Role of the loop L4,5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation.
Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L4,5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L4,5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L4,5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein-bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Madhuja
M
Samaddar
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Arvind Vittal
AV
Goswami
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Jaya
J
Purushotham
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Pushpa
P
Hegde
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India patrick@biochem.iisc.ernet.in.
Patrick
P
D'Silva
D000494:Allosteric Regulation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D063269:Mitochondrial Turnover; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000252:Calcium-Transporting ATPases