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 B ，Lucius AL 《-》

Application of the sequential n-step kinetic mechanism to polypeptide translocases.

Clp/Hsp100 proteins are essential motor proteins in protein quality control pathways in all organisms. Such enzymes couple the energy derived from ATP binding and hydrolysis to translocate and unfold polypeptide substrates. Often they perform this role in collaboration with proteases for protein removal or with other chaperones for protein disaggregation. Unlike other well-characterized motor proteins, fundamental parameters such as the microscopic rate constants and overall rate of translocation, step-size (amino acids translocated per step), processivity, and directionality are not available for many of these enzymes. We have recently developed a fluorescence stopped-flow method to elucidate these fundamental mechanistic details. In addition, we have developed a quantitative method to examine the single-turnover time courses that result from the rapid mixing experiments. With these two advances in hand, we have recently reported the first determination of the microscopic rate constants, overall rate of translocation, kinetic step-size, and processivity for the E. coli ClpA polypeptide translocase. Here, we report a description of both the fluorescence stopped-flow method to examine the mechanism of enzyme catalyzed polypeptide translocation and the mathematics required to quantitatively examine the resulting time courses.

Lucius AL ，Miller JM ，Rajendar B 《-》

E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP.

There are five known ATP-dependent proteases in Escherichia coli (Lon, ClpAP, ClpXP, HslUV, and the membrane-associated FtsH) that catalyze the removal of both misfolded and properly folded proteins in cellular protein quality control pathways. Hexameric ClpA rings associate with one or both faces of the cylindrically shaped tetradecameric ClpP protease. ClpA catalyzes unfolding and translocation of polypeptide substrates into the proteolytic core of ClpP for degradation through repeated cycles of ATP binding and hydrolysis at two nucleotide binding domains on each ClpA monomer. We previously reported a molecular mechanism for ClpA catalyzed polypeptide translocation in the absence of ClpP, including elementary rate constants, overall rate, and the kinetic step size. However, the potential allosteric effect of ClpP on the mechanism of ClpA catalyzed translocation remains unclear. Using single-turnover fluorescence stopped-flow methods, here we report that ClpA, when associated with ClpP, translocates polypeptide with an overall rate of ~35 aa s(-1) and, on average, traverses ~5 aa between two rate-limiting steps with reduced cooperativity between ATP binding sites in the hexameric ring. This is in direct contrast to our previously reported observation that, in the absence of ClpP, ClpA translocates polypeptide substrates with a maximum translocation rate of ~20 aa s(-1) with cooperativity between ATPase sites. Our results demonstrate that ClpP allosterically impacts the polypeptide translocation activity of ClpA by reducing the cooperativity between ATP binding sites.

Miller JM ，Lin J ，Li T ，Lucius AL ... -  《-》

ClpP hydrolyzes a protein substrate processively in the absence of the ClpA ATPase: mechanistic studies of ATP-independent proteolysis.

Jennings LD ，Lun DS ，Médard M ，Licht S ... -  《-》

The Escherichia coli ClpA molecular chaperone self-assembles into tetramers.

Veronese PK ，Stafford RP ，Lucius AL 《-》