Binding of MG132 or deletion of the Thr active sites in HslV subunits increases the affinity of HslV protease for HslU ATPase and makes this interaction nucleotide-independent.
HslVU is an ATP-dependent protease in bacteria consisting of HslV dodecamer and HslU hexamer. Upon ATP binding, HslU ATPase allosterically activates the catalytic function of HslV protease by 1-2 orders of magnitude. However, relatively little is known about the role of HslV in the control of HslU function. Here we describe the involvement of the N-terminal Thr active sites (Thr-1) of HslV in the communication between HslV and HslU. Binding of proteasome inhibitors to Thr-1 led to a dramatic increase in the interaction between HslV and HslU with a marked increase in ATP hydrolysis by HslU. Moreover, carbobenzoxy-leucyl-leucyl-leucinal (MG132) could bind to Thr-1 of free HslV, and this binding induced a tight interaction between HslV and HslU with the activation of HslU ATPase, suggesting that substrate-bound HslV can allosterically regulate HslU function. Unexpectedly, the deletion of Thr-1 also caused a dramatic increase in the affinity between HslV and HslU even in the absence of ATP. Furthermore, the increase in the number of the Thr-1 deletion mutant subunit in place of HslV subunit in a dodecamer led to a proportional increase in the affinity between HslV and HslU with gradual activation of HslU ATPase. Although the molecular mechanism elucidating how the Thr-1 deletion influences the interaction between HslV and HslU remains unknown, these results suggest an additional allosteric mechanism for the control of HslU function by HslV. Taken together, our findings indicate a critical involvement of Thr-1 of HslV in the reciprocal control of HslU function and, thus, for their communication.
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Eunyong
E
Park
Jung Wook
JW
Lee
Soo Hyun
SH
Eom
Jae Hong
JH
Seol
Chin Ha
CH
Chung
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D049071:Endopeptidase Clp / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007976:Leupeptins / Q000737:chemistry / Q000494:pharmacology*; D020836:Protein Structure, Quaternary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
C483566:ClpYQ protease, E coli; D015853:Cysteine Proteinase Inhibitors; D029968:Escherichia coli Proteins; D007976:Leupeptins; D021122:Protein Subunits; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde