@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 {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 {4935, title = {Advances in structural and functional analysis of membrane proteins by electron crystallography.}, journal = {Structure}, volume = {19}, year = {2011}, month = {2011 Oct 12}, pages = {1381-93}, abstract = {

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.

}, keywords = {Aquaporin 1, Archaea, Automatic Data Processing, Bacteriorhodopsins, Cell Membrane, Computer-Assisted, Cryoelectron Microscopy, Crystallography, Detergents, Dialysis, Humans, Image Processing, Lipid Bilayers, Membrane Proteins, Membrane Transport Proteins, Protein Conformation, Static Electricity}, issn = {1878-4186}, doi = {10.1016/j.str.2011.09.001}, author = {Wisedchaisri, Goragot and Reichow, Steve L and Gonen, Tamir} } @article {4925, title = {Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography.}, journal = {Structure}, volume = {19}, year = {2011}, month = {2011 Jul 13}, pages = {976-87}, abstract = {

In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.

}, keywords = {Crystallization, Crystallography, Electrons, Ligands, Membrane Proteins, Models, Molecular, Molecular Imaging, Peptide Fragments, Protein Conformation, X-Ray}, issn = {1878-4186}, doi = {10.1016/j.str.2011.04.008}, author = {Wisedchaisri, Goragot and Gonen, Tamir} } @article {3633, title = {Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR.}, journal = {Structure}, volume = {18}, year = {2010}, month = {2010 Nov 10}, pages = {1512-21}, abstract = {

Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins{\textendash}the nitrogen regulatory protein NrpR{\textendash}that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.

}, keywords = {Archaeal, Electron, Gene Expression Regulation, Ketoglutaric Acids, Methanococcus, Microscopy, Models, Molecular, Molecular Dynamics Simulation, Nitrogen, PII Nitrogen Regulatory Proteins, Protein Conformation, Quaternary Ammonium Compounds, Transcription Factors}, issn = {1878-4186}, doi = {10.1016/j.str.2010.08.014}, author = {Wisedchaisri, Goragot and Dranow, David M and Lie, Thomas J and Bonanno, Jeffrey B and Patskovsky, Yury and Ozyurt, Sinem A and Sauder, J Michael and Almo, Steven C and Wasserman, Stephen R and Burley, Stephen K and Leigh, John A and Gonen, Tamir} }