The conjugation protein TcpC from Clostridium perfringens is structurally related to the type IV secretion system protein VirB8 from Gram-negative bacteria.

Bacterial conjugation is important for the acquisition of virulence and antibiotic resistance genes. We investigated the mechanism of conjugation in Gram-positive pathogens using a model plasmid pCW3 from Clostridium perfringens. pCW3 encodes tetracycline resistance and contains the tcp locus, which is essential for conjugation. We showed that the unique TcpC protein (359 amino acids, 41 kDa) was required for efficient conjugative transfer, localized to the cell membrane independently of other conjugation proteins, and that membrane localization was important for its function, oligomerization and interaction with the conjugation proteins TcpA, TcpH and TcpG. The crystal structure of the C-terminal component of TcpC (TcpC(99-359)) was determined to 1.8-Å resolution. TcpC(99-359) contained two NTF2-like domains separated by a short linker. Unexpectedly, comparative structural analysis showed that each of these domains was structurally homologous to the periplasmic region of VirB8, a component of the type IV secretion system from Agrobacterium tumefaciens. Bacterial two-hybrid studies revealed that the C-terminal domain was critical for interactions with other conjugation proteins. The N-terminal region of TcpC was required for efficient conjugation, oligomerization and protein-protein interactions. We conclude that by forming oligomeric complexes, TcpC contributes to the stability and integrity of the conjugation apparatus, facilitating efficient pCW3 transfer.

Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer.

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.

Expression, purification and characterization of recombinant Z alpha(1)-antitrypsin--the most common cause of alpha(1)-antitrypsin deficiency.

Alpha(1)-antitrypsin (alpha(1)AT), the most abundant proteinase inhibitor circulating in the blood, protects extracellular matrix proteins of the lung against proteolytic destruction by neutrophil elastase. alpha(1)AT deficiency predisposes patients to emphysema, juvenile cirrhosis and hepatocellular carcinoma. Over 90% of clinical cases of severe alpha(1)AT deficiency are caused by the Z variant (E342K) of alpha(1)AT. The presence of the Z mutation results in misfolding and polymerization of alpha(1)AT. Due to its inherent propensity to polymerize there are no reported cases of recombinant Z alpha(1)AT production. This has created a major impediment to studying the effect of the Z mutation on alpha(1)AT. Here we report our attempts to produce recombinant Z alpha(1)AT using both Escherichia coli and Pichia pastoris as host systems. Using a range of expression vectors in E. coli we were unable to produce soluble active Z alpha(1)AT. Cytosolic expression of the Z alpha(1)AT gene in P. pastoris was successful. Monomeric and active recombinant Z alpha(1)AT was purified from the yeast cytosol using affinity chromatography and anion exchange chromatography. Biochemical analyses demonstrated that the recombinant Z alpha(1)AT has identical properties to its native counterpart purified from plasma of patients homozygous for the Z allele. A recombinant source of pathological Z alpha(1)AT will increase the chances of elucidating the mechanism of its polymerization and thus the development of therapeutic strategies.

Mechanisms of ataxin-3 misfolding and fibril formation: kinetic analysis of a disease-associated polyglutamine protein.

The polyglutamine diseases are a family of nine proteins where intracellular protein misfolding and amyloid-like fibril formation are intrinsically coupled to disease. Previously, we identified a complex two-step mechanism of fibril formation of pathologically expanded ataxin-3, the causative protein of spinocerebellar ataxia type-3 (Machado-Joseph disease). Strikingly, ataxin-3 lacking a polyglutamine tract also formed fibrils, although this occurred only via a single-step that was homologous to the first step of expanded ataxin-3 fibril formation. Here, we present the first kinetic analysis of a disease-associated polyglutamine repeat protein. We show that ataxin-3 forms amyloid-like fibrils by a nucleation-dependent polymerization mechanism. We kinetically model the nucleating event in ataxin-3 fibrillogenesis to the formation of a monomeric thermodynamic nucleus. Fibril elongation then proceeds by a mechanism of monomer addition. The presence of an expanded polyglutamine tract leads subsequently to rapid inter-fibril association and formation of large, highly stable amyloid-like fibrils. These results enhance our general understanding of polyglutamine fibrillogenesis and highlights the role of non-poly(Q) domains in modulating the kinetics of misfolding in this family.

Serpin acceleration of amyloid fibril formation: a role for accessory proteins.

Protein aggregation underlies an increasing number of human diseases. Recent experiments have shown that the aggregation reaction is exquisitely specific involving particular interactions between non-native proteins. However, aggregation of certain proteins, for example beta-amyloid, in vivo leads to the recruitment of other proteins into the aggregate. Antichymotrypsin, a non-fibril forming protein, is always observed to be associated with beta-amyloid plaques in Alzheimer's sufferers. The role of antichymotrypsin is controversial with studies showing it can either accelerate or inhibit the aggregation reaction. To investigate the role of antichymotrypsin in fibrillogenesis we have studied its interaction with apolipoprotein C-II, a well characterized model system for the study of fibrillogenesis. Our data demonstrate that sub-stoichiometric amounts of antichymotrypsin and its alternate structural forms can dramatically accelerate the aggregation of apolipoprotein C-II, whereas the presence of alpha(1)-antitrypsin, a structural homologue of antichymotrypsin, cannot. Sedimentation velocity experiments show more apolipoprotein C-II fibrils were formed in the presence of antichymotrypsin. Using pull-down assays and immuno-gold labeling we demonstrate an interaction between antichymotrypsin and apolipoprotein C-II fibrils that specifically occurs during fibrillogenesis. Taken together these data demonstrate an interaction between antichymotrypsin and apolipoprotein C-II that accelerates fibrillogenesis and indicates a specific role for accessory proteins in protein aggregation.

