Domain motion and interdomain hot spots in a multidomain enzyme

The aim of this article is to analyze conformational changes by comparing 10 different structures of Pseudomonas aeruginosa phosphomannomutase/ phosphoglucomutase (PMM/PGM), a four-domain enzyme in which both substrate binding and catalysis require substantial movement of the C-terminal domain. We focus on changes in interdomain and active site crevices using a method called computational solvent mapping rather than superimposing the structures. The method places molecular probes (i.e., small organic molecules containing various functional groups) around the protein to find hot spots. One of the most important hot spots is in the active site, consistent with the ability of the enzyme to bind both glucose and mannose phosphosugar substrates. The protein has eight additional hot spots at domain-domain interfaces and hinge regions. The locations and nature of six of these hot spots vary between the open, half-open, and closed conformers of the enzyme, in good agreement with the ligand-induced conformational changes. In the closed structures the number of probe clusters at the hinge region significantly depends on the position of the phosphorylated oxygen in the substrate (e.g., glucose 1-phosphate versus glucose 6-phosphate), but the protein remains almost unchanged in terms of the overall RMSD, indicating that computational solvent mapping is a more sensitive approach to detect changes in binding sites and interdomain crevices. Focusing on multidomain proteins we show that the subresolution conformational differences revealed by the mapping are in fact significant, and present a general statistical method of analysis to determine the significance of rigid body domain movements in X-ray structures. Published by Wiley-Blackwell.