The passage of virtually every molecule across the cell membrane is mediated by a class of proteins called transporters. Transporters are vital to the biology of all cells and a variety of diseases occur when these processes are perturbed or disrupted, as in several genetic disorders or the up-regulation of multidrug resistance transporters by tumor cells. The availability of high resolution structures of human transporters is essential to define the molecular structural basis of their mechanisms. We propose to establish a center for membrane protein structure determination, TransportPDB, with the objective of developing a comprehensive and efficient approach for pursuing the high-resolution x-ray crystal structures of 521 transporters in 48 families presently identified in humans and other targets from PSI-biology centers. For this purpose and to address the objectives of the PSI, we have the following specific aims: A1. An efficient pipeline will be established based on proven technologies and using our experience successfully crystallizing and solving the x-ray structures of integral membrane proteins. This pipeline will be based on several key principles: (a) target prioritization based on disease relevance and completing the protein-fold space coverage of human transporters, (b) the exclusive use of eukaryotic expression systems (Pichia pastoris and 293S mammalian cells) that have proven to deliver functional protein suitable for crystallization, (c) the cloning of constructs based on synthetic genes optimized for expression in both expression systems, and (d) using state-of-the-art data collection techniques for modestly diffracting crystals. The funnel-like organization will enable screening hundreds of human transporter targets and their close mammalian orthologs, driving towards the goal of successfully obtaining their x-ray crystal structures. A2. High-throughput methods and technology will be developed for functional and biophysical characterization of targets to rapidly identify conditions that maintain protein stability and function leading towards higher quality and better diffracting human transporter crystals. New crystal mounting methods, together with micro-beam/rastering technology and increased sensitivity in data collection (PILATUS detector), will be implemented that could be decisive for modestly diffracting membrane protein crystals. A3. Establish a resource for structural and functional data and other materials useful to the scientific community, including x-ray crystal structures of human transporters, codon-optimized clones, detergent solubilization conditions and corresponding stability properties of each target and homology models.
Transporters are vital to the biology of all cells and a variety of diseases occur when these processes are perturbed or disrupted, as in several genetic disorders or the up-regulation of multidrug resistance transporters by tumor cells. The development of effective therapeutics for the treatment of transport related diseases will be greatly accelerated by the availability of high resolution structures of human transporters, which are essential to understand the molecular structural basis of their mechanisms in detail.
|Vastermark, Ake; Saier Jr, Milton H (2014) Evolutionary relationship between 5+5 and 7+7 inverted repeat folds within the amino acid-polyamine-organocation superfamily. Proteins 82:336-46|
|Saier Jr, Milton H; Reddy, Vamsee S; Tamang, Dorjee G et al. (2014) The transporter classification database. Nucleic Acids Res 42:D251-8|
|Swartz, Douglas J; Mok, Leo; Botta, Sri K et al. (2014) Directed evolution of P-glycoprotein cysteines reveals site-specific, non-conservative substitutions that preserve multidrug resistance. Biosci Rep 34:|
|Västermark, Ake; Lunt, Bryan; Saier, Milton (2014) Major facilitator superfamily porters, LacY, FucP and XylE of Escherichia coli appear to have evolved positionally dissimilar catalytic residues without rearrangement of 3-TMS repeat units. J Mol Microbiol Biotechnol 24:82-90|
|Reddy, Abhinay; Cho, Jaehoon; Ling, Sam et al. (2014) Reliability of nine programs of topological predictions and their application to integral membrane channel and carrier proteins. J Mol Microbiol Biotechnol 24:161-90|
|Saier Jr, Milton H (2013) Microcompartments and protein machines in prokaryotes. J Mol Microbiol Biotechnol 23:243-69|
|Swartz, Douglas J; Weber, Joachim; Urbatsch, Ina L (2013) P-glycoprotein is fully active after multiple tryptophan substitutions. Biochim Biophys Acta 1828:1159-68|
|Zheng, Wei Hao; Vastermark, Ake; Shlykov, Maksim A et al. (2013) Evolutionary relationships of ATP-Binding Cassette (ABC) uptake porters. BMC Microbiol 13:98|
|Pieper, Ursula; Schlessinger, Avner; Kloppmann, Edda et al. (2013) Coordinating the impact of structural genomics on the human *-helical transmembrane proteome. Nat Struct Mol Biol 20:135-8|
|Bai, Jiangping; Swartz, Douglas J; Protasevich, Irina I et al. (2011) A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. PLoS One 6:e22577|