Modeling the intact form of the alpha 1-proteinase inhibitor

The structure of the intact form of the serpin alpha 1-proteinase inhibitor has been modeled based on the assumption that the central strand s4A of the six-stranded beta-sheet A of the cleaved inhibitor is not incorporated into the sheet of intact alpha 1-proteinase inhibitor. This strand was removed from its position in the center of the sheet by suitable rotations about the backbone dihedrals of Lys343 using molecular graphics. The resulting structure was then annealed using molecular dynamics (MD) while applying progressive distance restraints to the reactive peptide bond (Met358-Ser359) for 50 ps. During this time, the disrupted beta-sheet reformed to create a five-stranded beta-sheet with strands 3 and 5 in a parallel arrangement. This change and accompanying structural rearrangements are largely confirmed by the X-ray structure of plakalbumin, whose structure reflects the overall structure of intact serpins. The successful modeling experiment demonstrates the utility of MD for making gross structural predictions based on related structures. The binding loop of the intact form is modeled to allow docking with serine proteinases, in particular thrombin, which most highly constrains the possible conformations of the binding loop.     

Bypassing the kinetic trap of serpin protein folding by loop extension

The native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring-loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the most stable state is prevented, we designed mutations of alpha1-antitrypsin, a prototype serpin, which can bypass the folding barrier. Extending the reactive center loop of alpha1-antitrypsin converts the molecule into a more stable state. Remarkably, a 30-residue loop extension allows conversion into an extremely stable state, which is comparable to the relaxed cleaved form. Biochemical data strongly suggest that the strain release is due to the insertion of the reactive center loop into the major beta-sheet, A sheet, as in the known stable conformations of serpins. Our results clearly show that extending the reactive center loop is sufficient to bypass the folding barrier of alpha1-antitrypsin and suggest that the constrain held by polypeptide connection prevents the conversion of the native form into the lowest energy state.     

Analysis of the plasma elimination kinetics and conformational stabilities of native, proteinase-complexed, and reactive site cleaved serpins: comparison of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, antithrombin III, alpha 2-antiplasmin, angiotensinogen, and ovalbumin.

Proteinase inhibitors of the serpin superfamily may exist in one of three distinct conformations: the native form, a fully active protein with the reactive site loop intact; the proteolytically modified form in which inhibitory capacity is abolished; and the proteinase-complexed form, a stable equimolar complex between the inhibitor and a target proteinase. Here, the specificity and kinetics of the plasma elimination of different serpin conformations are compared. Proteinase-complexed serpins were rapidly cleared from the circulation. However, the native and modified forms were not cleared rapidly, indicating that the receptor-mediated pathways which recognize the complexes fail to recognize the native and modified forms. This result suggests that significant structural differences exist between modified and proteinase-complexed serpins. The structural differences were probed by using transverse urea gradient gel electrophoresis, a technique that allows comparisons of the conformational stabilities of proteins. With the exception of the noninhibitory serpins ovalbumin and angiotensinogen, the modified and proteinase-complexed serpins were both stabilized thermodynamically compared to the native forms. In addition, the proteinase component of the serpin-proteinase complex was usually thermodynamically stabilized. These data are used to compare the conformations of serpin-proteinase complexes with those of native and modified serpins; they are discussed in terms of a model whereby serpins inhibit proteinases in a manner similar to that described for other types of protein inhibitors of serine proteinases.     

Serpins Flex Their Muscle: II. STRUCTURAL INSIGHTS INTO TARGET PEPTIDASE RECOGNITION,POLYMERIZATION, AND TRANSPORT FUNCTIONS*

Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding.     

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation

Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.     

Conformational changes in serpins: I. The native and cleaved conformations of alpha(1)-antitrypsin

