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BBA - Biomembranes (v.1778, #9)

Editorial Board (pp. ii).

Structural proteomics of the cell envelope of Gram-negative bacteria by D. Peter Tieleman; Raymond J. Turner; Hans J. Vogel; Joel H. Weiner (pp. 1697-1697).

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis by Joel H. Weiner; Liang Li (pp. 1698-1713).

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, β-sheet motifs and stretches of α-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.

Keywords: Abbreviations; 2MEGA; dimethylation after lysine guanidination; ABC; ATP binding cassette; CNBr; cyanogen bromide; ESI; electrospray ionization; ICAT; isotope-coded affinity tag; IEF; isoelectric focusing; IMP; inner membrane protein; LC; liquid chromatography; MAAH; microwave-assisted acid hydrolysis; MALDI-TOF; matrix assisted laser desorption/ionization-time of flight; MS; mass spectrometry; OMP; outer membrane protein; ORF; open reading frame; PAGE; polyacrylamide gel electrophoresis; PMF; peptide mass fingerprinting; SDS; sodium dodecyl sulfate; TFA; trifluoroacetic acid; TMM; transmembraneCytoplasmic membrane; Periplasm; Outer membrane; Mass spectrometry; Proteomics; Polyacrylamide gel electrophoresis

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli by Waldemar Vollmer; Ute Bertsche (pp. 1714-1734).

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.

Keywords: Abbreviations; GlcNAc; N-acetylglucosamine; m-A; ₂; pm; meso-diaminopimelic acid; MurNAc; N-acetylmuramic acid; nPB; non-Penicillin-binding domain; PBP; Penicillin-binding protein; TG; transglycosylase; TP; transpeptidase; UDP; uridyl diphosphateMurein; Peptidoglycan; Sacculus; Penicillin-binding protein; Murein synthases and hydrolases; Bacterial cytoskeleton; Bacterial morphogenesis

Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—Distinct translocases and mechanisms by Paolo Natale; Thomas Brüser; Arnold J.M. Driessen (pp. 1735-1756).

In bacteria, two major pathways exist to secrete proteins across the cytoplasmic membrane. The general Secretion route, termed Sec-pathway, catalyzes the transmembrane translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane. The Twin- arginine translocation pathway, termed Tat-pathway, catalyses the translocation of secretory proteins in their folded state. Although the targeting signals that direct secretory proteins to these pathways show a high degree of similarity, the translocation mechanisms and translocases involved are vastly different.

Keywords: Secretion; SecA; SecY; Twin arginine; Tat

ATP-binding cassette transporters in Escherichia coli by Anastassiia Moussatova; Christian Kandt; Megan L. O'Mara; D. Peter Tieleman (pp. 1757-1771).

ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.

Keywords: ABC transporter; Homology; Periplasmic binding protein; BtuCD; Simulation; Importer; P-glycoprotein

The dynamics of the MBP–MalFGK₂ interaction: A prototype for binding protein dependent ABC-transporter systems by Brian H. Shilton (pp. 1772-1780).

This review is focused on the interaction between maltose binding protein (MBP) and the maltose transporter complex, MalFGK₂, which is a member of the ATP Binding Cassette (ABC) superfamily. The interaction between MBP and MalFGK₂ has a critical role in maltose transport, but a coherent description of the interaction is complicated because both MBP and MalFGK₂ can adopt multiple conformations. Drawing on genetic, structural, and biochemical data, the different conformations of MBP and MalFGK₂ are described and incorporated into a model for their interaction. The most important feature of this model is that ligand-bound MBP initiates the process of ATP-dependent maltose transport by stabilizing a high-energy conformation of MalFGK₂. In this model of the MBP–MalFGK₂ interaction, stabilization of a high-energy conformation of MalFGK₂ allows ATP to drive conformational changes in the system – in particular the opening of bound MBP – that leads to formation of a transition state for ATP hydrolysis. Such a role for ligand-bound MBP explains how MBP-independent MalFGK₂ mutants work, and represents a general mechanism for binding-protein dependent ABC import systems. In ABC export systems, which do not use a binding protein, the substrate itself is expected to play a role similar to ligand-bound MBP in the maltose transport system. The mechanistic model for the maltose transporter suggests that ABC-type import systems evolved to make use of a peripheral binding protein so that the transport process is essentially irreversible.

