Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB

The crystal structure of the unphosphorylated state of methylesterase CheB shows that the regulatory domain blocks access of substrate to the active s ite of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of a pproximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deut erium exchange. No changes in solvent accessibility were seen in the regula tory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two reg ions flank the active site and contain domain-domain contact residues. Comp arison with results from the isolated catalytic domain-containing C-termina l fragment of CheB (residues 147-349) showed that the increased solvent acc essibility was less than would have occurred upon detachment of the regulat ory domain. Thus, phosphorylation causes subtle changes in solvent accessib ility at the interdomain interface of CheB. (C) 2001 Academic Press.