@article {45904, title = {Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE.}, journal = {Nature Communication}, volume = {5}, year = {2014}, month = {2014 - Aug}, pages = {4521}, abstract = {

The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.

}, issn = {2041-1723}, doi = {10.1038/ncomms5521}, author = {Wisedchaisri, Goragot and Park, Min-Sun and Iadanza, Matthew G and Zheng, Hongjin and Gonen, Tamir} } @article {45372, title = {A type VI secretion-related pathway in bacteroidetes mediates interbacterial antagonism.}, journal = {Cell Host Microbe}, volume = {16}, year = {2014}, month = {2014 Aug 13}, pages = {227-36}, abstract = {

Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.

}, issn = {1934-6069}, doi = {10.1016/j.chom.2014.07.007}, author = {Russell, Alistair B and Wexler, Aaron G and Harding, Brittany N and Whitney, John C and Bohn, Alan J and Goo, Young Ah and Tran, Bao Q and Barry, Natasha A and Zheng, Hongjin and Peterson, S Brook and Chou, Seemay and Gonen, Tamir and Goodlett, David R and Goodman, Andrew L and Mougous, Joseph D} } @article {5958, title = {Crystal structure of a nitrate/nitrite exchanger.}, journal = {Nature}, volume = {497}, year = {2013}, month = {2013 May 12}, pages = {647-51}, abstract = {

Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.

}, issn = {1476-4687}, doi = {10.1038/nature12139}, author = {Zheng, Hongjin and Wisedchaisri, Goragot and Gonen, Tamir} } @article {6414, title = {Haemolysin coregulated protein is an exported receptor and chaperone of type VI secretion substrates.}, journal = {Molecular Cell}, volume = {51}, year = {2013}, month = {2013 Sep 12}, pages = {584-93}, abstract = {

Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp-effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2013.07.025}, author = {Silverman, Julie M and Agnello, Danielle M and Zheng, Hongjin and Andrews, Benjamin T and Li, Mo and Catalano, Carlos E and Gonen, Tamir and Mougous, Joseph D} } @article {65045, title = {Voltage sensor ring in a native structure of a membrane-embedded potassium channel.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {110}, year = {2013}, month = {2013 Feb 26}, pages = {3369-74}, abstract = {

Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid-protein interactions in general and for the mechanism of voltage sensing in particular.

}, keywords = {Aeropyrum, Cell Membrane, Cross-Linking Reagents, Crystallography, X-Ray, Electrons, Lipids, Models, Molecular, Potassium Channels, Voltage-Gated, Protein Structure, Secondary, Protein Structure, Tertiary}, issn = {1091-6490}, doi = {10.1073/pnas.1218203110}, author = {Shi, Liang and Zheng, Hongjin and Zheng, Hui and Borkowski, Brian A and Shi, Dan and Gonen, Tamir and Jiang, Qiu-Xing} } @article {3635, title = {Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system.}, journal = {mBio}, volume = {1}, year = {2010}, month = {2010}, abstract = {

The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.

}, keywords = {Bacterial Proteins, Membrane Transport Proteins, Models, Molecular, Protein Binding, Protein Multimerization, Salmonella typhimurium}, issn = {2150-7511}, doi = {10.1128/mBio.00158-10}, author = {Sanowar, Sarah and Singh, Pragya and Pfuetzner, Richard A and Andr{\'e}, Ingemar and Zheng, Hongjin and Spreter, Thomas and Strynadka, Natalie C J and Gonen, Tamir and Baker, David and Goodlett, David R and Miller, Samuel I} } @article {3637, title = {The prototypical H+/galactose symporter GalP assembles into functional trimers.}, journal = {Journal of Molecular Biology}, volume = {396}, year = {2010}, month = {2010 Feb 26}, pages = {593-601}, abstract = {

Glucose is a primary source of energy for human cells. Glucose transporters form specialized membrane channels for the transport of sugars into and out of cells. Galactose permease (GalP) is the closest bacterial homolog of human facilitated glucose transporters. Here, we report the functional reconstitution and 2D crystallization of GalP. Single particle electron microscopy analysis of purified GalP shows that the protein assembles as an oligomer with three distinct densities. Reconstitution assays yield 2D GalP crystals that exhibit a hexagonal array having p3 symmetry. The projection structure of GalP at 18 A resolution shows that the protein is trimeric. Each monomer in the trimer forms its own channel, but an additional cavity (10 approximately 15 A in diameter) is apparent at the 3-fold axis of the oligomer. We show that the crystalline GalP is able to selectively bind substrate, suggesting that the trimeric form is biologically active.

}, keywords = {Calcium-Binding Proteins, Computer-Assisted, Crystallization, Electron, Escherichia coli, Escherichia coli Proteins, Galactose, Image Processing, Microscopy, Monosaccharide Transport Proteins, Periplasmic Binding Proteins, Protein Binding, Protein Multimerization, Protein Structure, Quaternary}, issn = {1089-8638}, doi = {10.1016/j.jmb.2009.12.010}, author = {Zheng, Hongjin and Taraska, Justin and Merz, Alexey J and Gonen, Tamir} } @article {45965, title = {A conformational switch in bacteriophage p22 portal protein primes genome injection.}, journal = {Molecular Cell}, volume = {29}, year = {2008}, month = {2008 Feb 15}, pages = {376-83}, abstract = {

Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.

}, keywords = {Bacteriophage P22, Binding Sites, Capsid Proteins, DNA, Viral, Genome, Models, Molecular, Protein Binding, Protein Conformation, Viral Tail Proteins}, issn = {1097-2765}, doi = {10.1016/j.molcel.2007.11.034}, author = {Zheng, Hongjin and Olia, Adam S and Gonen, Melissa and Andrews, Simeon and Cingolani, Gino and Gonen, Tamir} }