Keywords: Abbreviations; ABC; ATP binding cassette; ABP; Arabinose binding protein; MalFGK; ₂; Maltose transporter integral membrane complex; MBP; Maltose binding protein; MBPi; MBP-independent; MM; Minimal maltose; PBP; Periplasmic binding protein; PDB; Protein data bank; SCOP; Structural classification of proteinsABC transporter; Maltose binding protein; Chemical–mechanical coupling; Reversibility; Protein–protein interaction; ATP hydrolysis; Transmembrane transport; Binding energy; Transition state stabilization

Structural biology of bacterial iron uptake by Karla D. Krewulak; Hans J. Vogel (pp. 1781-1804).

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.

Keywords: Structure; Iron; Heme; Bacterial iron uptake

A perspective on the structural studies of inner membrane electrochemical potential-driven transporters by M. Joanne Lemieux (pp. 1805-1813).

Electrochemical potential-driven transporters represent a vast array of proteins with varied substrate specificities. While diverse in size and substrate specificity, they are all driven by electrochemical potentials. Over the past five years there have been increasing numbers of X-ray structures reported for this family of transporters. Structural information is available for five subfamilies of electrochemical potential-driven transporters. No structural information exists for the remaining 91 subfamilies. In this review, the various subfamilies of electrochemical potential-driven transporters are discussed. The seven reported structures for the electrochemical potential-driven transporters and the methods for their crystallization are also presented. With a few exceptions, overall crystallization trends have been very similar for the transporters despite their differences in substrate specificity and topology. Also discussed is why the structural studies on these transporters were successful while others are not as fruitful. With the plethora of transporters with unknown structures, this review provides incentive for crystallization of transporters in the remaining subfamilies for which no structural information exists.

Keywords: Structural biology; Membrane protein; Transporter; X-ray crystallography; GlpT; LacY; EmrD; AcrB; Glt; _Ph; LeuT; NhaA; DAACS

Small multidrug resistance proteins: A multidrug transporter family that continues to grow by Denice C. Bay; Kenton L. Rommens; Raymond J. Turner (pp. 1814-1838).

The small multidrug resistance (SMR) protein family is a bacterial multidrug transporter family. As suggested by their title, SMR proteins are composed of four transmembrane α-helices of approximately 100–140 amino acids in length. Since their designation as a family, many homologues have been identified and characterized both structurally and functionally. In this review the topology, structure, drug resistance, drug binding, and transport mechanisms of the entire SMR protein family are examined. Additionally, updated bioinformatic analysis of predicted and characterized SMR protein family members was also conducted. Based on SMR sequence alignments and phylogenetic analysis of current members, we propose that this small multidrug resistance transporter family should be expanded into three subclasses: (i) the small multidrug pumps (SMP), (ii) suppressor of groEL mutation proteins (SUG), and a third group (iii) paired small multidrug resistance proteins (PSMR). The roles of these three SMR subclasses are examined, and the well-characterized members, such as Escherichia coli EmrE and SugE, are described in terms of their function and structural organization.

Keywords: Small multidrug resistance (SMR); EmrE; SugE; Paired SMR; Dual topology; Secondary drug transporter; Drug binding; Phylogenetic tree

Towards a systems biology approach to study type II/IV secretion systems by Bart Hazes; Laura Frost (pp. 1839-1850).

Many gram-negative bacteria produce thin protein filaments, named pili, which extend beyond the confines of the outer membrane. The importance of these pili is illustrated by the fact that highly complex, multi-protein pilus-assembly machines have evolved, not once, but several times. Their many functions include motility, adhesion, secretion, and DNA transfer, all of which can contribute to the virulence of bacterial pathogens or to the spread of virulence factors by horizontal gene transfer. The medical importance has stimulated extensive biochemical and genetic studies but the assembly and function of pili remains an enigma. It is clear that progress in this field requires a more holistic approach where the entire molecular apparatus that forms the pilus is studied as a system. In recent years systems biology approaches have started to complement classical studies of pili and their assembly. Moreover, continued progress in structural biology is building a picture of the components that make up the assembly machine. However, the complexity and multiple-membrane spanning nature of these secretion systems pose formidable technical challenges, and it will require a concerted effort before we can create comprehensive and predictive models of these remarkable molecular machines.

