Scanning Mutagenesis Identifies Amino Acid Residues Essential for the in Vivo Activity of the Escherichia coli DnaJ (Hsp40) J-Domain

Corresponding author: Pierre Genevaux, Centre Médical Universitaire, 1, rue Michel-Servet, CH-1211 Genève 4, Switzerland., pierre.genevaux{at}medecine.unige.ch (E-mail)

Communicating editor: A. L. SONENSHEIN

The DnaJ (Hsp40) cochaperone regulates the DnaK (Hsp70) chaperone by accelerating ATP hydrolysis in a cycle closely linked to substrate binding and release. The J-domain, the signature motif of the Hsp40 family, orchestrates interaction with the DnaK ATPase domain. We studied the J-domain by creating 42 mutant E. coli DnaJ variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage . Most mutants studied behaved like wild type in all assays. In addition to the ₃₃HisProAsp₃₅ (HPD) tripeptide found in all known functional J-domains, our study uncovered three new single substitution mutations (Y25A, K26A, and F47A) that totally abolish J-domain function. Furthermore, two glycine substitution mutants in an exposed flexible loop (R36G, N37G) showed partial loss of J-domain function alone and complete loss of function as a triple (RNQ-GGG) mutant coupled with the phenotypically silent Q38G. Interestingly, all the essential residues map to a small region on the same solvent-exposed face of the J-domain. Engineered mutations in the corresponding residues of the human Hdj1 J-domain grafted in E. coli DnaJ also resulted in loss of function, suggesting an evolutionarily conserved interaction surface. We propose that these clustered residues impart critical sequence determinants necessary for J-domain catalytic activity and reversible contact interface with the DnaK ATPase domain.

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