Mary C. Pearce

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.

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

α1‐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.

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 (TcpC99–359) was determined to 1.8‐Å resolution. TcpC99–359 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.

The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

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—tengpinΔ51—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 tengpinΔ51. 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.

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 β‐sheets, a region known to be important in maintaining antitrypsin stability.

Expression, purification and characterization of recombinant Z α1-Antitrypsin—The most common cause of α1-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.

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.