Keywords: Conjugation; Type IV secretion; Type II secretion; Type IV pili; Conjugative pili

Bringing order to a complex molecular machine: The assembly of the bacterial flagella by Dmitry Apel; Michael G. Surette (pp. 1851-1858).

The bacterial flagellum is an example of elegance in molecular engineering. Flagella dependent motility is a widespread and evolutionarily ancient trait. Diverse bacterial species have evolved unique structural adaptations enabling them to migrate in their environmental niche. Variability exists in the number, location and configuration of flagella, and reflects unique adaptations of the microorganism. The most detailed analysis of flagellar morphogenesis and structure has focused on Escherichia coli and Salmonella enterica. The appendage assembles sequentially from the inner to the outer-most structures. Additionally the temporal order of gene expression correlates with the assembly order of encoded proteins into the final structure. The bacterial flagellar apparatus includes an essential basal body complex that comprises the export machinery required for assembly of the hook and flagellar filament. A review outlining the current understanding of the protein interactions that make up this remarkable structure will be presented, and the associated temporal genetic regulation will be briefly discussed.

Keywords: Flagella; Motitlity; Basal body; Chemotaxis

Bacterial mechanosensitive channels: Experiment and theory by Ben Corry; Boris Martinac (pp. 1859-1870).

Since their discovery in Escherichia coli some 20 years ago, studies of bacterial mechanosensitive (MS) ion channels have been at the forefront of the MS channel research field. Two major events greatly advanced the research on bacterial MS channels: (i) cloning of MscL and MscS, the MS channels of Large and Small conductance, and (ii) solving their 3D crystal structure. These events enabled further experimental studies employing EPR and FRET spectroscopy in addition to patch clamp and molecular biological techniques that have successfully been used in characterization of the structure and function of bacterial MS channels. In parallel with the experimental studies computational modelling has been applied to elucidate the molecular dynamics of MscL and MscS, which has significantly contributed to our understanding of basic physical principles of the mechanosensory transduction in living organisms.

Keywords: MS channels; Patch clamp; Bilayer model; Mechanosensory transduction; EPR spectroscopy; FRET; molecular dynamics; Brownian dynamics

OmpA: Gating and dynamics via molecular dynamics simulations by Syma Khalid; Peter J. Bond; Timothy Carpenter; Mark S.P. Sansom (pp. 1871-1880).

Outer membrane proteins (OMPs) of Gram-negative bacteria have a variety of functions including passive transport, active transport, catalysis, pathogenesis and signal transduction. Whilst the structures of ∼25 OMPs are currently known, there is relatively little known about their dynamics in different environments. The outer membrane protein, OmpA from Escherichia coli has been studied extensively in different environments both experimentally and computationally, and thus provides an ideal test case for the study of the dynamics and environmental interactions of outer membrane proteins. We review molecular dynamics simulations of OmpA and its homologues in a variety of different environments and discuss possible mechanisms of pore gating. The transmembrane domain of E. coli OmpA shows subtle differences in dynamics and interactions between a detergent micelle and a lipid bilayer environment. Simulations of the crystallographic unit cell reveal a micelle-like network of detergent molecules interacting with the protein monomers. Simulation and modelling studies emphasise the role of an electrostatic-switch mechanism in the pore-gating mechanism. Simulation studies have been extended to comparative models of OmpA homologues from Pseudomonas aeruginosa (OprF) and Pasteurella multocida (PmOmpA), the latter model including the periplasmic C-terminal domain.

Keywords: Outer membrane protein; OmpA; Molecular dynamics; Homology model

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane by Russell E. Bishop (pp. 1881-1896).

