Asymmetric nucleotide transactions of the HslUV protease.
ATP binding and hydrolysis are critical for protein degradation by HslUV, a AAA(+) machine containing one or two HslU(6) ATPases and the HslV(12) peptidase. Although each HslU homohexamer has six potential ATP-binding sites, we show that only three or four ATP molecules bind at saturation and present evidence for three functional subunit classes. These results imply that only a subset of HslU and HslUV crystal structures represents functional enzyme conformations. Our results support an asymmetric mechanism of ATP binding and hydrolysis, and suggest that molecular contacts between HslU and HslV vary dynamically throughout the ATPase cycle. Nucleotide binding controls HslUV assembly and activity. Binding of a single ATP allows HslU to bind HslV, whereas additional ATPs must bind HslU to support substrate recognition and to activate ATP hydrolysis, which powers substrate unfolding and translocation. Thus, a simple thermodynamic hierarchy ensures that substrates bind to functional HslUV complexes, that ATP hydrolysis is efficiently coupled to protein degradation, and that working HslUV does not dissociate, allowing highly processive degradation.
Yakamavich
Joseph A
JA
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Baker
Tania A
TA
Sauer
Robert T
RT
D000251:Adenosine Triphosphatases / Q000378:metabolism / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D049071:Endopeptidase Clp / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D013379:Substrate Specificity
C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D005998:Glycine