Crystal structure analyses of uncomplexed ecotin in two crystal forms: Implications for its function and stability

Shin, Dongkun; Song, Hyun Kyu; Seong, Ihn Sik; Lee, Cheol Soon; Chung, Chin Ha; Suh, Se Won

doi:10.1002/pro.5560051110

Cited by 28 publications

(24 citation statements)

References 56 publications

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“…This pan-specificity of inhibition is due to formation of a heterotetrameric complex with target proteases. In a complex each ecotin monomer in the homodimer binds a protease via primary and secondary interactions [25–27]. The primary binding site, a protruding surface loop, interacts with the active cleft of a protease in a substrate-like manner typical for canonical inhibitors using the Laskowski mechanism of inhibition.…”

Section: - Characterization Of Prokaryote-derived Protein Inhibitmentioning

confidence: 99%

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Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

Kantyka¹,

Rawlings²,

Potempa³

2010

Biochimie

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In metazoan organisms protein inhibitors of peptidases are important factors essential for regulation of proteolytic activity. In vertebrates genes encoding peptidase inhibitors constitute up to 1% of genes reflecting a need for tight and specific control of proteolysis especially in extracellular body fluids. In stark contrast unicellular organisms, both prokaryotic and eukaryotic consistently contain only few, if any, genes coding for putative peptidase inhibitors. This may seem perplexing in the light of the fact that these organisms produce large numbers of proteases of different catalytic classes with the genes constituting up to 6% of the total gene count with the average being about 3%. Apparently, however, a unicellular life-style is fully compatible with other mechanisms of regulation of proteolysis and does not require protein inhibitors to control their intracellular and extracellular proteolytic activity. So in prokaryotes occurrence of genes encoding different types of peptidase inhibitors is infrequent and often scattered among phylogenetically distinct orders or even phyla of microbiota. Genes encoding proteins homologous to alpha-2-macroglobulin (family I39), serine carboxypeptidase Y inhibitor (family I51), alpha-1-peptidase inhibitor (family I4) and ecotin (family I11) are the most frequently represented in Bacteria. Although several of these gene products were shown to possess inhibitory activity, with an exception of ecotin and staphostatins, the biological function of microbial inhibitors is unclear. In this review we present distribution of protein inhibitors from different families among prokaryotes, describe their mode of action and hypothesize on their role in microbial physiology and interactions with hosts and environment. KeywordsBacteria; Archaea; proteolytic enzymes; proteolysis; regulation I -Overview of mechanisms regulating proteolysisRegardless of the complexity of the organism, peptidases in general are essential at every stage in the life of every individual cell. Apart from non-specific protein degradation, peptidases are involved in highly sophisticated essential biological processes, both in prokaryotic andCorresponding author: Jan Potempa, PhD, DSc, University of Louisville School of Dentistry\, Oral Health and Systemic Disease, Louisville, KY, USA, Fax: (+1) 502-852-5572, jspote01@louisville.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBiochimie. Author manuscript; available in PMC 2011 November 1. Published in final edited form as:Biochimie. 2010 November ; 92(11): 1644-1656. doi:...

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Section: - Characterization Of Prokaryote-derived Protein Inhibitmentioning

confidence: 99%

“…The secondary interaction site does show only minor alterations. Structures were obtained from the RCSB database (www.pdb.org) with accesion codes 1N8O [132] (chymotrypsin-ecotin complex) and 1ECY (free ecotin) [133]. …”

Section: Figures and Tablementioning

confidence: 99%

Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

Kantyka¹,

Rawlings²,

Potempa³

2010

Biochimie

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“…Numbering is according to E. coli ecotin. Secondary structure is shown above the sequence and was taken from the PDB file of uncomplexed ecotin, 1ECY[70]; arrows represent β-sheets and loops represent helices. The four main protease contact loops are labelled, and the P1 position (the primary specificity determinant) is designated by a downward arrow.…”

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confidence: 99%

The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase

Eggers

Murray

Delmar

et al. 2004

Biochemical Journal

102

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Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 microM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.

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“…Here, we report results obtained for the protein homodimer ecotin, a serine proteases inhibitor expressed by E. coli [10]. Each monomer is comprised of 142 amino acids arranged as an antiparallel seven-stranded ␤-barrel (Figure 1) [11]. The ecotin dimer has a dissociation constant of approximately 400 nM [12] and has been shown to retain its inhibitory activity after exposure to high temperatures (100°C) and highly acidic conditions (pH 1) [9, 13].…”

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Thermal dissociation of the protein homodimer ecotin in the gas phase

Felitsyn

Kitova

Klassen

2002

J. Am. Soc. Mass Spectrom.

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The influence of charge on the thermal dissociation of gaseous, protonated, homodimeric, protein ecotin ions produced by nanoflow electrospray ionization (nanoES) was investigated using the blackbody infrared radiative dissociation technique. Dissociation of the protonated dimer, (E 2 ϩ nH) nϩ § E 2 nϩ where n ϭ 14 -17, into pairs of monomer ions is the dominant reaction at temperatures from 126 to 175°C. The monomer pair corresponding to the most symmetric charge distribution is preferred, although 50 -60% of the monomer product ions correspond to an asymmetric partitioning of charge. The relative abundance of the different monomer ion pairs produced from E 2 14ϩ , E 2 15ϩ , and E 2 16ϩ depends on reaction time, with the more symmetric charge distribution pair dominating at longer times. The relative yield of monomer ions observed late in the reaction is independent of temperature indicating that proton transfer between the monomers does not occur during dissociation and that the different monomer ion pairs are formed from dimer ions which differ in the distribution of charge between the monomers. For E 2 17ϩ , the yield of monomer ions is independent of reaction time but does exhibit slight temperature dependence, with higher temperatures favoring the monomers corresponding to most symmetric charge distribution. The charge distribution in the E 2 15ϩ and E 2 16ϩ dimer ions influences the dissociation kinetics, with the more asymmetric distribution resulting in greater reactivity. In contrast, the charge distribution has no measurable effect on the dissociation kinetics and energetics of the E 2 17ϩ dimer. (J Am Soc Mass Spectrom 2002, 13, 1432-1442) © 2002 American Society for Mass Spectrometry P rotein complexes composed of two or more subunits are believed to constitute the bulk of soluble and membrane-bound proteins [1,2]. While the importance of protein assemblies in cellular function is well recognized, their structural characterization remains a significant analytical challenge. Mass spectrometry (MS), with its speed, sensitivity, and accurate mass capability holds tremendous potential as a tool for characterizing protein assemblies [3]. The use of multiple stages of MS with ion activation/dissociation (MS n ) to dissect gaseous assemblies into their constituent subunits represents an attractive, yet unproven, strategy for determining subunit composition and topology. In recent years, a number of MS n studies of gaseous protein dimers [4,5], tetramers [6], and pentamers [7] have been reported. Homo-and heterodimers are readily decomposed into their individual subunits, most generally by collision-induced dissociation (CID). An interesting feature of the dissociation behavior of the protein-protein complexes is the tendency for uneven sharing of charge between subunits, even in the case of homodimers. For example, the decomposition of four homodimers (human galectin I, E. coli glyoxalase I, horse heart cytochrome c, and hen egg lysozyme) yields a highly asymmetric charge distribution, with one of the subu...

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Crystal structure analyses of uncomplexed ecotin in two crystal forms: Implications for its function and stability

Cited by 28 publications

References 56 publications

Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

Prokaryote-derived protein inhibitors of peptidases: A sketchy occurrence and mostly unknown function

The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase

Thermal dissociation of the protein homodimer ecotin in the gas phase

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