Biochemistry of Energy-Dependent (Intracellular) Protein

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Research conducted in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of important cellular regulatory proteins and is an essential component of the protein quality control system as well. Most protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multi-component, molecular machines. The heart of the machine is an ATP-driven protein unfoldase that binds a specific protein target, disrupts its structure, and translocates the unfolded protein into the proteolytic chamber of a tightly associated self-compartmentalized endopeptidase. Our studies encompass structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from E. coli and their homologs in human mitochondria. In the last year, progress has been made in several areas. Based on our crystal structure of ClpP with a covalently bound peptide at the active site, we have generated mutants with altered substrate interactions. Substrates have a mobile binding mode in which polypeptides interact through main chain hydrogen bonding and can slide within an extended active site groove. Only the P1 residue has a defined binding pocket. We have mutated residues within the S1 pocket and found that the activity of ClpP is drastically affected. Studies are underway to determine whether the specificity of cleavage is affected in mutants in which the surface properties of the S1 pocket have been altered. We have initiated rapid kinetic measurements of ClpP and ClpAP peptidase and protease activity in order to determine the initial events in substrate unfolding and translocation and the mechanism by which substrates enter the ClpP chamber. We have obtained biochemical evidence of a dynamic change in ClpP ring contacts that results in dissociation of the tetradecamer into two heptameric rings. This dissociation should reflect a conformational change in ClpP that occurs during the catalytic cycle and could be related to substrate entry into the chamber or release of peptide products from the chamber after degradation. ClpS, an adaptor for ClpA, inhibits ClpA activity in vitro substrates such as GFP-SsrA; however, in vivo, ClpS is required for degradation of a class of substrates called "N-end rule" proteins, which have non-canonical amino terminal residues. Model N-end rule substrates are degraded by ClpA in the presence of ClpS and the N-domain, which is the binding site for ClpS, is required for this degradation. We are in the process of identifying endogenous N-end rule substrates by trapping them in vivo using ClpA and ClpS in combination with inactivated ClpP. In vitro, ClpS inhibits ClpA activity at a stoichiometry of one ClpS per hexamer. We are using a combination of cryo-electron microscopy (in collaboration with A. C. Steven, NIAMS) and biochemical methods to determine the effects of ClpS on the structure of ClpA and the distribution of the N-domains with ClpS bound. A specific anti-SsrA antibody prepared in our laboratory, has been used to measure the half-lives endogenous SsrA-tagged proteins in vivo and to demonstrate that ClpXP plays the major role in degradation of these proteins. Other ATP-dependent proteases (Lon and ClpAP) can degrade SsrA-tagged proteins but their contribution can be seen only in the absence of ClpX. We have found that over expression of many proteins in E. coli results in significant generation of multiple forms of the protein with SsrA-tags. These proteins are mostly degraded by ClpXP but can accumulate to significant levels when ClpXP activity is compromised. In our project aimed at obtaining the crystal structure of ClpA hexamers, we have generated ClpA mutants introducing cysteine residues at the subunit contact points expected in the hexamer.