The loss of tryptophan 194 in antichymotrypsin lowers the kinetic barrier to misfolding.

Antichymotrypsin, a member of the serpin superfamily, has been shown to form inactive polymers in vivo, leading to chronic obstructive pulmonary disease. At present, however, the molecular determinants underlying the polymerization transition are unclear. Within a serpin, the breach position is implicated in conformational change, as it is the first point of contact for the reactive center loop and the body of the molecule. W194, situated within the breach, represents one of the most highly conserved residues within the serpin architecture. Using a range of equilibrium and kinetic experiments, the contribution of W194 to proteinase inhibition, stability and polymerization was studied for antichymotrypsin. Replacement of W194 with phenylalanine resulted in a fully active inhibitor that was destabilized relative to the wild-type protein. The aggregation kinetics were significantly altered; wild-type antichymotrypsin exhibits a lag phase followed by chain elongation. The loss of W194 almost entirely removed the lag phase and accelerated the elongation phase. On the basis of our data, we propose that one of the main roles of W194 in antichymotrypsin is in preventing polymerization.

The roles of helix I and strand 5A in the folding, function and misfolding of α1-antitrypsin.

α(1)-Antitrypsin, the archetypal member of the serpin superfamily, is a metastable protein prone to polymerization when exposed to stressors such as elevated temperature, low denaturant concentrations or through the presence of deleterious mutations which, in a physiological context, are often associated with disease. Experimental evidence suggests that α(1)-Antitrypsin can polymerize via several alternative mechanisms in vitro. In these polymerization mechanisms different parts of the molecule are proposed to undergo conformational change. Both strand 5 and helix I are proposed to adopt different conformations when forming the various polymers, and possess a number of highly conserved residues however their role in the folding and misfolding of α(1)-Antitrypsin has never been examined. We have therefore created a range of α(1)Antitypsin variants in order to explore the role of these conserved residues in serpin folding, misfolding, stability and function. Our data suggest that key residues in helix I mediate efficient folding from the folding intermediate and residues in strand 5A ensure native state stability in order to prevent misfolding. Additionally, our data indicate that helix I is involved in the inhibitory process and that both structural elements undergo differing conformational rearrangements during unfolding and misfolding. These findings suggest that the ability of α(1)-Antitrypsin to adopt different types of polymers under different denaturing conditions may be due to subtle conformational differences in the transiently populated structures adopted prior to the I and M* states.

Production of recombinant serpins in Escherichia coli.

Serpins represent a diverse family of proteins that are found in a wide range of organisms and cellular locations. In order to study them, most need to be produced recombinantly, as isolation from their source is not always possible. Due to their relatively uncomplicated structure (single domain, few posttranslational modifications), the serpins are usually amenable to expression in Escherichia coli, which offers a fast and cost-effective solution for the generation of large amounts of protein. This chapter outlines the general procedures used in the expression and subsequent purification of serpins in E. coli, with a particular focus on the methods used for antitrypsin, the archetypal member of the family.

Kinetic instability of the serpin Z alpha1-antitrypsin promotes aggregation.

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is alpha(1)-antitrypsin (alpha(1)AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of alpha(1)AT, which accounts for 95% of all cases of alpha(1)AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z alpha(1)AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type alpha(1)AT and Z alpha(1)AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type alpha(1)AT and Z alpha(1)AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z alpha(1)AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.

Conformational change in the chromatin remodelling protein MENT.

Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is 'fine-tuned' in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding 'M-loop' and D-helix of MENT, but is independent of the protease specificity determining 'reactive centre loop'. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin.

Preventing serpin aggregation: the molecular mechanism of citrate action upon antitrypsin unfolding.

The aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. A similar reaction resulting in disease can occur in other human serpins, and collectively they are known as the serpinopathies. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation. The citrate ion has previously been shown to prevent antitrypsin aggregation and maintain the protein in an active conformation; its mechanism of action, however, is unknown. Here we demonstrate that the citrate ion prevents the initial misfolding of the native state to a polymerogenic intermediate in a concentration-dependent manner. Furthermore, we have solved the crystal structure of citrate bound to antitrypsin and show that a single citrate molecule binds in a pocket between the A and B beta-sheets, a region known to be important in maintaining antitrypsin stability.

Aeropin from the extremophile Pyrobaculum aerophilum bypasses the serpin misfolding trap.

Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins.

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.

DNA accelerates the inhibition of human cathepsin V by serpins.

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.

A common fold mediates vertebrate defense and bacterial attack.

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding.

The native serpin fold is metastable and possesses the inherent ability to convert into more stable, but inactive, conformations. In order to understand why serpins attain the native fold instead of other more thermodynamically favourable folds we have investigated the presence of residual structure within denatured antichymotrypsin (ACT). Through mutagenesis we created a single tryptophan variant of ACT in which a Trp residue (276) is situated on the H-helix, located within a region known as the B/C barrel. The presence of residual structure around Trp 276 in 5 M guanidine hydrochloride (GdnHCl) was shown by fluorescence and circular dichroism spectroscopy and fluorescence lifetime experiments. The residual structure was disrupted in the presence of 5 M guanidine thiocyanate (GdnSCN). Protein refolding studies showed that significant refolding could be achieved from the GdnHCl denatured state but not the GdnSCN denatured form. The implications of these data on the folding and misfolding of the serpin superfamily are discussed.