Plakalbumin, alpha 1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis

An old puzzle in protein biochemistry concerns the ready conversion of ovalbumin, by proteolysis, to the much more stable derivative, plakalbumin. Ovalbumin is now known to belong to the serpin superfamily, most of which are serine proteinase inhibitors. We report here studies of two such members of the family, the human plasma proteins alpha 1-antitrypsin and antithrombin, and show that they undergo a similar change in stability on selective proteolysis. This change, which is accompanied by a loss of inhibitory activity, can best be considered as an irreversible molecular transition from a native stressed (S) conformation, to a more ordered relaxed (R) form. The maintenance of the native S conformation, and hence the maintenance of inhibitory activity, is critically dependent on the integrity of an exposed loop of polypeptide. We propose that the susceptibility of this peptide loop to proteolytic cleavage gives it an incidental role as a physiological switch which allows the inactivation of individual inhibitors by specific proteolysis. The vulnerability of this exposed loop in each inhibitor also explains the pathological action of a number of venoms and toxins. In particular, the demonstration here of the cleavage of antithrombin, by leukocyte elastase, explains an observed change in blood coagulation that accompanies severe inflammation and which can result in fatal thrombosis.     

Structural basis of latency in plasminogen activator inhibitor-1.

Human plasminogen activator inhibitor-1 (PAI-1) is the fast-acting inhibitor of tissue plasminogen activator and urokinase and is a member of the serpin family of protease inhibitors. Serpins normally form complexes with their target proteases that dissociate very slowly as cleaved species and then fold into a highly stable inactive state in which the residues that flank the scissile bond (P1 and P1';) are separated by about 70 A. PAI-1 also spontaneously folds into a stable inactive state without cleavage; this state is termed 'latent' because inhibitory activity can be restored through denaturation and renaturation. Here we report the structure of intact latent PAI-1 determined by single-crystal X-ray diffraction to 2.6 A resolution. The three-dimensional structure reveals that residues on the N-terminal side of the primary recognition site are inserted as a central strand of the largest beta sheet, in positions similar to the corresponding residues in the cleaved form of the serpin alpha 1-proteinase inhibitor (alpha 1-PI). Residues C-terminal to the recognition site occupy positions on the surface of the molecule distinct from those of the corresponding residues in cleaved serpins or in the intact inactive serpin homologue, ovalbumin, and its cleavage product, plakalbumin. The structure of latent PAI-1 is similar to one formed after cleavage in other serpins, and the stability of both latent PAI-1 and cleaved serpins may be derived from the same structural features.     

Mobile reactive centre of serpins and the control of thrombosis

Two protease inhibitors in human plasma play a key part in the control of thrombosis: antithrombin inhibits coagulation and the plasminogen activator inhibitor PAI-1 inhibits fibrinolysis, the dissolving of clots. Both inhibitors are members of the serpin family and both exist in the plasma in latent or inactive forms. We show here that the reactive centre of the serpins can adopt varying conformations and that mobility of the reactive centre is necessary for the function of antithrombin and its binding and activation by heparin; the identification of a new locked conformation explains the latent inactive state of PAI-1. This ability to vary conformation not only allows the modulation of inhibitory activity but also protects the circulating inhibitor against proteolytic attack. Together these findings explain the retention by the serpins of a large and unconstrained reactive centre as compared to the small fixed peptide loop of other families of serine protease inhibitors.     

Role of corticosteroid-binding globulin in interaction of corticosterone with uterine and brain progesterone receptors

The central actions of the steroid hormone progesterone remain an enigma. However, there is no doubt that this hormone has a vital role in the control of sexual function and behaviour in many species, including man. Furthermore, progesterone may be involved in the premenstrual and postpartum syndromes. It would therefore be very useful to know the role and mechanism of action of progesterone in the brain. We now show that there are differences between progesterone receptors in brain and uterus, and possibly in the distribution of the serum progesterone-binding protein, corticosteroid-binding globulin (CBG), which may enter uterine but not brain cells.     

A redox switch in angiotensinogen modulates angiotensin release

Blood pressure is critically controlled by angiotensins, which are vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen-a non-inhibitory member of the serpin family of protease inhibitors. Although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1?? resolution show that the angiotensin cleavage site is inaccessibly buried in its amino-terminal tail. The conformational rearrangement that makes this site accessible for proteolysis is revealed in our 4.4?? structure of the complex of human angiotensinogen with renin. The co-ordinated changes involved are seen to be critically linked by a conserved but labile disulphide bridge. Here we show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidized sulphydryl-bridged form, which preferentially interacts with receptor-bound renin. We propose that this redox-responsive transition of angiotensinogen to a form that will more effectively release angiotensin at a cellular level contributes to the modulation of blood pressure. Specifically, we demonstrate the oxidative switch of angiotensinogen to its more active sulphydryl-bridged form in the maternal circulation in pre-eclampsia-the hypertensive crisis of pregnancy that threatens the health and survival of both mother and child.     

