Role of a conserved pore residue in the formation of a prehydrolytic high substrate affinity state in the AAA+ chaperone ClpA.
The AAA+ protease ClpAP, consisting of the ClpA chaperone and the ClpP protease, processively unfolds and translocates its substrates into its proteolytic core, where they are cleaved. Unfolding and efficient translocation require ATP-dependent conformational changes in ClpA's D2 loop, where the conserved GYVG motif resides. To explore the role of the essential tyrosine of this motif, we investigated how two mutations at this residue (Y540C and Y540A) affect the rate at which the enzyme processes unstructured substrates. The mutations decrease ClpA's ability to process unfolded or unstable protein substrates but have only mild effects on the rates of ATP hydrolysis or hydrolysis of small peptide substrates. The mutants' substrate binding properties were also characterized, using single molecule fluorescence microscopy. The single-molecule studies demonstrate that the conserved tyrosine is essential for the formation of the prehydrolytic, high substrate affinity conformation observed in wild-type ClpA. Together, the results support a model in which destabilization of the high substrate affinity conformation of ClpA makes translocation less efficient and uncouples it from ATP hydrolysis.
Farbman
Mary E
ME
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Gershenson
Anne
A
Licht
Stuart
S
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D001896:Boron Compounds / Q000494:pharmacology; D049071:Endopeptidase Clp / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D018832:Molecular Chaperones / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D021381:Protein Transport; D014443:Tyrosine / Q000737:chemistry
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010455:Peptides; D014443:Tyrosine; D000255:Adenosine Triphosphate; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases