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Huesgen P.F.,University of British Columbia | Huesgen P.F.,Jülich Research Center | Lange P.F.,University of British Columbia | Rogers L.D.,University of British Columbia | And 6 more authors.
Nature Methods | Year: 2014

To improve proteome coverage and protein C-terminal identification, we characterized the Methanosarcina acetivorans thermophilic proteinase LysargiNase, which cleaves before lysine and arginine up to 55 °C. Unlike trypsin, LysargiNase-generated peptides had N-terminal lysine or arginine residues and fragmented with b ion-dominated spectra. This improved protein C terminal-peptide identification and several arginine-rich phosphosite assignments. Notably, cleavage also occurred at methylated or dimethylated lysine and arginine, facilitating detection of these epigenetic modifications.


Arolas J.L.,Molecular Biology Institute of Barcelona | Arolas J.L.,University of Vienna | Goulas T.,Molecular Biology Institute of Barcelona | Pomerantsev A.P.,National Institute of Allergy and Infectious Diseases | And 2 more authors.
Structure | Year: 2016

Immune inhibitor A(InhA)-type metallopeptidases are potential virulence factors secreted by members of the Bacillus cereus group. Two paralogs from anthrax-causing Bacillus anthracis (BaInhA1 and BaInhA2) were shown to degrade host tissue proteins with broad substrate specificity. Analysis of their activation mechanism and the crystal structure of a zymogenic BaInhA2 variant revealed a ∼750-residue four-domain structure featuring a pro-peptide, a catalytic domain, a domain reminiscent of viral envelope glycoproteins, and a MAM domain grafted into the latter. This domain, previously found only in eukaryotes, is required for proper protein expression in B. anthracis and evinces certain flexibility. Latency is uniquely modulated by the N-terminal segment of the pro-peptide, which binds the catalytic zinc through its α-amino group and occupies the primed side of the active-site cleft. The present results further our understanding of the modus operandi of an anthrax secretome regulator. © 2016 Elsevier Ltd All rights reserved.


Trillo-Muyo S.,Molecular Biology Institute of Barcelona | Jasilionis A.,Vilnius University | Domagalski M.J.,University of Virginia | Chruszcz M.,University of South Carolina | And 5 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved.


Joshi R.S.,Molecular Biology Institute of Barcelona | Pina B.,CSIC - Institute of Environmental Assessment And Water Research | Roca J.,Molecular Biology Institute of Barcelona
Nucleic Acids Research | Year: 2012

The extent to which the DNA relaxation activities of eukaryotic topoisomerases (topo I and topo II) are redundant during gene transcription is unclear. Although both enzymes can often substitute for each other in vivo, studies in vitro had revealed that the DNA cross-inversion mechanism of topo II relaxes chromatin more efficiently than the DNA strand-rotation mechanism of topo I. Here, we show that the inactivation of topo II in budding yeast produces an abrupt decrease of virtually all polyA+ RNA transcripts of length above ∼3 kb, irrespective of their function. This reduction is not related to transcription initiation but to the stall of RNA polymerase II (Pol II) during elongation. This reduction does not occur in topo I mutants; and it is not avoided by overproducing yeast topo I or bacterial topo I, which relaxes (-) DNA supercoils. It is rescued by catalytically active topo II or a GyrBA enzyme, which relaxes (+) DNA supercoils. These findings demonstrate that DNA relaxation activities of topo I and topo II are not interchangeable in vivo. Apparently, only topo II relaxes efficiently the (+) DNA supercoils that stall the advancement of Pol II in long genes. A mechanistic model is proposed. © 2012 The Author(s).


Tallant C.,Molecular Biology Institute of Barcelona | Garcia-Castellanos R.,Molecular Biology Institute of Barcelona | Garcia-Castellanos R.,Barcelona Institute for Research in Biomedicine | Baumann U.,Molecular Biology Institute of Barcelona | And 2 more authors.
Journal of Biological Chemistry | Year: 2010

The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zinc-binding site, which is structurally and spatially conserved and invariantly shows a methionine at position 3 in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating 10 mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three-dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Gomis-Ruth F.X.,Molecular Biology Institute of Barcelona | Botelho T.O.,Molecular Biology Institute of Barcelona | Bode W.,Max Planck Institute For Biochemie
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2012