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 A 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.     

Serpin-resistant mutants of human tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) converts the inactive zymogen, plasminogen, into the powerful protease, plasmin, which then degrades the fibrin meshwork of thrombi. To prevent systemic activation of plasminogen, plasma contains several inhibitors of t-PA, the most important of which is plasminogen activator inhibitor-1 (PAI-1), a member of the serpin superfamily. As the ability to produce serpin-resistant variants of t-PA could increase the potential of this enzyme as a thrombolytic agent, we have used the known three-dimensional structure of the complex between trypsin and bovine pancreatic trypsin inhibitor (BPTI) to model the interactions between the active site of human t-PA and PAI-1. On the basis of this model we then altered by site-directed mutagenesis those amino acids of t-PA predicted to make contact with PAI-1 but not with the substrate plasminogen. We report here that although the resulting mutants have enzymatic properties similar to those of wild-type t-PA, they display significant resistance to inhibition by PAI-1. For example, following incubation with an amount of the serpin that completely inhibits the wild-type enzyme, one variant retains 95% of its initial activity. This mutant is also resistant to inhibition by the complex mixture of serpins present in human plasma.     

Structural basis for gate-DNA recognition and bending by type IIA topoisomerases

Type II topoisomerases disentangle DNA to facilitate chromosome segregation, and represent a major class of therapeutic targets. Although these enzymes have been studied extensively, a molecular understanding of DNA binding has been lacking. Here we present the structure of a complex between the DNA-binding and cleavage core of Saccharomyces cerevisiae Topo II (also known as Top2) and a gate-DNA segment. The structure reveals that the enzyme enforces a 150 degrees DNA bend through a mechanism similar to that of remodelling proteins such as integration host factor. Large protein conformational changes accompany DNA deformation, creating a bipartite catalytic site that positions the DNA backbone near a reactive tyrosine and a coordinated magnesium ion. This configuration closely resembles the catalytic site of type IA topoisomerases, reinforcing an evolutionary link between these structurally and functionally distinct enzymes. Binding of DNA facilitates opening of an enzyme dimerization interface, providing visual evidence for a key step in DNA transport.     

Homologues of Twisted gastrulation are extracellular cofactors in antagonism of BMP signalling

Twisted gastrulation (TSG) is involved in specifying the dorsal-most cell fate in Drosophila embryos, but its mechanism of action is poorly understood. TSG has been proposed to modify the action of Short gastrulation (SOG), thereby increasing signalling by the bone morphogenetic protein (BMP) Decapentaplegic. SOG, an inhibitor of BMP signalling, is in turn inactivated by the protease Tolloid. Here we identify Tsg gene products from human, mouse, Xenopus, zebrafish and chick. Expression patterns in mouse and Xenopus embryos are consistent with in vivo interactions between Tsg, BMPs and the vertebrate SOG orthologue, chordin. We show that Tsg binds both the vertebrate Decapentaplegic orthologue BMP4 and chordin, and that these interactions have multiple effects. Tsg increases chordin's binding of BMP4, potentiates chordin's ability to induce secondary axes in Xenopus embryos, and enhances chordin cleavage by vertebrate tolloid-related proteases at a site poorly used in Tsg's absence; also, the presence of Tsg enhances the secondary axis-inducing activity of two products of chordin cleavage. We conclude that Tsg acts as a cofactor in chordin's antagonism of BMP signalling.     

Molecular mechanisms of serpin deficiencies

The study of serpin deficiency is currently one of the most active areas in basic medical research. Recently, three hypotheses concerning serpin deficiency have been proposed, which are referred to as the conformational disturbance hypothesis (CDH) , loop-sheet polymerisation hypothesis (LSPH) and multiple binding site hypothesis (MB-SH) . CDH was put forward to explicit serpin deficiency due to conformational change of reactive loop of serpins as a result of mutations occurring away from the reactive site residues and LSPH was to explain deficient serpins due to the formation of polymers. MBSH was proposed to explain the mechanism of the formation of stable enzyme-serpin complex via more than one binding site and blockage or mutation in any of the sites resulting in serpin deficiency. A combination of these mechanisms may be critical in understanding the roles of the many documented mutations and autoimmunities which result in qualitative and quantitative serpin deficiency.     

