scholarly journals Prediction of the closed conformation and insights into the mechanism of the membrane enzyme LpxR

2018 ◽  
Author(s):  
Graham M Saunders ◽  
Hannah E Bruce Macdonald ◽  
Jonathan W Essex ◽  
Syma Khalid
Keyword(s):  
Outer Membrane ◽  
Membrane Lipids ◽  
Conformational Dynamics ◽  
Covalent Modification ◽  
Conserved Residues ◽  
Free Binding Energy ◽  
Scissile Bond ◽  
Membrane Models ◽  
Single Water Molecule ◽  
Key Residues

ABSTRACTCovalent modification of outer membrane lipids of Gram-negative bacteria can impact the ability of the bacterium to develop resistance to antibiotics as well as modulating the immune response of the host. The enzyme LpxR from Salmonella typhimurium is known to deacylate lipopolysaccharide molecules of the outer membrane, however the mechanism of action is unknown. Here we employ Molecular Dynamics and Monte Carlo simulations to study the conformational dynamics and substrate binding of LpxR in representative outer membrane models and also detergent micelles. We examine the roles of conserved residues and provide an understanding of how LpxR binds its substrate. Our simulations predict that the catalytic H122 must be Nε-protonated for a single water molecule to occupy the space between it and the scissile bond, with a free binding energy of -8.5 kcal mol-1. Furthermore, simulations of the protein within a micelle enable us to predict the structure of the putative ‘closed’ protein. Our results highlight the need for including dynamics, a representative environment and the consideration of multiple tautomeric and rotameric states of key residues in mechanistic studies; static structures alone do not tell the full story.

scholarly journals Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation

2008 ◽  
Vol 69 (2) ◽  
pp. 491-502 ◽  
Author(s):  
Lauren Mashburn-Warren ◽  
Jörg Howe ◽  
Patrick Garidel ◽  
Walter Richter ◽  
Frank Steiniger ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Lipids ◽  
Membrane Vesicle ◽  
Vesicle Formation

scholarly journals Lipid Headgroup Charge and Acyl Chain Composition Modulate Closure of Bacterial β-Barrel Channels

2019 ◽  
Vol 20 (3) ◽  
pp. 674 ◽  
Author(s):  
D. Perini ◽  
Antonio Alcaraz ◽  
María Queralt-Martín
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Protein Interactions ◽  
Membrane Lipids ◽  
Acyl Chain ◽  
Extensive Study ◽  
Electrophysiological Recordings ◽  
Sequential Closure ◽  
Lipid Protein Interactions ◽  
Outer Membrane Permeability

The outer membrane of Gram-negative bacteria contains β-barrel proteins that form high-conducting ion channels providing a path for hydrophilic molecules, including antibiotics. Traditionally, these proteins have been considered to exist only in an open state so that regulation of outer membrane permeability was accomplished via protein expression. However, electrophysiological recordings show that β-barrel channels respond to transmembrane voltages by characteristically switching from a high-conducting, open state, to a so-called ‘closed’ state, with reduced permeability and possibly exclusion of large metabolites. Here, we use the bacterial porin OmpF from E. coli as a model system to gain insight on the control of outer membrane permeability by bacterial porins through the modulation of their open state. Using planar bilayer electrophysiology, we perform an extensive study of the role of membrane lipids in the OmpF channel closure by voltage. We pay attention not only to the effects of charges in the hydrophilic lipid heads but also to the contribution of the hydrophobic tails in the lipid-protein interactions. Our results show that gating kinetics is governed by lipid characteristics so that each stage of a sequential closure is different from the previous one, probably because of intra- or intermonomeric rearrangements.

scholarly journals Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

Langmuir ◽  
2014 ◽  
Vol 31 (1) ◽  
pp. 404-412 ◽  
Author(s):  
Luke A. Clifton ◽  
Maximilian W. A. Skoda ◽  
Anton P. Le Brun ◽  
Filip Ciesielski ◽  
Ivan Kuzmenko ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Divalent Cation ◽  
Gram Negative ◽  
Bacterial Outer Membrane ◽  
Membrane Models

scholarly journals SalmonellaTol-Pal Reduces Outer Membrane Glycerophospholipid Levels for Envelope Homeostasis and Survival during Bacteremia

