A model of a transmembrane drug-efflux pump from Gram-negative bacteria.
TL;DR: Based on homology modelling and molecular docking, it is shown how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.
About: This article is published in FEBS Letters.The article was published on 2004-12-03. It has received 83 citations till now. The article focuses on the topics: Periplasmic space.
Citations
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TL;DR: Given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents less impacted by efflux and in targeting efflux directly with efflux inhibitors.
Abstract: Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.
979 citations
TL;DR: Functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase are presented, representing the phosphoenzyme intermediates associated withCa2+ binding, Ca2- translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively.
Abstract: The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells.
423 citations
Cites background or methods from "A model of a transmembrane drug-eff..."
...coli AcrB:AcrA:TolC efflux complex have been proposed by combining structures of the isolated components (Fernandez-Recio et al., 2004)....
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...open conformation of TolC (Fernandez-Recio et al., 2004) against the X-ray structure of the closed conformation (Koronakis et al....
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...For Figure 6, AcrB was docked manually onto TolC by superimposition of the hypothetical model for the complete effluxpump (Fernandez-Recio et al., 2004) onto this structure....
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...It is also significant that the handedness and magnitude of the rotation observed in the AcrB:YajC complex (Figures 4A and 4B) is consistent with the handedness and magnitude of the rotation suggested from a model of the open conformation of TolC (Fernandez-Recio et al., 2004) when compared with its closed structure (Figure 4D)....
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...For TolC (Figure 4D), the b-barrel domain of the closed X-ray structure (1EK9) was compared with that of the proposedopen conformation, kindly providedby Luisi andcolleagues (Fernandez-Recio et al., 2004), respectively....
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TL;DR: The 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-Nam(pentAPEptide) synthetic fragment of the cell wall is reported and a model for the bacterial cell wall has been proposed.
Abstract: The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples. Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed.
396 citations
TL;DR: In this article, the structure of the periplasmic protein AcrA was shown to have unsuspected conformational flexibility in the alpha-helical hairpin domain, which has potential mechanistic significance in coupling between acrA conformations and TolC channel opening.
276 citations
Additional excerpts
...Interestingly, modeling of just this interaction by docking and energy minimization required a hinge-like rearrangement of the α-helical hairpin domain of AcrA (Fernandez-Recio et al., 2004)....
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TL;DR: A flexible linear topology of the Adaptor allowed a multidomain docking approach to model the transporter–adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins.
Abstract: Bacteria like Escherichia coli and Pseudomonas aeruginosa expel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a β-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous β-roll, β-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter–adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin–TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA3-AcrB3-TolC3 model presents a 610,000-Da, 270-Å-long efflux pump crossing the entire bacterial cell envelope.
274 citations
Cites background or result from "A model of a transmembrane drug-eff..."
...5 differs substantially from previous purely computational models (13, 16, 30), contains complete structures of the 3 components, and is driven, and validated by a substantial number of site-specific in vivo cross-links....
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...loop and AcrB -turn even though it differs from previous models deriving from these data (18, 30)....
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References
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TL;DR: The PROCHECK suite of programs as mentioned in this paper provides a detailed check on the stereochemistry of a protein structure and provides an assessment of the overall quality of the structure as compared with well refined structures of the same resolution.
Abstract: The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures
22,829 citations
"A model of a transmembrane drug-eff..." refers methods in this paper
...After examinations of the alignments, 15 models were generated using MODELLER [30] and these were ranked by analysis of their stereochemistry using PROCHECK [31] and their sequence– environment compatibility using VERIFY 3D [32,33]....
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TL;DR: The MOLSCRIPT program as discussed by the authors produces plots of protein structures using several different kinds of representations, including simple wire models, ball-and-stick models, CPK models and text labels.
Abstract: The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
13,971 citations
"A model of a transmembrane drug-eff..." refers methods in this paper
...All figures have been prepared using the programs MOLSCRIPT [34] and rendered using Raster3D [35]....
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TL;DR: Raster3D is discussed, which is a suite of programs for molecular graphics, which must compromise the quality of rendered images to achieve rendering speeds high enough for useful interactive manipulation of three-dimensional objects.