The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles

Nitrate, the major nitrogen source for most plants, is widely used as a fertilizer and as a result has become a predominant freshwater pollutant. Plants need nitrate for growth and store most of it in the central vacuole. Some members of the chloride channel (CLC) protein family, such as the torpedo-fish ClC-0 and mammalian ClC-1, are anion channels, whereas the bacterial ClC-ec1 and mammalian ClC-4 and ClC-5 have recently been characterized as Cl-/H+ exchangers with unknown cellular functions. Plant members of the CLC family are proposed to be anion channels involved in nitrate homeostasis; however, direct evidence for anion transport mediated by a plant CLC is still lacking. Here we show that Arabidopsis thaliana CLCa (AtCLCa) is localized to an intracellular membrane, the tonoplast of the plant vacuole, which is amenable to electrophysiological studies, and we provide direct evidence for its anion transport ability. We demonstrate that AtCLCa is able to accumulate specifically nitrate in the vacuole and behaves as a NO3-/H+ exchanger. For the first time, to our knowledge, the transport activity of a plant CLC is revealed, the antiporter mechanism of a CLC protein is investigated in a native membrane system, and this property is directly connected with its physiological role.     

Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport

The ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization. This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export of yeast a-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR). Here we present a tertiary structure model of the ATP-binding cassettes characteristic of this class of transport system, based on similarities between the predicted secondary structures of members of this family and the previously determined structure of adenylate kinase. This model has implications for both the molecular basis of transport and cystic fibrosis and provides a framework for further experimentation.     

Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue

Neurotransmitter/Na(+) symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na(+) electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na(+) are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan, and the Na(+)-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na(+)-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ～10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30?? away, in a manner that allows the Na(+) gradient to power the transport mechanism.     

Crystal structure of a retroviral protease proves relationship to aspartic protease family

Retroviral gag, pol and env gene products are translated as precursor polyproteins, which are cleaved by virus-encoded proteases to produce the mature proteins found in virions. On the basis of the conserved Asp-Thr/Ser-Gly sequence at the putative protease active sites, and other biochemical evidence, retroviral proteases have been predicted to be in the family of pepsin-like aspartic proteases. It has been suggested that aspartic proteases evolved from a smaller, dimeric ancestral protein, and a recent model of the human immunodeficiency virus (HIV) protease postulated that a symmetric dimer of this enzyme is equivalent to a pepsin-like aspartic protease. We have now determined the crystal structure of Rous sarcoma virus (RSV) protease at 3-A resolution and find it is dimeric and has a structure similar to aspartic proteases. This structure should provide a useful basis for the modelling of the structures of other retroviral proteases, such as that of HIV, and also for the rational design of protease inhibitors as potential antiviral drugs.     

Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit beta-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3-5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.     

Crystal structure of the anthrax lethal factor.

Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.     

Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy

It is generally accepted that a globular protein in its native state adopts a single, well-defined conformation. However, there have been several reports that some proteins may exist in more than one distinct folded form in equilibrium. In the case of staphylococcal nuclease, evidence for multiple conformations has come from electrophoretic and NMR studies, although there has been some controversy as to whether these are actually interconvertible forms of the same molecular species. Recently, magnetization transfer (MT)-NMR has been developed as a means of studying the kinetics of conformational transitions in proteins. In the study reported here, this approach has been extended and used to demonstrate the presence of at least two native forms of nuclease in equilibrium and to study their interconversion with the unfolded state under the conditions of the thermal unfolding transition. The experiments reveal that two distinct native forms of the protein fold and unfold independently and that these can interconvert directly as well as via the unfolded state. The spectra of the different forms suggest that they are structurally similar but the MT experiments show that the kinetics of folding and unfolding are quite different. Characterization of this behaviour will, therefore, have important implications for our understanding of the relationship between structure and folding kinetics.     

Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease

The AAA domain, a conserved Walker-type ATPase module, is a feature of members of the AAA family of proteins, which are involved in many cellular processes, including vesicular transport, organelle biogenesis, microtubule rearrangement and protein degradation. The function of the AAA domain, however, has not been explained. Membrane-anchored AAA proteases of prokaryotic and eukaryotic cells comprise a subfamily of AAA proteins that have metal-dependent peptidase activity and mediate the degradation of non-assembled membrane proteins. Inactivation of an orthologue of this protease family in humans causes neurodegeneration in hereditary spastic paraplegia. Here we investigate the AAA domain of the yeast protein Yme1, a subunit of the iota-AAA protease located in the inner membrane of mitochondria. We show that Yme1 senses the folding state of solvent-exposed domains and specifically degrades unfolded membrane proteins. Substrate recognition and binding are mediated by the amino-terminal region of the AAA domain. The purified AAA domain of Yme1 binds unfolded polypeptides and suppresses their aggregation. Our results indicate that the AAA domain of Ymel has a chaperone-like activity and suggest that the AAA domains of other AAA proteins may have a similar function.     

Amino-terminal fragments of Escherichia coli lac repressor bind to DNA.

The N-terminal fragments (residues 1-51 and 1-59) obtained by selective tryptic cleavage of native lac repressor retain the ability to bind DNA. These fragments (headpieces) are monomeric and form complexes which resemble those of tetrameric repressor with non-operator DNA. But, they do not show the high specificity of repressor for operator sequences. The DNA binding has been demonstrated by filter-binding assay as well as in solution using absorption, circular dichroism, and fluorescence measurements.