Goragot Wisedchaisri

Crystal structure of a nitrate/nitrite exchanger.

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.

An improved protocol for rapid freezing of protein samples for long‐term storage

Freezing of purified protein drops directly in liquid nitrogen is a convenient technique for the long-term storage of protein samples. Although this enhances reproducibility in follow-up crystallization experiments, some protein samples are not amenable to this technique. It has been discovered that plunging PCR tubes containing protein samples into liquid nitrogen results in more rapid freezing of the samples and can safely preserve some proteins that are damaged by drop-freezing. The PCR-tube method can also be adapted to a PCR-plate freezing method with applications for high-throughput and structural genomics projects.

Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE.

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.

Crystal structures of the response regulator DosR from Mycobacterium tuberculosis suggest a helix rearrangement mechanism for phosphorylation activation

The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the worlds population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 A resolution and its C-terminal DNA-binding domain at 1.7 A resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected (beta alpha)(4) topology instead of the canonical (beta alpha)(5) fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix alpha 10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix alpha 10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.

Advances in Structural and Functional Analysis of Membrane Proteins by Electron Crystallography

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.

Crystal structures, metal activation, and DNA-binding properties of two-domain IdeR from Mycobacterium tuberculosis.

The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co2+ and Ni2+ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni2+ concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co2+ required for activation remains the same. We have determined the crystal structures of Ni2+-activated 2D-IdeR at 1.96 A resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 A and 2.6 A, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR-DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His6. This C-terminal tail promotes crystal contacts by forming a beta-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins.

Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography.

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.

Structural Underpinnings of Nitrogen Regulation by the Prototypical Nitrogen-Responsive Transcriptional Factor NrpR

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--the nitrogen regulatory protein NrpR--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.

Phasing electron diffraction data by molecular replacement: strategy for structure determination and refinement.

Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.