scholarly journals Peptides in BioNMR Research

2021 ◽  
Vol 75 (6) ◽  
pp. 505-507
Author(s):  
Oliver Zerbe ◽  
Christian Baumann ◽  
Matthias Schuster ◽  
Kerstin Moehle ◽  
Kathryn K. Oi ◽  
...  
Keyword(s):  
Adrenergic Receptor ◽  
Heteronuclear Nmr ◽  
Structural Data ◽  
Protein Assembly ◽  
Allosteric Modulator ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Isotope Labelling ◽  
Therapeutic Drugs ◽  
G Protein Coupled

Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ρ-TIA to the human G-protein coupled α1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics.

scholarly journals The gain-of-function allelebamAE470Kbypasses the essential requirement for BamD in β-barrel outer membrane protein assembly

2020 ◽  
Vol 117 (31) ◽  
pp. 18737-18743 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Meera Gupta ◽  
Martin Wühr ◽  
Thomas J. Silhavy
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Protein Assembly ◽  
Gram Negative Bacteria ◽  
Gain Of Function ◽  
Essential Requirement ◽  
Gram Negative ◽  
Selective Permeability ◽  
Catalytic Role ◽  
Global Defect

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral β-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the β-barrel assembly machine (BAM), which is composed of one OMP—BamA—and four lipoproteins—BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation,bamAE470K, that bypasses the requirement for BamD. AlthoughbamD::kanbamAE470Kcells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.

scholarly journals Membrane Protein Complex ExbB4-ExbD1-TonB1from Escherichia coli Demonstrates Conformational Plasticity

2015 ◽  
Vol 197 (11) ◽  
pp. 1873-1885 ◽  
Author(s):  
Aleksandr Sverzhinsky ◽  
Jacqueline W. Chung ◽  
Justin C. Deme ◽  
Lucien Fabre ◽  
Kristian T. Levey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Acquisition ◽  
Proton Translocation ◽  
Three Dimensional ◽  
Structural Data ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type

ABSTRACTIron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-taggedexbB-exbDand S-taggedtonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB fromEscherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstratein vitroevidence of ExbB4-ExbD1-TonB1complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3982-3991 ◽  
Author(s):  
Ana Sauri ◽  
Zora Soprova ◽  
David Wickström ◽  
Jan-Willem de Gier ◽  
Roel C. Van der Schors ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Critical Role ◽  
Structural Data ◽  
Intermediate Stage ◽  
Gram Negative Bacteria ◽  
Passenger Domain ◽  
Gram Negative ◽  
Periplasmic Chaperone ◽  
Folded Structures

Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms a β-barrel pore in the outer membrane implicated in translocation of the upstream ‘passenger’ domain across the outer membrane. However, recent structural data suggest that the diameter of the β-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage the β-domain was not inserted and folded as an integral β-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Bam (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane.

scholarly journals Mechanism of β2AR regulation by an intracellular positive allosteric modulator

Science ◽  
2019 ◽  
Vol 364 (6447) ◽  
pp. 1283-1287 ◽  
Author(s):  
Xiangyu Liu ◽  
Ali Masoudi ◽  
Alem W. Kahsai ◽  
Li-Yin Huang ◽  
Biswaranjan Pani ◽  
...  
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Helical Conformation ◽  
Allosteric Modulator ◽  
Intracellular Loop ◽  
Positive Allosteric Modulator ◽  
Transmembrane Segments ◽  
Allosteric Binding Sites ◽  
Loop 2 ◽  
G Protein Coupled

Drugs targeting the orthosteric, primary binding site of G protein–coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β2- over the β1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.

scholarly journals Outer membrane lipid homeostasis via retrograde phospholipid transport inEscherichia coli

2017 ◽  
Author(s):  
Rahul Shrivastava ◽  
Xiang’Er Jiang ◽  
Shu-Sin Chng
Keyword(s):  
Outer Membrane ◽  
Physiological Role ◽  
Protein Assembly ◽  
Membrane Lipid ◽  
Lipid Homeostasis ◽  
Gram Negative Bacteria ◽  
Inescherichia Coli ◽  
Lipid Asymmetry ◽  
Gram Negative ◽  
Phospholipid Transport

SummaryBiogenesis of the outer membrane (OM) in Gram-negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well-studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans-envelope Tol-Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol-Pal complex is required for PL transport and OM lipid homeostasis inEscherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess phospholipids (PLs) in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol-Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol-Pal complex may promote OM invagination during cell division.

