Structural biology and bioinformatic investigation of phosphatases and associated domains

Background:

The inositol polyphosphate 5-phosphatases are crucial human signalling enzymes that control numerous intracellular processes via removal of the 5-position phosphate group of various inositol substrates. In doing so they regulate important intracellular signalling pathways that control processes such as intracellular trafficking and receptor mediated signalling events. All 5-phosphatases contain a central catalytic domain that interacts with the target substrate. With the exception of the human type-1 5-phosphatase, each phosphatase contains several additional domains - the figure below details the domain structure of the 10 human 5-phosphatases identified to date. This work is being performed in collaboration with Prof. Chris Mitchell and Dr Harshal Nandurkar.

Selected references:

J.C. Whisstock, S. Romero, R. Gurung, H. Nandurkar, L. Ooms, S.P. Bottomley and C.A. Mitchell. "The inositol polyphosphate 5-phosphatases and the Apurinic/Apyrimidinic base excision repair endonucleases share a common mechanism for catalysis. J. Biol. Chem. 275: 37055-61. (2000).

F. Wiradjaja, L.M. Ooms, J.C. Whisstock, B.K. McColl, L. Helfenbaum, J.F. Sambrook, M.J. Gething and C.A. Mitchell. "The yeast inositol polyphosphate 5-phosphatase Inp54p localizes to the endoplasmic reticulum via a C-terminal hydrophobic anchoring-tail. Regulation of secretion from the endoplasmic reticulum." J. Biol. Chem. [epub ahead of print] (2000).

H. H. Nandurkar, K. K. Caldwell, J.C. Whisstock, M. Layton, E. A. Gaudet, F. A. Norris , P. W. Majerus, and C. A. Mitchell. "Characterisation of a 78 kDa adapter subunit to a phosphatidylinositol (3)P-3-phosphatase: identification of a myotubularin-related protein family lacking catalytic activity." Proc Natl Acad Sci U S A. in press.

Project 1 Determine the X-ray crystal structure of novel domains: The function of many of the domains associated with 5-phosphatases remains to be characterised. This project will focus upon the expression, purification and crystallosation of novel 5-phosphatase associated domains.

Project 2 Bioinformatic investigation of the 5-phosphatse superfamily: This project will investigate the phylogenetic relationships between the catalytic domain of all known members of the 5-phosphatase / AP endonuclease superfamily. Relationships identified will be supported by analysis of domain structure and intron / exon patterns. In addition we will attempt to identify additional novel family members.

Project 3 Function of novel E. coli phosphatase: The aim of the project will be to investigate the structure and function of a novel putative phosphatase from E. coli. The project will involve expression and purification of the putative phosphatase and investigation of substrate specificity. In addition, we will attempt to crystallise this molecule for structure determination.