Nucleotide-dependent substrate recognition by the AAA+ HslUV protease.
ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg(2+). Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.
Burton
Randall E
RE
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Baker
Tania A
TA
Sauer
Robert T
RT
D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D049071:Endopeptidase Clp / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology; D008274:Magnesium / Q000378:metabolism*; D011489:Protein Denaturation / Q000502:physiology; D017510:Protein Folding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D014764:Viral Proteins / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D008274:Magnesium