Repeating intermolecular protein association by means of beta-sheet expansion is the mechanism underlying a multitude of diseases including Alzheimer's, Huntington's and Parkinson's and the prion encephalopathies. A family of proteins, known as the serpins, also forms large stable multimers by ordered beta-sheet linkages leading to intracellular accretion and disease. These 'serpinopathies' include early-onset dementia caused by mutations in neuroserpin, liver cirrhosis and emphysema caused by mutations in alpha(1)-antitrypsin (alpha(1)AT), and thrombosis caused by mutations in antithrombin. Serpin structure and function are quite well understood, and the family has therefore become a model system for understanding the beta-sheet expansion disorders collectively known as the conformational diseases. To develop strategies to prevent and reverse these disorders, it is necessary to determine the structural basis of the intermolecular linkage and of the pathogenic monomeric state. Here we report the crystallographic structure of a stable serpin dimer which reveals a domain swap of more than 50 residues, including two long antiparallel beta-strands inserting in the centre of the principal beta-sheet of the neighbouring monomer. This structure explains the extreme stability of serpin polymers, the molecular basis of their rapid propagation, and provides critical new insights into the structural changes which initiate irreversible beta-sheet expansion.

The maintenance of normal blood flow depends completely on the inhibition of thrombin by antithrombin, a member of the serpin family. Antithrombin circulates at a high concentration, but only becomes capable of efficient thrombin inhibition on interaction with heparin or related glycosaminoglycans. The anticoagulant properties of therapeutic heparin are mediated by its interaction with antithrombin, although the structural basis for this interaction is unclear. Here we present the crystal structure at a resolution of 2.5 A of the ternary complex between antithrombin, thrombin and a heparin mimetic (SR123781). The structure reveals a template mechanism with antithrombin and thrombin bound to the same heparin chain. A notably close contact interface, comprised of extensive active site and exosite interactions, explains, in molecular detail, the basis of the antithrombotic properties of therapeutic heparin.

Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) are structurally related peptides found in all higher vertebrates. NPY is expressed exclusively in neurons, whereas PYY and PP are produced primarily in gut endocrine cells. Several receptor subtypes have been identified pharmacologically, but only the NPY/PYY receptor of subtype Y1 has been cloned. This is a heptahelix receptor that couples to G proteins. We utilized Y1 sequence information from several species to clone a novel human receptor with 43% amino acid sequence identity to human Y1 and 53% identity in the transmembrane regions. The novel receptor displays a pharmacological profile that distinguishes it from all previously described NPY family receptors. It binds PP with an affinity (Ki) of 13.8 pM, PYY with 1.44 nM, and NPY with 9.9 nM. Because these data may identify the receptor as primarily a PP receptor, we have named it PP1. In stably transfected Chinese hamster ovary cells the PP1 receptor inhibits forskolin-stimulated cAMP synthesis. Northern hybridization detected mRNA in colon, small intestine, pancreas, and prostate. As all three peptides are present in the gut through either endocrine release or innervation, all three peptides may be physiological ligands to the novel NPY family receptor PP1.

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Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10 000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparinactivated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFα and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.

The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend on the engagement of exosites on thrombin, either through direct interaction with a substrate, as with fibrinogen, or by binding to cofactors such as thrombomodulin. How thrombin specificity is controlled is of central importance to understanding normal hemostasis and how dysregulation causes bleeding or thrombosis. The binding of ligands to thrombin via exosite I and the coordination of Na þ have been associated with changes in thrombin conformation and activity. This phenomenon has become known as thrombin allostery, although direct evidence of conformational change, identification of the regions involved, and the functional consequences remain unclear. Here we investigate the conformational and dynamic effects of thrombin ligation at the active site, exosite I and the Na þ -binding site in solution, using modern multidimensional NMR techniques. We obtained full resonance assignments for thrombin in seven differently liganded states, including fully unliganded apo thrombin, and have created a detailed map of residues that change environment, conformation, or dynamic state in response to each relevant single or multiple ligation event. These studies reveal that apo thrombin exists in a highly dynamic zymogen-like state, and relies on ligation to achieve a fully active conformation. Conformational plasticity confers upon thrombin the ability to be at once selective and promiscuous.allostery | hemostasis | protease | structure | zymogen B lood coagulation (hemostasis) is the result of a cascade of events where zymogens are converted to active proteases through the specific action of a preceding protease (1, 2). This process is tightly regulated to ensure that stable blood clots form rapidly at the site of tissue damage. Dysregulation by several mechanisms is the cause of bleeding disorders, including hemophilia, and of thrombosis, the most common cause of morbidity and mortality in the industrialized world. Most hemostatic proteases have only a single target and are regulated by a single cofactor. However, thrombin (Fig. 1), the final protease in the cascade, has over a dozen substrates and at least five cofactors (3). Thrombin is critical for the initiation, propagation, and attenuation of the hemostatic response, and in each of these phases the activity of thrombin is directed by cofactors (4, 5). Of principal importance are the cofactors that convert thrombin between pro and anticoagulant activities. Thrombomodulin (TM) is an integral membrane protein that alters thrombin specificity from the procoagulant substrates such as PAR1, factors V and VIII, and fibrinogen to specific activation of the anticoagulant protease protein C (6). This change of function is thought to be due to the bloc...

Key Points The crystal structure of pro-pseutarin C reveals how the prothrombinase complex assembles and suggests a mechanism of prothrombin processing.