Identification and Characterization of residues involved in specific interaction between RapA phosphatase and its substrate Spo0F~P and its inhibitor PhrA in Bacillus subtilis

DIAZ, A.R; MORELLI, M; PEREGO,M

Congreso; Blast IX Meeting; 2007

The RapA belongs to a Rap family of proteins in Bacillus subtilis which encompass 11 members. But only three of them, RapA, RapB and RapE are known to target the sporulation phosphorelay by dephosphorylating the intermediate response regulator Spo0F~P. Phosphatase activity of these proteins is regulated at two levels: by transcriptional regulatory control of protein synthesis and by inhibition of their activity by specific pentapeptides generated from a precursor encoded by the phosphatase regulator phr genes which are actives after a complex export-import pathway. Rap proteins show an interesting structural feature because of have six tetratricopeptide repeat (TPR), a 34 amino acid degenerate sequence that form a pair of antiparallel a-helices motif. TPRs motifs occur in tandem arrays, where the individual TPRs stack on top of each other. The regular array that results can be visualized as a spiral staircase in which the individual TPR motifs are the steps. The pattern of sequence conservation imposed by the amphipathic a- helices of a TPR mediates interactions both within the TPR and between adjacent pairs, leaving few residues available for other interactions. In order to find residues involved in specific interaction between Rap A and its substrate Spo0F~P and its inhibitor PhrA we carried out a random mutagenesis and, in addition, residues proposed to be in a putative inner surface and oriented conveniently to interact with Spo0Fand /or PhrA were mutated to alanine by site directed mutagenesis. Residues Y224, N225, N228, H260 individually mutated to alanine give rise proteins which are resistant to inhibition by the PhrA in vivo and in vitro, but show phosphatase activity like wild type. Mutant proteins V159G, E185G, Y177D, L298S, L302R, Y310D, E317G, Y334D obtained by random mutagenesis give rise Spo+ phenotype. In vitro characterizations of these mutants are being carried out