The serpins (SERine Proteinase INhibitors) are a family of proteins with important physiological roles, including but not limited to the inhibition of chymotrypsin-like serine proteinases. The inhibitory mechan- ism involves a large conformational change known as the S-->R (stressed-->relaxed) transition. The largest structural differences occur in a region around the scissile bond called the reactive centre loop: In the native (S) state, the reactive centre is exposed, and is free to interact with proteinases. In inhibitory serpins, in the cleaved (R) state the reactive centre loop forms an additional strand within the beta-sheet. The latent state is an uncleaved state in which the intact reactive centre loop is integrated into the A sheet as in the cleaved form, to give an alternative R state.The serpin structures illustrate detailed control of conformation within a single protein. Serpins are also an unusual family of proteins in which homologues have native states with different folding topologies. Determination of the structures of inhibitory serpins in multiple conformational states permits a detailed analysis of the mechanism of the S-->R transition, and of the way in which a single sequence can form two stabilised states of different topology.Here we compare the conformations of alpha(1)-antitrypsin in native and cleaved states. Many protein conformational changes involve relative motions of large rigid subunits. We determine the rigid subunits of alpha(1)-antitrypsin and analyse the changes in their relative position and orientation. Knowing that the conformational change is initiated by cleavage at the reactive centre, we describe a mechanism of the S-->R transition as a logical sequence of mechanical effects, even though the transition likely proceeds in a concerted manner.     

Conformational changes in serpins: I. The native and cleaved conformations of alpha(1)-antitrypsin

The serpins (SERine Proteinase INhibitors) are a family of proteins with important physiological roles, including but not limited to the inhibition of chymotrypsin-like serine proteinases. The inhibitory mechan- ism involves a large conformational change known as the S-->R (stressed-->relaxed) transition. The largest structural differences occur in a region around the scissile bond called the reactive centre loop: In the native (S) state, the reactive centre is exposed, and is free to interact with proteinases. In inhibitory serpins, in the cleaved (R) state the reactive centre loop forms an additional strand within the beta-sheet. The latent state is an uncleaved state in which the intact reactive centre loop is integrated into the A sheet as in the cleaved form, to give an alternative R state.The serpin structures illustrate detailed control of conformation within a single protein. Serpins are also an unusual family of proteins in which homologues have native states with different folding topologies. Determination of the structures of inhibitory serpins in multiple conformational states permits a detailed analysis of the mechanism of the S-->R transition, and of the way in which a single sequence can form two stabilised states of different topology.Here we compare the conformations of alpha(1)-antitrypsin in native and cleaved states. Many protein conformational changes involve relative motions of large rigid subunits. We determine the rigid subunits of alpha(1)-antitrypsin and analyse the changes in their relative position and orientation. Knowing that the conformational change is initiated by cleavage at the reactive centre, we describe a mechanism of the S-->R transition as a logical sequence of mechanical effects, even though the transition likely proceeds in a concerted manner.     

How serpins change their fold for better and for worse

The serpins differ from the many other families of serine protease inhibitors in that they undergo a profound change in topology in order to entrap their target protease in an irreversible complex. The solving of the structure of this complex has now provided a video depiction of the changes involved. Cleavage of the exposed reactive centre of the serpin triggers an opening of the five-stranded A-sheet of the molecule, with insertion of the cleaved reactive loop as an additional strand in the centre of the sheet. The drastic displacement of the acyl-linked protease grossly disrupts its active site and gives an overall loss of 40% of ordered structure. This ability to provide effectively irreversible inhibition explains the selection of the serpins to control the proteolytic cascades of higher organisms. The conformational mechanism provides another advantage in its potential to modulate activity. Sequential crystallographic structures now provide clear depictions of the way antithrombin is activated on binding to the heparans of the microcirculation, and how evolution has utilized this mobile mechanism for subtle variations in activity. The complexity of these modulatory mechanisms is exemplified by heparin cofactor II, where the change in fold is seen to trigger multiple allosteric effects. The downside of the mobile mechanism of the serpins is their vulnerability to aberrant intermolecular beta-linkages, resulting in various disorders from cirrhosis to thrombosis. These provide a well defined structural prototype for the new entity of the conformational diseases, including the common dementias, as confirmed by the recent identification of the familial neuroserpin dementias.     

Use of NMR to study serpin function

Two-dimensional heteronuclear [1H,15N] single quantum correlation NMR spectra of serpins show dramatic changes between native and loop-inserted conformations, making them very sensitive reporters of the serpin conformation. Much of the spectral overlap that arises when all amides are 15N labelled can be removed by use of selective labelling of a single type of amino acid, such as alanine. The method allows ready determination of whether loop insertion is present, and to what extent, as well as providing information on motional freedom of components of the complex and of the reactive center loop. With label introduced separately into the proteinase, information can also be obtained on the conformational changes brought about in that moiety by complex formation. In addition, with the use of cryoprobes, high field spectrometers, TROSY-based signal detection and deuteration, samples as small as 1-2mg can easily be examined, making it applicable to a wide range of serpins, including those that can only be expressed in mammalian cells.     

