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 site of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of approximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deuterium exchange. No changes in solvent accessibility were seen in the regulatory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two regions flank the active site and contain domain-domain contact residues. Comparison with results from the isolated catalytic domain-containing C-terminal fragment of CheB (residues 147-349) showed that the increased solvent accessibility was less than would have occurred upon detachment of the regulatory domain. Thus, phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of CheB.
Hughes
C A
CA
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA, 92093-0359, USA
Mandell
J G
JG
Anand
G S
GS
Stock
A M
AM
Komives
E A
EA
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D006859:Hydrogen / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000577:Amides; D001426:Bacterial Proteins; D012997:Solvents; D006859:Hydrogen; D002265:Carboxylic Ester Hydrolases; C041144:chemotactic protein methylesterase