2018 ◽  
Vol 86 (7) ◽  
Author(s):  
Revathi Masilamani ◽  
Melina B. Cian ◽  
Zachary D. Dalebroux
Keyword(s):  
Outer Membrane ◽  
Membrane Lipids ◽  
Lipid Homeostasis ◽  
Content Type ◽  
Bacterial Morphology ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Rifampin Resistance ◽  
Mutant Phenotypes ◽  
Acyl Coenzyme A

ABSTRACTSalmonellae regulate membrane lipids during infection, but the exact proteins and mechanisms that promote their survival during bacteremia remain largely unknown. Mutations in genes encoding the conservedSalmonella entericaserovar Typhimurium (S. Typhimurium) Tol-Pal apparatus caused the outer membrane (OM) sensor lipoprotein, RcsF, to become activated. The capsule activation phenotype for the mutants suggested that Tol-Pal might influence envelope lipid homeostasis. The mechanism involves reducing OM glycerophospholipid (GPL) levels, since the mutant salmonellae similarly accumulated phosphatidylglycerols (PGl) and phosphatidylethanolamines (PE) within the OM in comparison to the wild type. The data support theEscherichia colimodel, whereby Tol-Pal directs retrograde GPL translocation across the periplasm. TheS. Typhimurium mechanism involves contributions from YbgC, a cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase, and CpoB, a periplasmic TolA-binding protein. The functional relationship between Tol-Pal and YbgC and CpoB was previously unresolved. TheS. Typhimurium Tol-Pal proteins contribute similarly toward promoting OM-GPL homeostasis and Rcs signaling inactivity but differently toward promoting bacterial morphology, rifampin resistance, survival in macrophages, and survival in mice. For example,tolQ,tolR,tolA, andcpoBmutants were significantly more attenuated thanybgC,tolB, andpalmutants in a systemic mouse model of disease. Therefore, key roles exist for TolQ, TolR, TolA, and CpoB during murine bacteremia, which are independent of maintaining GPL homeostasis. The ability of TolQR to channel protons across the inner membrane (IM) is necessary forS. Typhimurium TolQRA function, since mutating conserved channel-facing residues rendered TolQ ineffective at rescuing deletion mutant phenotypes. Therefore, Tol-Pal promotesS. Typhimurium survival during bacteremia, in part, by reducing OM GPL concentrations, while TolQRA and CpoB enhance systemic virulence by additional mechanisms.

Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

scholarly journals Importance of Conserved Residues of the Serine Protease Autotransporter β-Domain in Passenger Domain Processing and β-Barrel Assembly

2010 ◽  
Vol 78 (8) ◽  
pp. 3516-3528 ◽  
Author(s):  
Yihfen T. Yen ◽  
Casey Tsang ◽  
Todd A. Cameron ◽  
Dennis O. Ankrah ◽  
Athina Rodou ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Serine Protease ◽  
Cell Fractionation ◽  
Passenger Domain ◽  
Double Mutation ◽  
Conserved Residues ◽  
Virulence Proteins ◽  
Secretion Pathway ◽  
Residue Substitution ◽  
Cellular Compartments

ABSTRACT Serine protease autotransporters of the family Enterobacteriaceae (SPATE) comprise a family of virulence proteins secreted by enteric Gram-negative bacteria via the autotransporter secretion pathway. A SPATE polypeptide contains a C-terminal translocator domain that inserts into the bacterial outer membrane as a β-barrel structure and mediates secretion of the passenger domain to the extracellular environment. In the present study, we examined the role of conserved residues located in the SPATE β-barrel-forming region in passenger domain secretion. Thirty-nine fully conserved residues in Tsh were mutated by single-residue substitution, and defects in their secretion phenotypes were assessed by cell fractionation and immunochemistry. A total of 22 single mutants exhibited abnormal phenotypes in different cellular compartments. Most mutants affecting secretion are charged residues with side chains pointing into the β-barrel interior. Seven mutants showed notable abnormalities in processing (constructs with the E1231A, E1249A, and R1374A mutations) and β-barrel assembly or insertion into the outer membrane (constructs with the G1158Y, F1360A, Y1375A, and F1377A mutations). The phenotypes of the β-barrel assembly/insertion mutants and the presence of a processed Tsh passenger domain in the periplasm support the possibility that the translocator domain must undergo extensive folding prior to insertion into the outer membrane. Results from double-mutation experiments further demonstrate that F1360 and F1377 affect β-barrel insertion/assembly at different times. In light of these new data, a more refined model for the mechanism of SPATE secretion is presented.

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