A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

The conserved protein-conducting channel, referred to as the Sec61 channel in eukaryotes or the SecY channel in eubacteria and archaea, translocates proteins across cellular membranes and integrates proteins containing hydrophobic transmembrane segments into lipid bilayers. Structural studies illustrate how the protein-conducting channel accomplishes these tasks. Three different mechanisms, each requiring a different set of channel binding partners, are employed to move polypeptide substrates: The ribosome feeds the polypeptide chain directly into the channel, a ratcheting mechanism is used by the eukaryotic endoplasmic reticulum chaperone BiP, and a pushing mechanism is utilized by the bacterial ATPase SecA. We review these translocation mechanisms, relating biochemical and genetic observations to the structures of the protein-conducting channel and its binding partners.

Gram-negative bacteria and their complex cell envelope comprising an outer and inner membrane are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular penetration of small molecules, including nutrients and antibacterial agents. The synergistic action between relatively slow porin-mediated passive uptake across the outer membrane and active efflux transporters in the inner membrane creates a permeability barrier that reinforces the enzymatic modification barrier, which efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in small molecule translocation in Enterobacteriaceae and consider the crucial role of porins in antibiotic resistance. Commented [w1]: Is this specification necessary here?, in my opinion it deviates, better to put later… Commented [JP2]: Editor request... porins represent the preferred route for the entry of β-lactams, including cephalosporins, penicillins and carbapenems 14-16. The clinical relevance of membrane-associated mechanisms (MAMs) of resistance (i.e. porin defects and/or overexpression of multidrug efflux pumps) has been well established for these antibiotics. The Influx and Efflux rates control the internal concentration of antibiotics and represent the first lane (mechanical barrier) protecting the bacterial cells against therapeutic treatment 1-3,6. Consequently, studies on bacterial porins are receiving a renewed interest due to their key role in the bacterial susceptibility towards clinically used antibiotics. In combination with the expression of antibiotic-modifying enzymes expressed in the periplasm (e.g. β-lactamases), porins play a key role in β-lactam resistance 4,17. In this review, we discuss recent advances in our understanding of the molecular and functional roles of classic porins in antibiotic translocation in Enterobacteriaceae. We explore structural aspects and the insights gained into permeation and the pore translocation process, the regulation of porin expression as well as the role of porins in the emergence of antibiotic susceptibility. Enterobacterial general porins Structural aspects The crystal structures of a general porin from Rhodobacter capsulatus 18 , the OmpF and PhoE porins from E. coli 19 and other E. coli OmpF structures including mutants 20,21 were the first to be solved. Only a limited number of other enterobacterial porin structures have been reported, i.e. E. coli OmpC, K. pneumoniae OmpK36 and Salmonella typhi OmpF 22-24. The lack of data has hindered attempts to relate structure to function. Recently, the structures of two porins from P. stuartii as well as the structures of the OmpF and OmpC orthologs of K. pneumoniae, E. aerogenes and E. cloacae have been reported 12,25,26. Another recent study reported th...

Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.

Characterization of a large family of outer membrane channels from gram-negative bacteria suggest how they can thrive in nutrient-poor environments and how channel inactivation can contribute to antibiotic resistance.

The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota, which has an important role in human health and nutrition. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA-SusG. Glycan degradation mainly occurs intracellularly and depends on the import of oligosaccharides by an outer membrane protein complex composed of an extracellular SusD-like lipoprotein and an integral membrane SusC-like TonB-dependent transporter. The presence of the partner SusD-like lipoprotein is the major feature that distinguishes SusC-like proteins from previously characterized TonB-dependent transporters. Many sequenced gut Bacteroides spp. encode over 100 SusCD pairs, of which the majority have unknown functions and substrate specificities. The mechanism by which extracellular substrate binding by SusD proteins is coupled to outer membrane passage through their cognate SusC transporter is unknown. Here we present X-ray crystal structures of two functionally distinct SusCD complexes purified from Bacteroides thetaiotaomicron and derive a general model for substrate translocation. The SusC transporters form homodimers, with each β-barrel protomer tightly capped by SusD. Ligands are bound at the SusC-SusD interface in a large solvent-excluded cavity. Molecular dynamics simulations and single-channel electrophysiology reveal a 'pedal bin' mechanism, in which SusD moves away from SusC in a hinge-like fashion in the absence of ligand to expose the substrate-binding site to the extracellular milieu. These data provide mechanistic insights into outer membrane nutrient import by members of the microbiota, an area of major importance for understanding human-microbiota symbiosis.

Escherichia coli OmpW belongs to a family of small outer membrane proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. To gain insight into the function of these proteins we have determined the crystal structure of E. coli OmpW to 2.7-Å resolution. The structure shows that OmpW forms an 8-stranded ␤-barrel with a long and narrow hydrophobic channel that contains a bound n-dodecyl-N,N-dimethylamine-N-oxide detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of n-dodecyl-N,Ndimethylamine-N-oxide. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial outer membrane. The outer membrane (OM)2 of Gram-negative bacteria is a protective barrier that hinders the permeability of both hydrophilic and hydrophobic compounds, because of the presence of lipopolysaccharide (LPS) within the outer leaflet of the OM (1). To obtain nutrients and other molecules that are necessary for growth and function of the cell, Gramnegative bacteria have channels within their OM that facilitate uptake of these molecules. With respect to the transport of small, hydrophilic substances, these channels can be divided in three classes, based on their mode of transport (1): general porins, substrate-specific transporters, and active transporters. A wealth of structural and functional information is available for many of these OM channel proteins, which form monomeric or trimeric barrels that are each composed of 12-22 antiparallel ␤-strands. In addition to OM proteins with established transport functions, the OM also contains a considerable number of smaller, monomeric ␤-barrels that are composed of 8 or 10 ␤-strands. These proteins have been implicated in a wide range of functions including OM lipid metabolism, cell adhesion, and structural functions. One of these small OM proteins is OmpA from Escherichia coli, which belongs to a protein family with a number of established and putative functions, the most important of which is to provide structural stability to the cell via interactions of its C-terminal domain with the periplasmic peptidoglycan layer (1). Another member of the small OM protein family is NspA from Neisseria meningitidis, which belongs to the Opa family of proteins that are thought to mediate adhesion to host cells (2).A fundamental question is whether these small barrels can function as transport channels. Arguing against this possibility are the crystal structures that have been determined for several of these proteins, and which do not show continuous channels that would be consistent with transport functions. On the other hand, it has been shown that, at least in vitro, OmpA forms both small and large ion channels (3) and is permeable to larger uncha...