Visualization of three-dimensional structures is essential to the transmission of information to the general reader and the comparison of related structures. Therefore, it would be useful to provide a common framework. Based on the work of Schechter and Berger, and the finding that most peptidases bind their substrates in extended conformation, we suggest a "standard orientation" for the overall description of metallopeptidases (MPs) as done before for peptidases of other classes. This entails a frontal view of the horizontally-aligned active-site cleft. A substrate is bound N- to C-terminally from left (on the non-primed side of the cleft) to right (on the primed side), and the catalytic metal ion resides at the cleft bottom at roughly half width. This view enables us to see that most metalloendopeptidases are bifurcated into an upper and a lower sub-domain by the cleft, whose back is framed by a nearly horizontal "active-site helix." The latter comprises a short zinc-binding consensus sequence, either HEXXH or HXXEH, which provides two histidines to bind the single catalytic metal and the general-base/acid glutamate required for catalysis. In addition, an oblique "backing helix" is observed behind the active-site helix, and a mixed β-sheet of at least three strands is positioned in the upper sub-domain paralleling the cleft. The lowermost "upper-rim" strand of the sheet runs antiparallel to the substrate bound in the cleft and therefore contributes both to delimitating the cleft top and to binding of the substrate main-chain on its non-primed side through β-ribbon-like interactions. In contrast, in metalloexopeptidases, which chop off N- or C-terminal residues only, extensive binding on both sides of the cleft is not required and a different overall scaffold is generally observed. This consists of an αβα- sandwich, which is reminiscent of, but clearly distinct from, the archetypal α/β-hydrolase fold. Metalloexopeptidases have their active sites at the C-terminal end of a central, eight-stranded twisted β-sheet, and can contain one or two catalytic metal ions. As the zinc-binding site and the residues engaged in substrate binding and catalysis are mainly provided by loops connecting the β-sheet strands and the helices on either side, the respective standard orientations vary with respect to the position of the sheets. The standard orientation of eight prototypic MP structures is presented and discussed. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. © 2011 Elsevier B.V. All rights reserved.


Tallant C.,Molecular Biology Institute of Barcelona | Marrero A.,Molecular Biology Institute of Barcelona | Gomis-Ruth F.X.,Molecular Biology Institute of Barcelona
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2010

Matrix metalloproteinases (MMPs) are zinc-dependent protein and peptide hydrolases. They have been almost exclusively studied in vertebrates and 23 paralogs are present in humans. They are widely involved in metabolism regulation through both extensive protein degradation and selective peptide-bond hydrolysis. If MMPs are not subjected to exquisite spatial and temporal control, they become destructive, which can lead to pathologies such as arthritis, inflammation, and cancer. The main therapeutic strategy to combat the dysregulation of MMPs is the design of drugs to target their catalytic domains, for which purpose detailed structural knowledge is essential. The catalytic domains of 13 MMPs have been structurally analyzed so far and they belong to the "metzincin" clan of metalloendopeptidases. These compact, spherical, ~165-residue molecules are divided by a shallow substrate-binding crevice into an upper and a lower sub-domain. The molecules have an extended zinc-binding motif, HEXXHXXGXXH, which contains three zinc-binding histidines and a glutamate that acts as a general base/acid during catalysis. In addition, a conserved methionine lying within a "Met-turn" provides a hydrophobic base for the zinc-binding site. Further earmarks of MMPs are three α-helices and a five-stranded β-sheet, as well as at least two calcium sites and a second zinc site with structural functions. Most MMPs are secreted as inactive zymogens with an N-terminal ~80-residue pro-domain, which folds into a three-helix globular domain and inhibits the catalytic zinc through a cysteine imbedded in a conserved motif, PRCGXPD. Removal of the pro-domain enables access of a catalytic solvent molecule and substrate molecules to the active-site cleft, which harbors a hydrophobic S1&core;-pocket as main determinant of specificity. Together with the catalytic zinc ion, this pocket has been targeted since the onset of drug development against MMPs. However, the inability of first- and second-generation inhibitors to distinguish between different MMPs led to failures in clinical trials. More recent approaches have produced highly specific inhibitors to tackle selected MMPs, thus anticipating the development of more successful drugs in the near future. Further strategies should include the detailed structural characterization of the remaining ten MMPs to assist in achieving higher drug selectivity. In this review, we discuss the general architecture of MMP catalytic domains and its implication in function, zymogenic activation, and drug design. © 2009 Elsevier B.V.


