ALANINE SCANNING MUTAGENESIS OF THE SWITCH-I REGION IN THE ATPASE SITE OF DICTYOSTELIUM-DISCOIDEUM MYOSIN-II

In order to determine the functional roles of the conserved sequence ( NXNSSRFG) of the ''switch I'' loop (residues 233-240 in Dictyostelium myosin II), alanine scanning mutagenesis was performed on Dictyosteliu m myosin II. N233A and S237A mutant myosins did not bind a fluorescent analog of ADP, mant-deoxyADP, at the low concentration range (micromo lar and had low level of ATPase activities. They were nonmotile when e xamined by the in vitro motility assay. Dictyostelium cells expressing these myosins showed worse phenotypes than that of myosin-null cells. In contrast to these mutant myosins, R238A myosin tightly bound mant- deoxyADP. However, the mutant had a defect in the ATP hydrolysis step and exhibited the lowest ATPase activities among the mutants examined here. The R238A myosin was nonmotile. R238C or R238H mutations, which mimic the Usher syndrome mutations, generated myosins with similar fun ctional defects to those of the R238A mutation. Cells expressing the R 238A myosin exhibited the phenotype similar to that of the myosin-null cells. N235A, S236A, F239A, and G240A myosins retained moderate level s of ATPase activities and could drive sliding of actin filaments at v arious speeds. Phenotypes of cells expressing them were very similar t o that of the wild-type cells. Taken together, these results suggest t hat side chains of N233 and S237 may play essential roles in holding a nucleotide in the ATPase pocket and that R238 may play crucial roles in the ATP hydrolysis step, while those of the other residues in the s witch I loop are not essential for the process.