Specific interactions of serpins in their native forms attenuate their conformational transitions

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein superfamily. Serpins are unique in that their native forms are not the most thermodynamically stable conformation; instead, a more stable, latent conformation exists. During the transition to the latent form, the first strand of beta-sheet C (s1C) in the serpin is peeled away from the beta-sheet, and the reactive center loop (RCL) is inserted into beta-sheet A, rendering the serpin inactive. To elucidate the contribution of specific interactions in the metastable native form to the latency transition, we examined the effect of mutations at the s1C of PAI-1, specifically in positions P4' through P10'. Several mutations strengthened the interactions between these residues and the core protein, and slowed the transition of the protein from the metastable native form to the latent form. In particular, anchoring of the strand to the protein's hydrophobic core at the beginning (P4' site) and center of the strand (P8' site) greatly retarded the latency transition. Mutations that weakened the interactions at the s1C region facilitated the conformational conversion of the protein to the latent form. PAI-1's overall structural stability was largely unchanged by the mutations, as evaluated by urea-induced equilibrium unfolding monitored via fluorescence emission. Therefore, the mutations likely exerted their effects by modulating the height of the energy barrier from the native to the latent form. Our results show that interactions found only in the metastable native form of serpins are important structural features that attenuate folding of the proteins into their latent forms.     

Interactions causing the kinetic trap in serpin protein folding

Conformational transition is fundamental to the mechanism of functional regulation in proteins, and serpins (serine protease inhibitors) can provide insight into this process. Serpins are metastable in their native forms, and they ordinarily undergo conformational transition to a stable state only when they form a tight complex with target proteases. The metastable native form is thus considered to be a kinetically trapped folding intermediate. We sought to understand the nature of the serpin kinetic trap as a step toward discovering how conformational transition is regulated. We found that mutations of the B/C beta-barrel of native alpha(1)-antitrypsin, a prototypical serpin, allowed conversion of the molecule into a more stable state. A 2.2 A resolution crystal structure of the stable form (PDB code, ) showed that the reactive site loop is inserted into an A beta-sheet, as in the latent plasminogen activator inhibitor-1. Mutational analyses suggest strongly that interactions not found in the final stable form cause the kinetic trap in serpin protein folding.     

Structural factors affecting the choice between latency transition and polymerization in inhibitory serpins

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) protein family, is unique among the serpins in its conformational lability. This lability allows spontaneous conversion of the active form to a more stable, latent conformation under physiological conditions. In other serpins, polymerization, rather than latency transition, is induced under pathological conditions or upon heat treatment. To identify specific factors promoting latency conversion in PAI-1, we mutated PAI-1 at various positions and compared the effects with those of equivalent mutations in alpha(1)-antitrypsin, the archetypal serpin. Mutations that improved interactions with the turn between helix F and the third strand of beta-sheet A (thFs3A) or the fifth strand of beta-sheet A (s5A), which are near the site of latency transition-associated insertion of the reactive center loop, retarded latency conversion but did not greatly increase structural stability. Mutations that decreased interactions with s2C facilitated conformational conversion, possibly by releasing the reactive center loop from beta-sheet C. Mutations of Thr93 that filled a hydrophobic surface pocket on s2A dramatically increased structural stability but had a negligible effect on the conformational transition. Our results suggest that the structural features controlling latency transition in PAI-1 are highly localized, whereas the conformational strain of the native forms of other inhibitory serpins is distributed throughout the molecule and induces polymerization.     

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.     

Probing the local conformational change of alpha1-antitrypsin

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of beta-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in alpha(1)-antitrypsin, which would lock the displacement of thFs3A from beta-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of alpha(1)-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into beta-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.     

How the serpin α1-proteinase inhibitor folds

Serpins are remarkable and unique proteins in being able to spontaneously fold into a metastable conformation without the aid of a chaperone or prodomain. This metastable conformation is essential for inhibition of proteinases, so that massive serpin conformational change, driven by the favorable energetics of relaxation of the metastable conformation to the more stable one, can kinetically trap the proteinase-serpin acylenzyme intermediate. Failure to direct folding to the metastable conformation would lead to inactive, latent serpin. How serpins fold into such a metastable state is unknown. Using the ability of component peptides from the serpin α(1)PI to associate, we have now elucidated the pathway by which this serpin efficiently folds into its metastable state. In addition we have established the likely structure of the polymerogenic intermediate of the Z variant of α(1)PI.     