Gomis-Ruth F.X.,Molecular Biology Institute of Barcelona | Trillo-Muyo S.,Molecular Biology Institute of Barcelona | Stocker W.,Matrix
Biological Chemistry | Year: 2012

The astacins are a family of multi-domain metallopeptidases with manifold functions in metabolism. They are either secreted or membrane-anchored and are regulated by being synthesized as inactive zymogens and also by colocalizing protein inhibitors. The distinct family members consist of N-terminal signal peptides and pro-segments, zincdependent catalytic domains, further downstream extracellular domains, transmembrane anchors, and cytosolic domains. The catalytic domains of four astacins and the zymogen of one of these have been structurally characterized and shown to comprise compact ~200-residue zinc-dependent moieties divided into an N-terminal and a C-terminal sub-domain by an active-site cleft. Astacins include an extended zinc-binding motif (HEXXHXXGXXH) which includes three metal ligands and groups them into the metzincin clan of metallopeptidases. In mature, unbound astacins, a conserved tyrosine acts as an additional zinc ligand, which is swung out upon substrate or inhibitor binding in a 'tyrosine switch' motion. Other characteristic structural elements of astacin catalytic domains are three large α-helices and a five-stranded β-sheet, as well as two or three disulfi de bonds. The N-terminal pro-segments are variable in length and rather unstructured. They inhibit the catalytic zinc following an 'aspartate-switch' mechanism mediated by an aspartate embedded in a conserved motif (FXGD). Removal of the pro-segment uncovers a deep and extended active-site cleft, which in general shows preference for aspartate residues in the specifi city pocket (S 1′). Furthermore, astacins undergo major rearrangement upon activation within an 'activation domain,' and show a slight hinge movement when binding substrates or inhibitors. In this review, we discuss the overall architecture of astacin catalytic domains and their involvement in function and zymogenic activation. Copyright © 2011-2012 by Walter de Gruyter.


Guevara T.,Molecular Biology Institute of Barcelona | Yiallouros I.,Matrix | Kappelhoff R.,Matrix | Kappelhoff R.,University of British Columbia | And 3 more authors.
Journal of Biological Chemistry | Year: 2010

Proteolysis is regulated by inactive (latent) zymogens, with a prosegment preventing access of substrates to the active-site cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a "cysteine switch" mechanism involves a conserved prosegment motif with a cysteine residue that coordinates the catalytic zinc ion. Another family of metzincins, the astacins, do not possess a cysteine switch, so latency is maintained by other means. We have solved the high resolution crystal structure of proastacin from the European crayfish, Astacus astacus. Its prosegment is the shortest structurally reported for a metallopeptidase, and it has a unique structure. It runs through the active-site cleft in reverse orientation to a genuine substrate. Moreover, a conserved aspartate, projected by a wide loop of the prosegment, coordinates the zinc ion instead of the catalytic solvent molecule found in the mature enzyme. Activation occurs through two-step limited proteolysis and entails major rearrangement of a flexible activation domain, which becomes rigid and creates the base of the substrate-binding cleft. Maturation also requires the newly formed N terminus to be precisely trimmed so that it can participate in a buried solvent-mediated hydrogen-bonding network, which includes an invariant active-site residue. We describe a novel mechanism for latency and activation, which shares some common features both with other metallopeptidases and with serine peptidases. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.


Cerda-Costa N.,Molecular Biology Institute of Barcelona | Gomis-Ruth F.X.,Molecular Biology Institute of Barcelona
Protein Science | Year: 2014

The cleavage of peptide bonds bymetallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in allmajor physiological processes, and so their deregulation leads to diseases ranging fromcancer andmetastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders.MPs cleave their substrateswithout a covalent intermediate in a singlestep reaction involving a solventmolecule, a general base/acid, and a mono- or dinuclear catalyticmetal site.MostmonometallicMPs comprise a short metal-bindingmotif (HEXXH), which includes two metalbinding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe ofMPs. The latter divides mainly into the gluzincin andmetzincin clans. Metzincins consist of globular ~130-270- residue catalytic domains,which are usually preceded byN-terminal pro-segments, typically required for folding and latency maintenance. The catalytic domains are often followed byC-terminal domains for substrate recognition and other protein-protein interactions, anchoring tomembranes, oligomerization, and compartmentalization.Metzincin catalytic domains consist of a structurally conserved N-terminal subdomain spanning a five-stranded b-sheet, a backing helix, and an active-site helix. The latter contains most of the metal-binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C-terminal subdomains are generally shorter, differmore amongmetzincins, and mainly share a conserved loop-the Met-turn-and a C-terminal helix. The accumulated structural data frommore than 300 deposited structures of the 12 currently characterize dmetzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of theirworking mechanisms, and form the basis for the design of novel drugs. © 2013 The Protein Society.

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