Structural bases for substrate and inhibitor recognition by matrix metaloproteinases

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases which are involved in the proteolytic processing of several components of the extracellular matrix. As a consequence, MMPs are implicated in several physiological and pathological processes, like skeletal growth and remodelling, wound healing, cancer, arthritis, and multiple sclerosis, raising a very widespread interest toward this class of enzymes as potential therapeutic targets. Here, structure-function relationships are discussed to highlight the role of different MMP domains on substrate/inhibitor recognition and processing and to attempt the formulation of advanced guidelines, based on natural substrates, for the design of inhibitors more efficient in vivo. © 2008 Bentham Science Publishers Ltd

Structural, antigenic and immunogenic features of respiratory syncytial virus glycoproteins relevant for vaccine development

Extraordinary progress in the structure and immunobiology of the human respiratory syncytial virus glycoproteins has been accomplished during the last few years. Determination of the fusion (F) glycoprotein structure folded in either the prefusion or the postfusion conformation was an inspiring breakthrough not only to understand the structural changes associated with the membrane fusion process but additionally to appreciate the antigenic intricacies of the F protein. Furthermore, these developments have opened new avenues for structure-based designs of promising hRSV vaccine candidates. Finally, recent advances in our knowledge of the attachment (G) glycoprotein and its interaction with cell-surface receptors have revitalized interest in this molecule as a vaccine, as well as its role in hRSV immunobiology.Work in the Madrid lab is currently funded by grant SAF2015-67033-R from Plan Nacional de I+D+I. J.S.M is supported in part by award P20GM113132 from the National Institute of General Medical Sciences of the National Institutes of Health.S

A novel mode of translocation for cytolethal distending toxin

Thermal instability in the toxin catalytic subunit may be a common property of toxins that exit the endoplasmic reticulum (ER) by exploiting the mechanism of ER-associated degradation (ERAD). The Haemophilus ducreyi cytolethal distending toxin (HdCDT) does not utilize ERAD to exit the ER, so we predicted the structural properties of its catalytic subunit (HdCdtB) would differ from other ER-translocating toxins. Here, we document the heat-stable properties of HdCdtB which distinguish it from other ER-translocating toxins. Cell-based assays further suggested that HdCdtB does not unfold before exiting the ER and that it may move directly from the ER lumen to the nucleoplasm. These observations suggest a novel mode of ER exit for HdCdtB. (c) 2008 Elsevier B.V. All rights reserved

Paradigms of receptor kinase signaling in plants

Plant receptor kinases (RKs) function as key plasma-membrane localized receptors in the perception of molecular ligands regulating development and environmental response. Through the perception of diverse ligands, RKs regulate various aspects throughout the plant life cycle from fertilization to seed set. Thirty years of research on plant RKs has generated a wealth of knowledge on how RKs perceive ligands and activate downstream signaling. In the present review, we synthesize this body of knowledge into five central paradigms of plant RK signaling: (1) RKs are encoded by expanded gene families, largely conserved throughout land plant evolution; (2) RKs perceive many different kinds of ligands through a range of ectodomain architectures; (3) RK complexes are typically activated by co-receptor recruitment; (4) post-translational modifications fulfill central roles in both the activation and attenuation of RK-mediated signaling; and, (5) RKs activate a common set of downstream signaling processes through receptor-like cytoplasmic kinases (RLCKs). For each of these paradigms, we discuss key illustrative examples and also highlight known exceptions. We conclude by presenting five critical gaps in our understanding of RK function

Investigating The Functional Relationships Of RND Efflux Pumps In Methicillin Resistant Staphylococcus Aureus

The RND superfamily of efflux pumps plays vital roles in the intrinsic defence mechanisms of bacterial pathogens. The staphylococci have two genes encoding RND transporters; farE, which promotes efflux of antimicrobial fatty acids, and an uncharacterized gene we have named femT. Although RND pumps are known to contribute to maintenance of cellular homeostasis and antimicrobial resistance, the function of FemT, and the relationship between FemT and FarE in staphylococci have not been identified. Using established assays, we have tested the phenotype of a femT deletion mutant. Here, we show that this mutant is more susceptible to lysostaphin, vancomycin and oxacillin, and grows faster in Mueller-Hinton broth. Most notably, when evaluating the relationship between these transporters, inducible expression of FarE in fatty acids was abolished in FemT-deficient mutants. These findings suggest an interplay between the two transporters, and cumulatively, represent the first description of both systems operating together in S. aureus