Conformational changes in serpins: II. The mechanism of activation of antithrombin by heparindagger

Antithrombin, uniquely among plasma serpins acting as proteinase inhibitors in the control of the blood coagulation cascade, circulates in a relatively inactive form. Its activation by heparin, and specifically by a pentasaccharide core of heparin, has been shown to involve release of the peptide loop containing the reactive centre from partial insertion in the A sheet of the molecule. Here we compare the structures of the circulating inactive form of antithrombin with the activated structure in complex with heparin pentasaccharide. We show that the rearrangement of the reactive centre loop that occurs upon activation is part of a widespread conformational change involving a realignment of the two major domains of the molecule. We also examine natural mutants that possess high affinity for heparin pentasaccharide, and relate the kinetics of their interaction with heparin pentasaccharide to the structural transitions occuring in the activation process.     

Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition

CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution. The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases. However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA.     

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles

Serine proteinase inhibitors (Serpins) are irreversible suicide inhibitors of proteases that regulate diverse physiological processes such as coagulation, fibrinolysis, complement activation, angiogenesis, apoptosis, inflammation, neoplasia and viral pathogenesis. The molecular structure and physical properties of serpins permit these proteins to adopt a number of variant conformations under physiological conditions including the native inhibitory form and several inactive, non-inhibitory forms, such as complexes with protease or other ligands, cleaved, polymerised and oxidised. Alterations of a serpin which affect its structure and/or secretion and thus reduce its functional levels may result in pathology. Serpin dysfunction has been implicated in thrombosis, emphysema, liver cirrhosis, immune hypersensitivity and mental disorders. The loss of inhibitory activity of serpins necessarily results in an imbalance between proteases and their inhibitors, but it may also have other physiological effects through the generation of abnormal concentrations of modified, non-inhibitory forms of serpins. Although these forms of inhibitory serpins are detected in tissues and fluids recovered from inflammatory sites, the important questions of which conditions result in generation of different molecular forms of serpins, what biological function these forms have, and which of them are directly linked to pathologies and/or may be useful markers for characterisation of disease states, remain to be answered. Elucidation of the biological activities of non-inhibitory forms of serpins may provide useful insights into the pathogenesis of diseases and suggest new therapeutic strategies.     

Importance of the amino-acid composition of the shutter region of plasminogen activator inhibitor-1 for its transitions to latent and substrate forms.

The serpins are of general protein chemical interest due to their ability to undergo a large conformational change consisting of the insertion of the reactive centre loop (RCL), which becomes strand 4, into the central beta sheet A. To make space for the incoming RCL, the 'shutter region' opens by the beta strands 3A and 5A sliding apart over the underlying alpha helix B. Loop insertion occurs during the formation of complexes of serpins with their target serine proteinases and during latency transition. This type of loop insertion is unique to plasminogen activator inhibitor-1 (PAI-1). We report here that amino-acid substitutions in a buried cluster of three residues forming a hydrogen bonding network in the shutter region drastically accelerate PAI-1 latency transition; that the rate was in all cases normalized by the PAI-1 binding protein vitronectin; and that substitution of an adjacent beta strand 5A Lys residue, believed to anchor beta strand 5A to other secondary structural elements, had differential effects on the rates of latency transition in the absence and the presence of vitronectin, respectively. An overlapping, but not identical set of substitutions resulted in an increased tendency to substrate behaviour of PAI-1 at reaction with its target proteinases. These findings show that vitronectin regulates the movements of the RCL through conformational changes of the shutter region and beta strand 5A, are in agreement with RCL insertion proceeding by different routes during latency transition and complex formation, and contribute to the biochemical basis for the potential use of PAI-1 as a therapeutic target in cancer and cardiovascular diseases.     