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel β-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three β-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipid∷lipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane β-barrel enzyme PagL, a lipid A 3- O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two β-barrel enzymes of unknown structure; namely, the Salmonella enterica 3′-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O₂ to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how β-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger β-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.

Keywords: Abbreviations; l; -Ara4N; 4-amino-4-deoxy-; l; -arabinose; GroPEtn; glycerophosphoethanolamine; GPL; glycerophospholipid; O; _Ag; O-antigen; PtdEtn; phosphatidylethanolamineOMPLA; OmpT; PagP; PagL; LpxR; LpxQ

The prokaryotic complex iron–sulfur molybdoenzyme family by Richard A. Rothery; Gregory J. Workun; Joel H. Weiner (pp. 1897-1929).

Bacterial genomes encode an extensive range of respiratory enzymes that enable respiratory metabolism with a diverse group of reducing and oxidizing substrates under both aerobic and anaerobic growth conditions. An important class of enzymes that contributes to this broad diversity is the complex iron–sulfur molybdoenzyme (CISM) family. The architecture of this class comprises the following subunits. (i) A molybdo- bis(pyranopterin guanine dinucleotide) (Mo- bisPGD) cofactor-containing catalytic subunit that also contains a cubane [Fe-S] cluster (FS0). (ii) A four-cluster protein (FCP) subunit that contains 4 cubane [Fe-S] clusters (FS1–FS4). (iii) A membrane anchor protein (MAP) subunit which anchors the catalytic and FCP subunits to the cytoplasmic membrane. In this review, we define the CISM family of enzymes on the basis of emerging structural and bioinformatic data, and show that the catalytic and FCP subunit architectures appear in a wide range of bacterial redox enzymes. We evaluate evolutionary events involving genes encoding the CISM catalytic subunit that resulted in the emergence of the complex I (NADH:ubiquinone oxidoreductase) Nqo3/NuoG subunit architecture. We also trace a series of evolutionary events leading from a primordial Cys-containing peptide to the FCP architecture. Finally, many of the CISM archetypes and related enzymes rely on the tat translocon to transport fully folded monomeric or dimeric subunits across the cytoplasmic membrane. We have used genome sequence data to establish that there is a bias against the presence of soluble periplasmic molybdoenzymes in bacteria lacking an outer membrane.

Keywords: Abbreviations; CISM; complex iron–sulfur molybdoenzyme; DmsABC; E. coli; DMSO reductase; DorA; soluble; Rhodobacter; DMSO reductase; E; _m; midpoint potential; EPR; electron paramagnetic resonance; ETR; electron transfer relay; EXAFS; extended X-ray absorption fine structure; FCP; four cluster protein; FHL; formate hydrogen lyase; FdnGHI; E. coli; formate dehydrogenase N; MAP; membrane anchor protein; Mo-; bis; PGD; molybdo-; bis; (pyranopterin guanine dinucleotide); NarGHI; E. coli; nitrate reductase A; PDB; protein data bank; rmsd; root–mean–square deviation; Sec; selenocysteine; SSM; secondary structure matching; TCP; three-cluster protein; TM; transmembrane; TorA; periplasmic TMAO reductaseNitrate reductase; Formate dehydrogenase; Iron sulfur; Molybdenum cofactor; Structural biology; Cell envelope

Signaling mechanisms for activation of extracytoplasmic function (ECF) sigma factors by Benjamin E. Brooks; Susan K. Buchanan (pp. 1930-1945).

A variety of mechanisms are used to signal extracytoplasmic conditions to the cytoplasm. These mechanisms activate extracytoplasmic function (ECF) sigma factors which recruit RNA-polymerase to specific genes in order to express appropriate proteins in response to the changing environment. The two best understood ECF signaling pathways regulate σ^E-mediated expression of periplasmic stress response genes in Escherichia coli and FecI-mediated expression of iron–citrate transport genes in E. coli. Homologues from other Gram-negative bacteria suggest that these two signaling mechanisms and variations on these mechanisms may be the general schemes by which ECF sigma factors are regulated in Gram-negative bacteria.

Keywords: ECF; Sigma factor; Anti-sigma factor; Sigma E; FecI

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BBA - Biomembranes (v.1778, #9)