Polymerization of plasminogen activator inhibitor-1

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into beta-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and beta-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-A-sheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving beta-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet.     

The native metastability and misfolding of serine protease inhibitors

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.     

The native metastable fold of C1-inhibitor is stabilized by disulfide bonds

C1-inhibitor is a member of the serpin family of proteinase inhibitors and is an important inhibitor of complement and contact system proteinases. The native protein has the characteristic serpin feature of being in a kinetically trapped metastable state rather than in the most stable state it could adopt. A consequence of this is that it readily forms loop-sheet dimers and polymers, by a mechanism believed to be the same as observed with other serpins. An unusual feature of C1-inhibitor is that it has a unique amino-terminal domain, of unknown function, held to the serpin domain by two disulfide bonds not found in other serpins. We report here that reduction of these bonds by DTT, causes a conformational change such that the reactive center loop inserts into beta-sheet A. This form of C1-inhibitor is less stable to heat and urea than the native protein, and is more susceptible to extensive degradation by trypsin. These data show that the disulfide bonds in C1-inhibitor are required for the protein to be stabilized in the metastable state with the reactive center loop expelled from beta-sheet A.     

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.     

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 role of strand 1 of the C β-sheet in the structure and function of α1-antitrypsin

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.     

Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state.

The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of alpha(1)-antitrypsin. alpha(1)-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B beta-sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to alpha(1)-antitrypsin. The denaturation of alpha(1)-antitrypsin and its variants was extremely similar when followed by far-UV CD, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A beta-sheet is expanded in the intermediate state whilst no structural change around the B beta-sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed.     

Cleaved serpin refolds into the relaxed state via a stressed conformer

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly(1)-Ala(352)) and light (Ser(353)-Pro(385)) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T(m) = 68.4 degrees C, whereas cl-R339T refolded into the relaxed form with T(m) = 85.5 degrees C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1'-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure.     

The N terminus of the serpin, tengpin, functions to trap the metastable native state

Serpins fold to a metastable native state and are susceptible to undergoing spontaneous conformational change to more stable conformers, such as the latent form. We investigated conformational change in tengpin, an unusual prokaryotic serpin from the extremophile Thermoanaerobacter tengcongensis. In addition to the serpin domain, tengpin contains a functionally uncharacterized 56-amino-acid amino-terminal region. Deletion of this domain creates a variant--tengpinDelta51--which folds past the native state and readily adopts the latent conformation. Analysis of crystal structures together with mutagenesis studies show that the N terminus of tengpin protects a hydrophobic patch in the serpin domain and functions to trap tengpin in its native metastable state. A 13-amino-acid peptide derived from the N terminus is able to mimick the role of the N terminus in stabilizing the native state of tengpinDelta51. Therefore, the function of the N terminus in tengpin resembles protein cofactors that prevent mammalian serpins from spontaneously adopting the latent conformation.     

Molecular gymnastics: serpin structure, folding and misfolding

The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding.     

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.     

The structural basis of serpin polymerization studied by hydrogen/deuterium exchange and mass spectrometry

The serpinopathies are a group of inherited disorders that share as their molecular basis the misfolding and polymerization of serpins, an important class of protease inhibitors. Depending on the identity of the serpin, conditions arising from polymerization include emphysema, thrombosis, and dementia. The structure of serpin polymers is thus of considerable medical interest. Wild-type alpha(1)-antitrypsin will form polymers upon incubation at moderate temperatures and has been widely used as a model system for studying serpin polymerization. Using hydrogen/deuterium exchange and mass spectrometry, we have obtained molecular level structural information on the alpha(1)-antitrypsin polymer. We found that the flexible reactive center loop becomes strongly protected upon polymerization. We also found significant increases in protection in the center of beta-sheet A and in helix F. These results support a model in which linkage between serpins is achieved through insertion of the reactive center loop of one serpin into beta-sheet A of another. We have also examined the heat-induced conformational changes preceding polymerization. We found that polymerization is preceded by significant destabilization of beta-sheet C. On the basis of our results, we propose a mechanism for polymerization in which beta-strand 1C is displaced from the rest of beta-sheet C through a binary serpin/serpin interaction. Displacement of strand 1C triggers further conformational changes, including the opening of beta-sheet A, and allows for subsequent polymerization.     

The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted

Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1' residues acting as a "bait" for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into beta-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor-donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in beta-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into beta-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.