scholarly journals Structure of protease-cleavedEscherichia coliα-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

2015 ◽  
Vol 71 (7) ◽  
pp. 1478-1486 ◽  
Author(s):  
Cameron D. Fyfe ◽  
Rhys Grinter ◽  
Inokentijs Josts ◽  
Khedidja Mosbahi ◽  
Aleksander W. Roszak ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Change ◽  
Domain Architecture ◽  
Activation Mechanism ◽  
Protease Inhibition ◽  
Intrinsically Disordered ◽  
Bait Region ◽  
Thioester Bond ◽  
Interdomain Interactions ◽  
Putative Mechanism

Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleavedEscherichia coliα-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintainE. coliα-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture ofEscherichia coliα-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

scholarly journals The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

1987 ◽  
Vol 243 (1) ◽  
pp. 47-54 ◽  
Author(s):  
L J Larsson ◽  
P Lindahl ◽  
C Hallén-Sandgren ◽  
I Björk
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Cross Linking ◽  
Bait Region ◽  
Close Proximity ◽  
Tryptophan Residues ◽  
Thioester Bond ◽  
Bond Region

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent ‘bait’ region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

scholarly journals Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition

2015 ◽  
Vol 112 (27) ◽  
pp. 8290-8295 ◽  
Author(s):  
Irene Garcia-Ferrer ◽  
Pedro Arêde ◽  
Josué Gómez-Blanco ◽  
Daniel Luque ◽  
Stephane Duquerroy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Large Cell ◽  
Structural Rearrangement ◽  
Gram Negative Bacteria ◽  
Human Gut ◽  
Bait Region ◽  
Α2 Macroglobulin ◽  
Thioester Bond ◽  
Peptidase Inhibitor

The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric “snap trap.”

scholarly journals Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Michael Puljung ◽  
Natascia Vedovato ◽  
Samuel Usher ◽  
Frances Ashcroft
Keyword(s):  
Conformational Change ◽  
Ionic Currents ◽  
Nucleotide Binding ◽  
Activation Mechanism ◽  
Time Resolved ◽  
K Channels ◽  
Novel Approach ◽  
Binding Event ◽  
Fluorescent Nucleotides

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

scholarly journals Autotransporters of Escherichia coli: a sequence-based characterization

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2459-2469 ◽  
Author(s):  
Timothy J. Wells ◽  
Makrina Totsika ◽  
Mark A. Schembri
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Domain Architecture ◽  
Bioinformatic Analysis ◽  
Tissue Tropism ◽  
Genome Sequences ◽  
E Coli ◽  
Disease Outcomes ◽  
High Degree ◽  
Encoding Genes

Autotransporter (AT) proteins are found in all Escherichia coli pathotypes and are often associated with virulence. In this study we took advantage of the large number of available E. coli genome sequences to perform an in-depth bioinformatic analysis of AT-encoding genes. Twenty-eight E. coli genome sequences were probed using an iterative approach, which revealed a total of 215 AT-encoding sequences that represented three major groups of distinct domain architecture: (i) serine protease AT proteins, (ii) trimeric AT adhesins and (iii) AIDA-I-type AT proteins. A number of subgroups were identified within each broad category, and most subgroups contained at least one characterized AT protein; however, seven subgroups contained no previously described proteins. The AIDA-I-type AT proteins represented the largest and most diverse group, with up to 16 subgroups identified from sequence-based comparisons. Nine of the AIDA-I-type AT protein subgroups contained at least one protein that possessed functional properties associated with aggregation and/or biofilm formation, suggesting a high degree of redundancy for this phenotype. The Ag43, YfaL/EhaC, EhaB/UpaC and UpaG subgroups were found in nearly all E. coli strains. Among the remaining subgroups, there was a tendency for AT proteins to be associated with individual E. coli pathotypes, suggesting that they contribute to tissue tropism or symptoms specific to different disease outcomes.

scholarly journals A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system

FEBS Journal ◽  
2005 ◽  
Vol 272 (11) ◽  
pp. 2773-2783 ◽  
Author(s):  
Tomoaki Kato ◽  
Daizo Hamada ◽  
Takashi Fukui ◽  
Makoto Hayashi ◽  
Takeshi Honda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Change ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Escherichia Coli O157 ◽  
Type Iii ◽  
Ph Dependent

scholarly journals Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 517
Author(s):  
Seigo Usuki ◽  
Yoshiaki Yasutake ◽  
Noriko Tamura ◽  
Tomohiro Tamura ◽  
Kunikazu Tanji ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Intramolecular Interaction ◽  
Protein Secondary Structure ◽  
Potential Interaction ◽  
Activation Mechanism ◽  
Receptor Interaction ◽  
Intrinsically Disordered ◽  
Hydroxyl Fatty Acids

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and α-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of μM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand–receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

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