Effects of Temperature and ATP on the Kinetic Step-Size for E. Coli ClpA Translocation

Abstract

The E. coli chaperone, ClpA belongs to the AAA+ super-family of proteins. Many AAA+ proteins interact with functional partners to form complex molecular machines. ClpAP/ClpXP has been shown to recognize and bind specifically to the 11 amino acid SsrA sequence (AANDENYALAA) at the carboxy terminus of substrate proteins. When ClpA is associated with ClpP, ClpA translocates SsrA-tagged substrates through its own central channel and into the ClpP cavity for proteolytic degradation. The kinetic mechanism by which E. coli ClpA translocates was studied using a fluorescence stopped-flow method. Single-turnover ClpA translocation experiments were performed using a series of fluorescently labeled polypeptide substrates containing the 11 amino acid SsrA sequence at the carboxy terminus which serves as the specific binding site for ClpA. From the single-turnover experiments, we reported that E. coli ClpA, in the absence of the proteolytic component ClpP, processively and directionally steps along the polypeptide backbone with a kinetic step size of ∼14 amino acids per step, with a rate of ∼ 19 AA s−1 at saturating [ATP]. In order to probe how the kinetic step size might relate to a mechanical step size, we performed single-turnover experiments as a function of [ATP] and temperature. The average kinetic-step size of ∼14 amino acids per step is independent of [ATP] and temperature. The implications of the constancy of the measured kinetic-step size for the mechanism of ClpA catalyzed polypeptide translocation are discussed.