Molecular mechanism of polypeptide translocation catalyzed by the Escherichia coli ClpA protein translocase.
The removal of damaged or unneeded proteins by ATP-dependent proteases is crucial for cell survival in all organisms. Integral components of ATP-dependent proteases are motor proteins that unfold stably folded proteins that have been targeted for removal. These protein unfoldases/polypeptide translocases use ATP to unfold the target proteins and translocate them into a proteolytic component. Despite the central role of these motor proteins in cell homeostasis, a number of important questions regarding the molecular mechanisms of enzyme catalyzed protein unfolding and translocation remain unanswered. Here, we demonstrate that Escherichia coli ClpA, in the absence of the proteolytic component ClpP, processively and directionally steps along the polypeptide backbone with a kinetic step size of approximately 14 amino acids, independent of the concentration of ATP with a rate of approximately 19 amino acids s(-1) at saturating concentrations of ATP. In contrast to earlier studies by others, we have developed single-turnover fluorescence stopped-flow methods that allow us to quantitatively examine the molecular mechanism of the motor component ClpA decoupled from the proteolytic component ClpP. For the first time, we reveal that in the absence of ClpP ClpA translocates polypeptides directionally, processively and in discrete steps similar to other motor proteins that translocate vectorially on a linear lattice, such as nucleic acid helicases and kinesin. We believe that the methods employed here will be generally applicable to the examination of other AAA+ protein translocases involved in a variety of important biological functions where the substrate is not covalently modified; for example, membrane fusion, membrane transport, protein disaggregation, and protein refolding.
Rajendar
Burki
B
The University of Alabama at Birmingham, Department of Chemistry, 1530 3rd Avenue South, Birmingham, AL 35294-1240, USA.
Lucius
Aaron L
AL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D021381:Protein Transport
D029968:Escherichia coli Proteins; D010455:Peptides; D000255:Adenosine Triphosphate; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp