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 cellular regulatory proteins and is an essential part of the protein quality control system. Protein degradation within the cytosol is carried out by ATP-dependent proteases. The core 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 bacteria and from human mitochondria and assays of their biological activities. We have focused on three major areas: the structural basis for substrate selection and engagement by ClpAP;the relationship between the double-ringed structure of ClpP and its ability to allow unfolded proteins to enter the degradation chamber;and the role of human ClpXP in mitochondrial function and signaling under conditions of stress. We have also worked with collaborators to obtain high resolution structural information on Lon protease. ClpAP is responsible for degradation of N-end rule proteins in bacterial cells. This activity is mediated by an adaptor protein, ClpS, which binds to the N-domains of ClpA and binds to the N-terminus of proteins bearing N-degrons (hydrophobic amino acids that mark proteins for degradation). ClpS is absolutely required when proteins have no other degradation signals other than the N-degron. Peptides with N-degrons inhibit ClpS-mediated degradation but have no effect on ClpA itself. We have found that a single molecule of ClpS is preferentially bound to ClpA hexamers, despite the presence of six N-domains that should be available for interaction with ClpS. This finding can be explained by two alternative models: one, binding of the first molecule of ClpS reorganizes the N-domains of ClpA in a way that limits the accessibility of other sites for binding ClpS, or, two, the first ClpS molecule binds to an N-domain and a portion of the ClpS occupies the axial channel, providing increased affinity and occluding the binding of other ClpS molecules. We have shown that the N-terminal 20 amino acids of ClpS is needed for enhanced binding and activity of ClpS and future studies will address the interaction site where the N-terminus of ClpS binds to ClpA. To learn how proteins with N-degrons arise in cells, we will first measure the total amount of N-degrons in cells under different growth conditions and in cells subjected to various kinds of stress. To look for specific proteins with N-degrons, we have constructed mutants lacking other proteases that will retain the ability to specifically target N-degron proteins to ClpAP. We have expressed inactive ClpP to trap N-degron proteins, identify them by tandem mass spectrometry. In other studies of ClpA, we have generated ClpA mutants altered in the Walker B consensus (part of the catalytic site) of the D1 and D2 domains and showed that they bind ATP and assemble into stable complexes. D2 mutants in particular are deficient in ATPase and other activities. By assembling mixed hexamers of wild type ClpA and D2 mutants, we have found that allosteric interactions between the D2 domains of ClpA are modulated by substrate binding. Also, negative interactions between the D2 and D1 sites suggest that communication between the two domains during the ATPase cycle is needed to coordinate substrate processing. Mutants defective in ATP hydrolysis in D1 are completely unable to unfold stable proteins, demonstrating that the D1 domain prepares substrates for efficient translocation by D2. In collaborative studies, high resolution cryo electron microscopy has been used to detect the highly mobile axial loops in the D2 domain of ClpA. Mobility is likely required to enable the protein interaction sites within the channel to engage and release substrates during translocation. We have also visualized the opening of the axial pore of ClpP when ClpA binds and detected the loop of ClpA that engages the docking site on ClpP. These studies set the stage for analysis of the dynamics of ClpAP in the process of substrate binding and translocation. In studies of ClpP, we found that the N-terminal peptide of ClpP influences the stability of the tetradecamer. ClpP is a complex of two heptamers and the closed tetradecamer is needed for activity. The N-termini of ClpP line the axial channel and protrude from the apical surface. Interactions of the N-termini either with the surface or the axial channel affect activity and influence the stability of the tetradecamer, and peptide binding also affects the interaction between the two heptamers. These effects have implications for the way in which substrates passing through the axial channel can affect the conformation of ClpP and the activity of the catalytic site and also for the manner in which entering substrates might affect the discharge of peptide degradation products to clear the chamber for new protein substrates. In collaborative studies, we have found that an acyldepsipeptide antibiotic (ADEP) binds ClpP and affects stability of the tetradecamer. ADEP activates protein degradation to levels comparable to the ATP-dependent degradation observed with ClpA and ClpX complexes. However, only unfolded proteins can be taken up by ClpP in the presence of ADEP. Cross-linked ClpP, in which the heptamers cannot separate, also are activated for protein degradation by ADEP, implying that proteins must enter through the axial channel and not between the rings and further implying that ADEP binding changes the positions of the N-termini in a manner that enlarges the axial channel to permit proteins to enter. Studies of human ClpXP have confirmed that over expression of human ClpP affects the timing and extent of apoptotic cell death in response to DNA damage, death receptor binding, and kinase inhibition. The similarity in response to three divergent stress signaling pathways suggests that hClpP alters the basal structure or physiology of mitochondria. We have found that hClpP protein is lost from nutritionally deprived cells and from cells that are stressed following blockage of hClpP expression with siRNA. Cells lacking hClpP lose mitochondrial membrane integrity. These data suggest that hClpP is needed in mitochondrial during growth but is eliminated under non-growing conditions. Depletion of hClpX following treatment with siRNA also leads to cell death. A mitochondria-specific unfolded protein response elicited by down regulation of hClpX acts through the JNK1 and JNK2 pathways. We have expressed a novel substrate for hClpXP, GFP-SsrA, in mitochondria and shown that its levels are increased when either hClpP or hClpX is depleted from mitochondria. However, studies with purified hClpXP show that GFP-SsrA is a very poor substrate for wild type and mutant forms of hClpXP, suggesting that other factors in the mitochondria are needed to help mediate degradation. Studies are now directed at identifying an adaptor protein that can activate specific protein degradation by hClpXP. In collaborative studies, we have obtained the first X-ray crystal structure of an intact hexamer of Lon protease. This structure reveals several novel aspects of Lon protease and provides a clear model for the compartmentation o [summary truncated at 7800 characters]
