to mediate this activity. To understand this function on the molecular level, we have solved the three-dimensional structure of Maspin to 3.1 A resolution. The molecular structure shows the characteristic features of the serpin fold, but the RCL of Maspin is unique in length, composition, and placement. Although the RCL of Maspin is accessible and cleavable by some proteinases, it functions in the uncleaved, constrained conformation observed here. These structural results will contribute to our understanding of the mechanism by which Maspin suppresses tumors.     

Anti-hemostatic effects of a serpin from the saliva of the tick Ixodes ricinus

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.     

Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents.

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serpin family of serine proteinase inhibitors. Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P1 residue of the reactive centre loop (RCL) of the serpin, followed by insertion of the RCL into beta-sheet A of the serpin. Concomitantly, there are conformational changes in the flexible joint region lateral to beta-sheet A. We have now, by site-directed mutagenesis, mapped the epitope for a monoclonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acting on beta-sheet A and the flexible joint region. Curiously, the epitope is localized in alpha-helix C and the loop connecting alpha-helix I and beta-strand 5A, on the side of PAI-1 opposite to beta-sheet A and distantly from the flexible joint region. By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody-induced conformational change from the epitope to the rest of the molecule. We have thus provided evidence for communication between secondary structural elements not previously known to interact in serpins.     

Fluorescence correlation spectroscopic study of serpin depolymerization by computationally designed peptides

Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found.     

Is the binding of β-amyloid protein to antichymotrypsin in Alzheimer plaques mediated by a β-strand insertion?

A growing body of experimental evidence demonstrates that the serpin antichymotrypsin plays a regulatory role in Alzheimer plaque physiology by interacting with the 42 residue β-amyloid protein, and we have used molecular modeling and energy minimization techniques to study this interaction. Based on the unique plasticity of β-sheet elements in antichymotrypsin (as well as other serpins), we conclude that the interaction of the two proteins is mediated by insertion of the N-terminus of β-amyloid into β-sheet C of antichymotrypsin as a pseudo-strand s1C. This β-strand insertion requires the displacement of native antichymotrypsin strand s1C, which is known to occur partially or completely at different stages of serpin function. Thus, the association of the two proteins in vivo may be facilitated by a particular functional state of the serpin, e.g., the native or protease-complexed state. © 1996 Wiley-Liss, Inc.     

Serpins: implications of a mobile reactive centre.

The serpins are unique among the families of serine proteinase inhibitors in having a reactive centre that is situated on a mobile loop. The structures of three alternative conformations are now known, and it can be deduced that the active form involves the partial insertion of the loop into the A sheet of the molecule. The ability of the loop to move in and out of this sheet has been adapted by evolution to allow the modulation of inhibitory activity. Manipulation of the structure of the loop and of other functional domains in the serpin superfamily enables the production of serpins with tailor-made activities. The ability of the loop to lock in latent conformations or to take part in intermolecular polymerization has implications for the production and stabilization of recombinant serpins. This review has been adapted from Current Opinion in Structural Biology 1992, 2:438-446.     

Mutational analysis of plasminogen activator inhibitor-1.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue-type and urokinase-type plasminogen activator and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis. PAI-1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop (RCL) as strand 4 in beta-sheet A, thereby converting to the so-called latent state. We have investigated the importance of the amino acid sequence of alpha-helix F (hF) and the connecting loop to s3A (hF/s3A-loop) for the rate of latency transition. We grafted regions of the hF/s3A-loop from antithrombin III and alpha1-protease inhibitor onto PAI-1, creating eight variants, and found that one of these reversions towards the serpin consensus decreased the rate of latency transition. We prepared 28 PAI-1 variants with individual residues in hF and beta-sheet A replaced by an alanine. We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case. Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complex formation with target proteases. The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop-sheet interactions in serpins.     

Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity

Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352–358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API RCL, but also in that of other serpins.     

Mutant recombinant serpins as highly specific inhibitors of human kallikrein 14

The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of alpha1-antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kündig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607-613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display-selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgo?o CA, Cloutier SM, Kündig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291-298], we studied the importance of the scaffold, serpins alpha1-antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACT(E8), ACT(G9), AAT(E8) and AAT(G9) showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8-based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin-like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.