Peter G. W. Gettins

Pathogenic alpha(1)-antitrypsin polymers are formed by reactive loop-beta-sheet A linkage

α1-Antitrypsin is the most abundant circulating protease inhibitor and the archetype of the serine protease inhibitor or serpin superfamily. Members of this family may be inactivated by point mutations that favor transition to a polymeric conformation. This polymeric conformation underlies diseases as diverse as α1-antitrypsin deficiency-related cirrhosis, thrombosis, angio-edema, and dementia. The precise structural linkage within a polymer has been the subject of much debate with evidence for reactive loop insertion into β-sheet A or C or as strand 7A. We have used site directed cysteine mutants and fluorescence resonance energy transfer (FRET) to measure a number of distances between monomeric units in polymeric α1-antitrypsin. We have then used a combinatorial approach to compare distances determined from FRET with distances obtained from 2.9 × 106 different possible orientations of the α1-antitrypsin polymer. The closest matches between experimental FRET measurements and theoretical structures show conclusively that polymers of α1-antitrypsin form by insertion of the reactive loop into β-sheet A.

The ternary complex of antithrombin–anhydrothrombin–heparin reveals the basis of inhibitor specificity

Antithrombin, the principal physiological inhibitor of the blood coagulation proteinase thrombin, requires heparin as a cofactor. We report the crystal structure of the rate-determining encounter complex formed between antithrombin, anhydrothrombin and an optimal synthetic 16-mer oligosaccharide. The antithrombin reactive center loop projects from the serpin body and adopts a canonical conformation that makes extensive backbone and side chain contacts from P5 to P6′ with thrombins restrictive specificity pockets, including residues in the 60-loop. These contacts rationalize many earlier mutagenesis studies on thrombin specificity. The 16-mer oligosaccharide is just long enough to form the predicted bridge between the high-affinity pentasaccharide-binding site on antithrombin and the highly basic exosite 2 on thrombin, validating the design strategy for this synthetic heparin. The protein-protein and protein-oligosaccharide interactions together explain the basis for heparin activation of antithrombin as a thrombin inhibitor.

Active Site Distortion Is Sufficient for Proteinase Inhibition by Serpins STRUCTURE OF THE COVALENT COMPLEX OF α1-PROTEINASE INHIBITOR WITH PORCINE PANCREATIC ELASTASE

We report here the x-ray structure of a covalent serpin-proteinase complex, α1-proteinase inhibitor (α1PI) with porcine pancreatic elastase (PPE), which differs from the only other x-ray structure of such a complex, that of α1PI with trypsin, in showing nearly complete definition of the proteinase. α1PI complexes with trypsin, PPE, and human neutrophil elastase (HNE) showed similar rates of deacylation and enhanced susceptibility to proteolysis by exogenous proteinases in solution. The differences between the two x-ray structures therefore cannot arise from intrinsic differences in the inhibition mechanism. However, self-proteolysis of purified complex resulted in rapid cleavage of the trypsin complex, slower cleavage of the PPE complex, and only minimal cleavage of the HNE complex. This suggests that the earlier α1 PI-trypsin complex may have been proteolyzed and that the present structure is more likely to be representative of serpin-proteinase complexes. The present structure shows that active site distortion alone is sufficient for inhibition and suggests that enhanced proteolysis is not necessarily exploited in vivo.

Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor.

Pigment epithelium-derived factor (PEDF), a noninhibitory member of the serpin superfamily, is the most potent inhibitor of angiogenesis in the mammalian ocular compartment. It also has neurotrophic activity, both in the retina and in the central nervous system, and is highly up-regulated in young versus senescent fibroblasts. To provide a structural basis for understanding its many biological roles, we have solved the crystal structure of glycosylated human PEDF to 2.85 Å. The structure revealed the organization of possible receptor and heparin-binding sites, and showed that, unlike any other previously characterized serpin, PEDF has a striking asymmetric charge distribution that might be of functional importance. These results provide a starting point for future detailed structure/function analyses into possible mechanisms of PEDF action that could lead to development of therapeutics against uncontrolled angiogenesis.

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, α1-proteinase inhibitor (α1PI) 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 α1PI 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 α1PI 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 α1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.

NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein is a member of the low density lipoprotein receptor family and contains clusters of cysteine-rich complement-like repeats of about 42 residues that are present in all members of this family of receptors. These clusters are thought to be the principal binding sites for protein ligands. We have expressed one complement-like repeat, CR8, from the cluster in lipoprotein receptor-related protein that binds certain proteinase inhibitor-proteinase complexes and used three-dimensional NMR on the13C/15N-labeled protein to determine the structure in solution of the calcium-bound form. The structure is very similar in overall fold to repeat 5 from the low density lipoprotein receptor (LB5), with backbone root mean square deviation of 1.5 Å. The calcium-binding site also appears to be homologous, with four carboxyl and two backbone carbonyl ligands. However, differences in primary structure are such that equivalent surfaces that might represent the binding interfaces are very different from one another, indicating that different domains will have very different ligand specificities.

Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. a paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors

Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin-antithrombin-proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors.

Canonical inhibitor-like interactions explain reactivity of α-PI Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, α1-proteinase inhibitor (α1PI) 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 α1PI 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 α1PI 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 α1PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.

The F-helix of serpins plays an essential, active role in the proteinase inhibition mechanism

Proteinase inhibition by serpins requires a 70 Å translocation of the proteinase, circumvention of the blocking helix F, and a crushing of the proteinase to render it catalytically incompetent. I propose that temporary displacement of the F‐helix during proteinase transit, and its subsequent return after complete passage of the proteinase, not only allows the proteinase to reach its final location, but provides an absolutely essential coupling mechanism for making the final proteinase crushing step energetically favorable. The F‐helix is therefore not a passive impediment to proteinase translocation, but a critical, active element in permitting the serpin inhibition mechanism to operate successfully.

Exosite Determinants of Serpin Specificity

Serpins form an enormous superfamily of 40–60-kDa proteins found in almost all types of organisms, including humans. Most are one-use suicide substrate serine and cysteine proteinase inhibitors that have evolved to finely regulate complex proteolytic pathways, such as blood coagulation, fibrinolysis, and inflammation. Despite distinct functions for each serpin, there is much redundancy in the primary specificity-determining residues. However, many serpins exploit additional exosites to generate the exquisite specificity that makes a given serpin effective only when certain other criteria, such as the presence of specific cofactors, are met. With a focus on human serpins, this minireview examines use of exosites by nine serpins in the initial complex-forming phase to modulate primary specificity in either binary serpin-proteinase complexes or ternary complexes that additionally employ a protein or other cofactor. A frequent theme is down-regulation of inhibitory activity unless the exosite(s) are engaged. In addition, the use of exosites by maspin and plasminogen activator inhibitor-1 to indirectly affect proteolytic processes is considered.