Here we describe the crystal structure of the N-terminal domain of the FK506-binding protein (FKBP) from wheat (wFKBP73), which is the first structure presenting three FK domains (wFK73_1, wFK73_2 and wFK73_3). The crystal model includes wFK73_2 and wFK73_3 domains and only part of the wFK73_1 domain. The wFK73_1 domain is responsible for binding FK506 and for peptidyl prolyl cis/trans isomerase (PPIase) activity, while the wFK73_2 and wFK73_3 domains lack these activities. A structure-based sequence comparison demonstrated that the absence of a large enough hydrophobic pocket important for PPIase activity, and of the conserved residues necessary for drug binding in the wFK73_2 and wFK73_3 domains explains the lack of these activities in these domains. Sequence and structural comparison between the three wFKBP73 domains suggest that the wFK73_2 domain is the most divergent. A structural comparison of the FK domains of wFKBP73 with other FKBPs containing more than one FK domain, revealed that while the overall architecture of each of the three FK domains displays a typical FKBP fold, their relative arrangement in space is unique and may have important functional implications. We suggest that the existence of FKBPs with three FK domains offers additional interactive options for these plant proteins enlarging the overall regulatory functions of these proteins.
The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cellfree E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory informationmanagement system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.
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