scholarly journals Salt-Resistant Alpha-Helical Cationic Antimicrobial Peptides

1999 ◽  
Vol 43 (7) ◽  
pp. 1542-1548 ◽  
Author(s):  
Carol Friedrich ◽  
Monisha G. Scott ◽  
Nedra Karunaratne ◽  
Hong Yan ◽  
Robert E. W. Hancock
Keyword(s):  
Cytoplasmic Membrane ◽  
Salt Resistance ◽  
Tryptophan Residue ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Therapeutic Drugs ◽  
C Terminus ◽  
Helical Content ◽  
Charged Derivative ◽  
Positively Charged

ABSTRACT Analogues based on the insect cecropin–bee melittin hybrid peptide (CEME) were studied and analyzed for activity and salt resistance. The new variants were designed to have an increase in amphipathic α-helical content (CP29 and CP26) and in overall positive charge (CP26). The α-helicity of these peptides was demonstrated by circular dichroism spectroscopy in the presence of liposomes. CP29 was shown to have activity against gram-negative bacteria that was similar to or better than those of the parent peptides, and CP26 had similar activity. CP29 had cytoplasmic membrane permeabilization activity, as assessed by the unmasking of cytoplasmic β-galactosidase, similar to that of CEME and its more positively charged derivative named CEMA, whereas CP26 was substantially less effective. The activity of the peptides was not greatly attenuated by an uncoupler of membrane potential, carbonyl cyanide-m-chlorophenylhydrazone. The tryptophan residue in position 2 was shown to be necessary for interaction with cell membranes, as demonstrated by a complete lack of activity in the peptide CP208. Peptides CP29, CEME, and CEMA were resistant to antagonism by 0.1 to 0.3 M NaCl; however, CP26 was resistant to antagonism only by up to 160 mM NaCl. The peptides were generally more antagonized by 3 and 5 mM Mg2+ and by the polyanion alginate. It appeared that the positively charged C terminus in CP26 altered its ability to permeabilize the cytoplasmic membrane of Escherichia coli, although CP26 maintained its ability to kill gram-negative bacteria. These peptides are potential candidates for future therapeutic drugs.

scholarly journals Determination of the molecular basis for coprogen import by Gram-negative bacteria

IUCrJ ◽  
2019 ◽  
Vol 6 (3) ◽  
pp. 401-411 ◽  
Author(s):  
Rhys Grinter ◽  
Trevor Lithgow
Keyword(s):  
Outer Membrane ◽  
Molecular Basis ◽  
Structural Data ◽  
Structural Basis ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Highly Active ◽  
Hydroxamate Siderophores ◽  
Membrane Barrier

In order to survive in mixed microbial communities, some species of fungi secrete coprogens, siderophores that facilitate capture of the scarce nutrient iron. The TonB-dependent transporter FhuE is integrated in the outer membrane of Gram-negative bacteria and has been reported to scavenge these fungally produced coprogens. In this work, anEscherichia colistrain was engineered that is dependent solely on FhuE for its access to siderophore-sequestered iron. Using this tool, it is shown that while FhuE is highly active in the import of coprogens, it has some level of promiscuity, acting as a low-affinity transporter for related siderophores. The crystal structure of FhuE in complex with coprogen was determined, providing a structural basis to explain this selective promiscuity. The structural data, in combination with functional analysis, presented in this work show that FhuE has evolved to specifically engage with planar siderophores. A potential evolutionary driver, and a critical consequence of this selectivity, is that it allows FhuE to exclude antibiotics that mimic nonplanar hydroxamate siderophores: these toxic molecules could otherwise cross the outer membrane barrier through a Trojan horse mechanism.

Bacterial-Mucosal Cell Junctional Complexes

1974 ◽  
Vol 32 ◽  
pp. 144-145
Author(s):  
Roger C. Wagner
Keyword(s):  
Intestinal Wall ◽  
Rat Colon ◽  
Mucosal Cell ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Mucosal Cells ◽  
Mucosal Lining ◽  
Degenerative Alterations ◽  
Junctional Complexes ◽  
Intestinal Mucosal Cells

Bacteria exhibit the ability to adhere to the apical surfaces of intestinal mucosal cells. These attachments either precede invasion of the intestinal wall by the bacteria with accompanying inflammation and degeneration of the mucosa or represent permanent anchoring sites where the bacteria never totally penetrate the mucosal cells.Endemic gram negative bacteria were found attached to the surface of mucosal cells lining the walls of crypts in the rat colon. The bacteria did not intrude deeper than 0.5 urn into the mucosal cells and no degenerative alterations were detectable in the mucosal lining.

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