Robin W. Carrell

Structure of a serpin-protease complex shows inhibition by deformation.

The serpins have evolved to be the predominant family of serine-protease inhibitors in man. Their unique mechanism of inhibition involves a profound change in conformation, although the nature and significance of this change has been controversial. Here we report the crystallographic structure of a typical serpin–protease complex and show the mechanism of inhibition. The conformational change is initiated by reaction of the active serine of the protease with the reactive centre of the serpin. This cleaves the reactive centre, which then moves 71 Å to the opposite pole of the serpin, taking the tethered protease with it. The tight linkage of the two molecules and resulting overlap of their structures does not affect the hyperstable serpin, but causes a surprising 37% loss of structure in the protease. This is induced by the plucking of the serine from its active site, together with breakage of interactions formed during zymogen activation. The disruption of the catalytic site prevents the release of the protease from the complex, and the structural disorder allows its proteolytic destruction. It is this ability of the conformational mechanism to crush as well as inhibit proteases that provides the serpins with their selective advantage.

What do dysfunctional serpins tell us about molecular mobility and disease

Proteinase inhibitors of the serpin family have a unique ability to regulate their activity by changing the conformation of their reactive-centre loop. Although this may explain their evolutionary success, the dependence of function on structural mobility makes the serpins vulnerable to the effects of mutations. Here, we describe how studies of dysfunctional variants, together with crystal structures of serpins in different forms, provide insights into the molecular functions and remarkable folding properties of this family. In particular, comparisons of variants affecting different serpins allow us to define the domains which control this folding and show how spontaneous but inappropriate changes in conformation cause diverse diseases.

Hormone binding globulins undergo serpin conformational change in inflammation

A surprising recent finding is that thyroxine binding globulin (TBG)1 and cortisol binding globulin (CBG)2, are members of the serine protease inhibitor (serpin) superfamily3. Apparently evol-ution has completely adapted the serpin structure for its new role in these proteins as a transport agent, as there is no evidence of any retained protease inhibitory activity. This drastic change in function raises the question as to why such a complex molecular framework has been selected for the relatively simple task of hormone transport? To function as inhibitors the serpins have a native stressed (S) conformation that makes them vulnerable to proteolytic cleavage, the cleavage being accompanied by an irreversible transition to a stable relaxed (R) form4,5. We demon-strate here that TBG and CBG have retained the stressed native structure typical of the inhibitor members of the family and we provide evidence that the S–R transition has been adapted to allow altered hormone delivery at inflammatory sites.

Biological implications of a 3 å structure of dimeric antithrombin

BACKGROUND Antithrombin, a member of the serpin family of inhibitors, controls coagulation in human plasma by forming complexes with thrombin and other coagulation proteases in a process greatly accelerated by heparin. The structures of several serpins have been determined but not in their active conformations. We have determined the structure of intact antithrombin in order to study its mechanism of activation, particularly with respect to heparin, and the dysfunctions of this mechanism that predispose individuals to thrombotic disease. RESULTS The crystal structure of a dimer of one active and one inactive molecule of antithrombin has been determined at 3 A. The first molecule has its reactive-centre loop in a predicted active conformation compatible with initial entry of two residues into the main beta-sheet of the molecule. The inactive molecule has a totally incorporated loop as in latent plasminogen activator inhibitor-1. The two molecules are linked by the reactive loop of the active molecule which has replaced a strand from another beta-sheet in the latent molecule. CONCLUSION The structure, together with identified mutations affecting its heparin affinity, allows the placement of the heparin-binding site on the molecule. The conformation of the two forms of antithrombin demonstrates the extraordinary mobility of the reactive loop in the serpins and provides insights into the folding of the loop required for inhibitory activity together with the potential modification of this by heparin. The mechanism of dimerization is relevant to the polymerization that is observed in diseases associated with variant serpins.

Crystal structure of uncleaved ovalbumin at 1.95 A resolution.

Ovalbumin, the major protein in avian egg-white, is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily. The crystal structure of uncleaved, hen ovalbumin was solved by the molecular replacement method using the structure of plakalbumin, a proteolytically cleaved form of ovalbumin, as a starting model. The final refined model, including four ovalbumin molecules, 678 water molecules and a single metal ion, has a crystallographic R-factor of 17.4% for all reflections between 6.0 and 1.95 A resolution. The root-mean-square deviation from ideal values in bond lengths is 0.02 A and in bond angles is 2.9 degrees. This is the first crystal structure of a member of the serpin family in an uncleaved form. Surprisingly, the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation. The implications for the mechanism of inhibition of the inhibitory members of the family is discussed.

Prevalence of antithrombin deficiency in the healthy population

. In a cohort of 9669 blood donors we have identified 16 cases of congenital AT deficiency (1 in 600) by way of family studies and AT gene analysis. Two donors had type I AT deficiency (prevalence 0.21 per 1000; 95% CI = 0.03/1000 to 0.75/1000), their families displaying a symptomatic phenotype. 14 donors had a type II deficiency (prevalence 1.45 per 1000; 95% CI = 0.79/1000 to 2.43/1000): one recurring and three unique mutations. None of these type II deficiencies appeared to confer a high thrombotic risk despite many of the affected individuals having experienced potentially prothrombotic challenges. The high frequency of these relatively asymptomatic variants may reflect a selection bias in the study population. However, their existence should not only add to our understanding of structure‐function relationships of AT but may also influence our management of asymptomatic deficient individuals identified in epidemiological or presurgical screening programmes.

Familial dementia caused by polymerization of mutant neuroserpin

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutation, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

Mutation of antitrypsin to antithrombin: α1-antitrypsin Pittsburgh (358 Met→Arg), a fatal bleeding disorder

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant proteins being an acute-phase reactant, the level of which increased several-fold after trauma.

Serpinopathies and the conformational dementias

The serpin superfamily of serine proteinase inhibitors has a central role in controlling proteinases in many biological pathways in a wide range of species. The inhibitory function of the serpins involves a marked conformational transition, but this inherent molecular flexibility also renders the serpins susceptible to point mutations that result in aberrant intermolecular linkage and polymer formation. The effects of such protein aggregation are cumulative, with a progressive loss of cellular function that results in diseases as diverse as cirrhosis and emphysema. The recent recognition that mutations in a serpin can also result in late-onset dementia provides insights into changes that underlie other conformational diseases, such as the amyloidoses, the prion encephalopathies and Huntington and Alzheimer diseases.

How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration

The interaction of the plasma protein vitronectin with plasminogen activator inhibitor-1 (PAI-1) is central to human health. Vitronectin binding extends the lifetime of active PAI-1, which controls hemostasis by inhibiting fibrinolysis and has also been implicated in angiogenesis. The PAI-1–vitronectin binding interaction also affects cell adhesion and motility. For these reasons, elevated PAI-1 activities are associated both with coronary thrombosis and with a poor prognosis in many cancers. Here we show the crystal structure at a resolution of 2.3 Å of the complex of the somatomedin B domain of vitronectin with PAI-1. The structure of the complex explains how vitronectin binds to and stabilizes the active conformation of PAI-1. It also explains the tissue effects of PAI-1, as PAI-1 competes for and sterically blocks the interaction of vitronectin with cell surface receptors and integrins. Structural understanding of the essential biological roles of the interaction between PAI-1 and vitronectin opens the prospect of specifically designed blocking agents for the prevention of thrombosis and treatment of cancer.