Abstract: Publisher Summary This chapter discusses Raster3D, which is a suite of programs for molecular graphics. Crystallographers were among the first and most avid consumers of graphics workstations. Rapid advances in computer hardware, and particularly in the power of specialized computer graphics boards, have led to successive generations of personal workstations with ever more impressive capabilities for interactive molecular graphics. For many years, it was standard practice in crystallography laboratories to prepare figures by photographing directly from the workstation screen. No matter how beautiful the image on the screen, however, this approach suffers from several intrinsic limitations. Among these is the inherent limitation imposed by the effective resolution of the screen. Use of the graphics hardware in a workstation to generate images for later presentation can also impose other limitations. Designers of workstation hardware must compromise the quality of rendered images to achieve rendering speeds high enough for useful interactive manipulation of three-dimensional objects.
3,735 citations
"A model of a transmembrane drug-eff..." refers methods in this paper
...All figures have been prepared using the programs MOLSCRIPT [34] and rendered using Raster3D [35]....
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TL;DR: It is shown that an effective test of the accuracy of a 3D protein model is a comparison of the model to its own amino-acid sequence, using a3D profile, computed from the atomic coordinates of the structure 3D profiles of correct protein structures match their own sequences with high scores, in contrast,3D profiles for protein models known to be wrong score poorly.
Abstract: As methods for determining protein three-dimensional (3D) structure develop, a continuing problem is how to verify that the final protein model is correct. The revision of several protein models to correct errors has prompted the development of new criteria for judging the validity of X-ray and NMR structures, as well as the formation of energetic and empirical methods to evaluate the correctness of protein models. The challenge is to distinguish between a mistraced or wrongly folded model, and one that is basically correct, but not adequately refined. We show that an effective test of the accuracy of a 3D protein model is a comparison of the model to its own amino-acid sequence, using a 3D profile, computed from the atomic coordinates of the structure 3D profiles of correct protein structures match their own sequences with high scores. In contrast, 3D profiles for protein models known to be wrong score poorly. An incorrectly modelled segment in an otherwise correct structure can be identified by examining the profile score in a moving-window scan. The accuracy of a protein model can be assessed by its 3D profile, regardless of whether the model has been derived by X-ray, NMR or computational procedures.
3,213 citations
"A model of a transmembrane drug-eff..." refers methods in this paper
...After examinations of the alignments, 15 models were generated using MODELLER [30] and these were ranked by analysis of their stereochemistry using PROCHECK [31] and their sequence– environment compatibility using VERIFY 3D [32,33]....
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TL;DR: Three-dimensional profiles computed from correct protein structures match their own sequences with high scores, and can be verified by its 3D profile, regardless of whether the model has been derived by X-ray, nuclear magnetic resonance (NMR), or computational procedures.
Abstract: Publisher Summary The three-dimensional (3D) profile of a protein structure is a table computed from the atomic coordinates of the structure that can be used to score the compatibility of the 3D structure model with any amino acid sequence. Three-dimensional profiles computed from correct protein structures match their own sequences with high scores. An incorrectly modeled segment in an otherwise correct structure can be identified by examining the profile score in a moving-window scan. Thus, the correctness of a protein model can be verified by its 3D profile, regardless of whether the model has been derived by X-ray, nuclear magnetic resonance (NMR), or computational procedures. For this reason, 3D profiles are useful in the evaluation of undetermined protein models, based on low-resolution electron-density maps, on NMR spectra with inadequate distance constraints, or on computational procedures. An advantage of using 3D profiles for testing models is that profiles have not themselves been used in the determination of the structure. Traditional R-factor tests in X-ray analysis depend on the comparison of observed properties—that is, the X-ray structure factor magnitudes with the same property calculated from the final protein model.
1,851 citations
"A model of a transmembrane drug-eff..." refers methods in this paper
...After examinations of the alignments, 15 models were generated using MODELLER [30] and these were ranked by analysis of their stereochemistry using PROCHECK [31] and their sequence– environment compatibility using VERIFY 3D [32,33]....
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