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Scott A Lesley

Publications and source records attributed to Scott A Lesley.

At least 19 recordsLinked to original sources
PubMed2006

Comparative structural analysis of a novel glutathioneS-transferase (ATU5508) from Agrobacterium tumefaciens at 2.0 A resolution.

Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.

Agrobacterium tumefaciens↗
PubMed2006

Crystal structure of the human TRPV2 channel ankyrin repeat domain.

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.

Amino Acid Sequence↗
PubMed2006

Crystal structure of a divalent metal ion transporter CorA at 2.9 angstrom resolution.

CorA family members are ubiquitously distributed transporters of divalent metal cations and are considered to be the primary Mg2+ transporter of Bacteria and Archaea. We have determined a 2.9 angstrom resolution structure of CorA from Thermotoga maritima that reveals a pentameric cone-shaped protein. Two potential regulatory metal binding sites are found in the N-terminal domain that bind both Mg2+ and Co2+. The structure of CorA supports an efflux system involving dehydration and rehydration of divalent metal ions potentially mediated by a ring of conserved aspartate residues at the cytoplasmic entrance and a carbonyl funnel at the periplasmic side of the pore.

Amino Acid Sequence↗
PubMed2006

Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination.

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.

Circular Dichroism↗
PubMed2005

Structural insights into enzyme regulation for inositol 1,4,5-trisphosphate 3-kinase B.

D-Myoinositol 1,4,5-trisphophate 3-kinases (IP(3)-3Ks) play important roles in metazoan cellular signaling. It has been demonstrated that mice without a functional version of IP(3)-3K isoform B are deficient in peripheral T-cells, indicating that IP(3)-3KB is essential to the developing immune system. The recent apo IP(3)-3KA structure exhibited a helix at the catalytic domain N-terminus exhibited a helix at the N-terminus of the catalytic domain, with a tryptophan indole moiety mimicking the binding mode of the substrate ATP purine ring, suggesting a mechanism of autoinhibition. Here we present the structure of the complete catalytic domain of IP(3)-3KB, including the CaM binding domain in complex with Mg(2+) and ATP. The crystal structure reveals a homodimeric arrangement of IP(3)-3KB catalytic domains, mediated via an intermolecular antiparallel beta-sheet formed from part of the CaM binding region. Residues from the putative autoinhibitory helix are rearranged into a loop configuration, with extensive interactions with the bound ATP. Mutagenesis of residues from this region reveals that substitution of the putative autoinhibitory tryptophan generates a hyperactive enzyme which retains Ca(2+)/CaM sensitivity. The IP(3)-3KB structure suggests a mechanism of enzyme activation, and raises the possibility that an interaction between IP(3)-3KB molecules may occur as part of the catalytic or regulatory cycle.

Adenosine Triphosphate↗