Serine and cysteine proteases are translocated to similar extents upon formation of covalent complexes with serpins. Fluorescence perturbation and fluorescence resonance energy transfer mapping of the protease binding site in CrmA complexes with granzyme B and caspase-1

CrmA is a "cross-class" serpin family inhibitor of the proapoptotic serine protease, granzyme B, as well as cysteine proteases of the caspase family. To determine whether crmA inhibits these structurally diverse proteases by a common conformational trapping mechanism, we mapped the position of the protease in crmA complexes with granzyme B or caspase-1 by fluorescence perturbation and fluorescence resonance energy transfer (FRET) analyses of site-specific fluorophore-labeled crmAs. A reactive loop P6 NBD label underwent similar large fluorescence enhancements (>200%) either upon reactive loop cleavage by AspN protease or complex formation with granzyme B or caspase-1, consistent with the insertion of the cleaved reactive loop into sheet A in both types of crmA-protease complexes. NBD labels on the noninserting part of the reactive loop docking site for protease (P1' residue) or midway between the two ends of sheet A (helix F residue 101) showed no significant perturbations due to protease complexation. By contrast, labels at positions 68 and 261, lying at the end of sheet A most distal from the reactive loop, showed marked perturbations distinct from those induced by AspN cleavage and thus ascribable to granzyme B or caspase-1 proximity in the complexes. Substantial FRET between protease tryptophans and 5-dimethylaminonaphthalene-1-sulfonyl-labeled crmAs occurred in protease complexes with crmAs labeled at the 68 and 261 positions, but not the P1' position. These results suggest that granzyme B and caspase-1 are inhibited by crmA by a common mechanism involving full reactive loop insertion into sheet A and translocation of the protease to the distal end of the sheet as previously found for inhibition of other serine proteases by serpins.     

The 1.5 A crystal structure of a prokaryote serpin: controlling conformational change in a heated environment

Serpins utilize conformational change to inhibit target proteinases; the price paid for this conformational flexibility is that many undergo temperature-induced polymerization. Despite this thermolability, serpins are present in the genomes of thermophilic prokaryotes, and here we characterize the first such serpin, thermopin. Thermopin is a proteinase inhibitor and, in comparison with human alpha(1)-antitrypsin, possesses enhanced stability at 60 degrees C. The 1.5 A crystal structure reveals novel structural features in regions implicated in serpin folding and stability. Thermopin possesses a C-terminal "tail" that interacts with the top of the A beta sheet and plays an important role in the folding/unfolding of the molecule. These data provide evidence as to how this unusual serpin has adapted to fold and function in a heated environment.     

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.     

Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates.

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747-2754]. Selected substrates were then transplanted into the reactive site loop of alpha1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific alpha1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.     

Loop-inserted and thermostabilized structure of P1-P1' cleaved ovalbumin mutant R339T.

Ovalbumin is a member of a superfamily of serine proteinase inhibitors, known as the serpins. It is, however, non-inhibitory towards serine proteinases, and lacks the loop insertion mechanism common to the serpins due to unknown structural factors. Mutant ovalbumin, R339T, in which the P14 hinge residue is replaced, was produced and analyzed for its thermostability and three-dimensional structure. Differential scanning calorimetry revealed that the mutant ovalbumin, but not the wild-type protein, undergoes a marked thermostabilization (DeltaT(m)=15.8 degrees C) following the P1-P1' cleavage. Furthermore, the crystal structure, solved at 2.3 A resolution, clearly proved that the P1-P1' cleaved form assumes the fully loop-inserted conformation as seen in serpin that possess inhibitory activity. We therefore conclude that ovalbumin acquires the structural transition mechanism into the loop-inserted, thermostabilized form by the single hinge mutation. The mutant protein does not, however, possess inhibitory activity. The solved structure displays the occurrence of specific interactions that may prevent the smooth motion, relative to sheet A, of helices E and F and of the loop that follows helix F. These observations provide crucial insights into the question why R339T is still non-inhibitory.     

Ab initio construction of all-atom loop conformations

In this study, a new ab initio method named CLOOP has been developed to build all-atom loop conformations. In this method, a loop main-chain conformation is generated by sampling main-chain dihedral angles from a restrained varphi/psi set, and the side-chain conformations are built randomly. The CHARMM all-atom force field was used to evaluate the loop conformations. Soft core potentials were used to treat the non-bond interactions, and a designed energy-minimization technique was used to close and optimize the loop conformations. It is shown that the two strategies improve the computational efficiency and the loop-closure rate substantially compared to normal minimization methods. CLOOP was used to construct the conformations of 4-, 8-, and 12-residue loops in Fiser's test set. The average main-chain root-mean-square deviations obtained in 1,000 trials for the 10 different loops of each size are 0.33, 1.27, and 2.77 A, respectively. CLOOP can build all-atom loop conformations with a sampling accuracy comparable with previous loop main-chain construction algorithms. [Figure: see text].