Comparing sequence and structure of falcipains and human homologs at prodomain and catalytic active site for malarial peptide-based inhibitor design:

Falcipains are major cysteine proteases of Plasmodium falciparum involved in haemoglobin degradation and remain attractive anti-malarial drug targets. Several inhibitors against these proteases have been identified, yet none of them has been approved for malaria treatment. Other Plasmodium species also possess highly homologous proteins to falcipains. For selective therapeutic targeting, identification of sequence and structure differences with homologous human cathepsins is necessary. The substrate processing activity of these proteins is tightly controlled via a prodomain segment occluding the active site which is chopped under low pH conditions exposing the catalytic site. Current work characterizes these proteases to identify residues mediating the prodomain regulatory function for the design of peptide based anti-malarial inhibitors

The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling

Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a ‘defective’ docking domain may be a primary structural role of H2A.Bbd in chromatin

Structural determinants of peroxidase activities

Horseradish peroxidase (HRP) is a robust enzyme with commercial applications as an immunodiagnostic reporter enzyme and in the catalysis of difficult chemical transformations. The commercial enzyme is still isolated from the roots of the horseradish plant Armoracia rusticana, and has been studied as a model haem enzyme system since the early 1940’s. Following the development of methods to produce the active recombinant enzyme in E.coli (Smith et al., 1990) and completion of the crystallographic structure in 1997 (Gajhede et al., 1997) it has been possible to identify the structural requirements for activity and to extend these activities by protein engineering techniques. Three aspects relating to the enhancement of the ‘normal’ and ‘designed’ activities of selected variants have been explored at the structure function level in this work. Earlier work (Gajhede et al., 1997; White et al., 1997) highlighted residues that interact with aromatic substrates but that also potentially occlude access to the reactive haem edge by larger bulky substrates of potential commercial interest. The X-ray structure of the HRP-C* A140G/F179A variant was solved to 2.0Å. A larger engineered cavity at the haem edge was observed consistent with the ability of the variant to oxidise luminol directly, a property not seen in the wild-type enzyme. The structure factors for other residues in the haem access channel were not significantly affected. The structure of the S167M HRP variant was also solved, because of interest in its ability to form a novel sulphonium linkage to the haem vinyl group (K. Cali, DPhil thesis, University of Sussex). The sulphur to β vinyl distance was found to be 3.15Å, compared to 1.7Å in the natural sulphonium linkage of myeloperoxidase. This implies that significant thermal motion in the structure is required for the haem-protein crosslink to form, accounting for the relatively slow autocatalytic modification process observed in the presence of hydrogen peroxide. Previous work (Ngo and Smith, Int. Pat. No. WO/2007/020428) has shown that HRP engineered with a more open distal haem pocket (mimicking that of chloroperoxidase or cytochrome P450s) with a weak surrogate base (provided by a Glu residue as in chloroperoxidase) was capable of both entantioselective sulphoxidation and epoxidation. Building on this work, an alternative variant was designed in which the location of the weak base, provided by a Glu or Asp residue, has been varied within the active site. In particular, the HRP variant R38E:F41A:H42A (EAA) catalyses the production of the 1 and 2 naphthol from naphthalene at a rate of 124±4 min-1, suggesting the generation of an epoxide intermediate in the active site. The wild-type enzyme does not catalyse this reaction or the sulphoxidation reactions described for earlier variants. This is believed to be the first report of aromatic C-H bond activation by an engineered plant peroxidase and is unusual in that C-H bond activation of this type normally requires a P450-type thiolate ligated haem system. Equilibrium binding studies show that naphthalene binds to the engineered haem cavity with an estimated Kd of 30±2 μM. Unfortunately, crystals of HRP variants described by Ngo and Smith (2007) and of the new EAA variant described here could not be obtained, despite many crystallisation attempts under a wide range of conditions

Structural and functional aspects of the multidrug efflux pump AcrB

The tripartite efflux system AcrA/AcrB/TolC is the main pump in Escherichia coli for the efflux of multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB is central to substrate recognition and energy transduction and acts as a proton/drug antiporter. Recent structural studies show that homotrimeric AcrB can adopt different monomer conformations representing consecutive states in an allosteric functional rotation transport cycle. The conformational changes create an alternate access drug transport tunnel including a hydrophobic substrate binding pocket in one of the cycle intermediate