James A. Irving

Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis

Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.

The Evolution of Enzyme Specificity in Fasciola spp.

Fasciola spp., commonly known as liver fluke, are significant trematode parasites of livestock and humans. They secrete several cathepsin L-like cysteine proteases, some of which differ in enzymatic properties and timing of expression in the parasites life cycle. A detailed sequence and evolutionary analysis is presented, based on 18 cathepsin L-like enzymes isolated from Fasciola spp. (including a novel clone identified in this study). The enzymes form a monophyletic group which has experienced several gene duplication events over the last ~135 million years, giving rise to the present-day enzymatic repertoire of the parasite. This timing of these duplications appears to correlate with important points in the evolution of the mammalian hosts. Furthermore, the dates suggest that Fasciola hepatica and Fasciola gigantica diverged around 19 million years ago. A novel analysis, based on the pattern of amino acid diversity, was used to identify sites in the enzyme that are predicted to be subject to positive adaptive evolution. Many of these sites occur within the active site cleft of the enzymes, and hence would be expected to lead to differences in substrate specificity. Using homology modeling, with reference to previously obtained biochemical data, we are able to predict S2 subsite specificity for these enzymes: specifically those that can accommodate bulky hydrophobic residues in the P2 position and those that cannot. A number of other positions subject to evolutionary pressure and potentially significant for enzyme function are also identified, including sites anticipated to diminish cystatin binding affinity.

Cloning and Expression of the Major Secreted Cathepsin B-Like Protein from Juvenile Fasciola hepatica and Analysis of Immunogenicity following Liver Fluke Infection

ABSTRACT The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation.

MENT (Myeloid andErythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.

Protein structural alignments and functional genomics.

Structural genomics—the systematic solution of structures of the proteins of an organism—will increasingly often produce molecules of unknown function with no close relative of known function. Prediction of protein function from structure has thereby become a challenging problem of computational molecular biology. The strong conservation of active site conformations in homologous proteins suggests a method for identifying them. This depends on the relationship between size and goodness‐of‐fit of aligned substructures in homologous proteins. For all pairs of proteins studied, the root‐mean‐square deviation (RMSD) as a function of the number of residues aligned varies exponentially for large common substructures and linearly for small common substructures. The exponent of the dependence at large common substructures is well correlated with the RMSD of the core as originally calculated by Chothia and Lesk (EMBO J 1986;5:823–826), affording the possibility of reconciling different structural alignment procedures. In the region of small common substructures, reduced aligned subsets define active sites and can be used to suggest the locations of active sites in homologous proteins. Proteins 2001;42:378–382.

The Murine Orthologue of Human Antichymotrypsin A STRUCTURAL PARADIGM FOR CLADE A3 SERPINS

Antichymotrypsin (SERPINA3) is a widely expressed member of the serpin superfamily, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure of a murine antichymotrypsin orthologue to 2.1 Å, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A β-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90° to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 ± 0.9 × 106 m-1 s-1) and cathepsin G (ka: 7.9 ± 0.9 × 105 m-1 s-1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4–P6′ region of the RCL, the distal hinge, and the loop between strands 4B and 5B.

MUSTANG-MR Structural Sieving Server: Applications in Protein Structural Analysis and Crystallography

Background A central tenet of structural biology is that related proteins of common function share structural similarity. This has key practical consequences for the derivation and analysis of protein structures, and is exploited by the process of “molecular sieving” whereby a common core is progressively distilled from a comparison of two or more protein structures. This paper reports a novel web server for “sieving” of protein structures, based on the multiple structural alignment program MUSTANG. Methodology/Principal Findings “Sieved” models are generated from MUSTANG-generated multiple alignment and superpositions by iteratively filtering out noisy residue-residue correspondences, until the resultant correspondences in the models are optimally “superposable” under a threshold of RMSD. This residue-level sieving is also accompanied by iterative elimination of the poorly fitting structures from the input ensemble. Therefore, by varying the thresholds of RMSD and the cardinality of the ensemble, multiple sieved models are generated for a given multiple alignment and superposition from MUSTANG. To aid the identification of structurally conserved regions of functional importance in an ensemble of protein structures, Lesk-Hubbard graphs are generated, plotting the number of residue correspondences in a superposition as a function of its corresponding RMSD. The conserved “core” (or typically active site) shows a linear trend, which becomes exponential as divergent parts of the structure are included into the superposition. Conclusions The application addresses two fundamental problems in structural biology: First, the identification of common substructures among structurally related proteins—an important problem in characterization and prediction of function; second, generation of sieved models with demonstrated uses in protein crystallographic structure determination using the technique of Molecular Replacement.

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation.

Most serpins are associated with protease inhibition, and their ability to form loop–sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non‐histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage‐specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA‐binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop–sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non‐histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop–sheet linkages.

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.