Overview
Protein function and folding studied using biochemistry, design, structural biophysics, and molecular genetics. Mechanisms of AAA+ protein unfolding and degradation machines, protease systems that signal between cell compartments, and co-translational tmRNA-mediated ribosome rescue and protein tagging.

Research Summary
We study the relationships between protein structure, sequence, folding, and function, with particular attention to ATP-dependent machines that catalyze protein destruction and cellular systems that target proteins for destruction.

AAA+ degradation machines: Organisms ranging from bacteria to humans contain ATP-dependent proteases that consist of an AAA+ ATPase and an associated compartmentalized peptidase. The AAA+ subunits recognize native protein substrates, unfold these proteins, and translocate the unfolded polypeptide into the proteolytic chamber of an associated barrel-shaped peptidase for degradation. We study several ATP-dependent bacterial proteases, including ClpXP, ClpAP, and HslUV. The ClpX, ClpA, and HslU components of these proteases are hexameric-ring ATPases that share homologous AAA+ domains. ClpP and HslV are non-homologous double-ring peptidases. In collaborative studies with Tania Baker’s lab, we are probing the rules of substrate recognition, studying regulatory factors that enhance or inhibit recognition of specific substrates, and dissecting the mechanism by which these enzymes catalyze protein denaturation.

For energy-dependent bacterial proteases, unstructured peptide tags provide a site for the AAA+ ATPase to initially grasp protein substrates. There are many different classes of peptide targeting sequences but how ClpX or HslU bind these “degradation tags” is a major unsolved structural problem. Substrate selection can be also be regulated by interaction of the protease and/or substrate with adaptor proteins. For example, the dimeric SspB adaptor binds substrates bearing the ssrA-degradation tag and tethers them to ClpXP for efficient degradation. We have redesigned the ssrA tag to allow more efficient SspB-mediated substrate delivery, and are actively working to construct an SspB-dependent system as well as orthogonal systems to allow targeted and temporally controlled degradation of any desired protein in bacterial cells. SspB variants that are monomeric or that assemble only as obligate heterodimers have been engineered using computational protein design, and should be useful for these studies. We are also examining degradation of model substrates by ClpXP and ClpAP in E. coli to understand how proteolytic capacity changes depending upon environmental conditions and the levels of specific adaptor proteins.

Unfolding of native proteins by AAA+ ATPases is an active mechanical process that is an essential step in degradation and also provides a way for these enzymes to disassemble macromolecular complexes. Very stable proteins—those for which spontaneous unfolding can take months or even years—are unfolded in a few minutes or less in the enzymatic reaction. Current evidence suggests that, following binding of the peptide tag, the hexameric enzyme begins to translocate this tag through a central pore, generating a denaturation force when the attached native protein cannot pass through this narrow aperture. Denaturation of native protein substrates by AAA+ machines depends both on ATP hydrolysis and on the local stability of the substrate structure immediately adjacent to the degradation tag. By studying the degradation of model substrates varying in stability, we have shown that ClpX applies an unfolding force iteratively. Depending on the substrate, enzymatic unfolding of a single substrate can require as few as 10 or as many as 5000 cycles of ATP hydrolysis. This dramatic variation in the thermodynamic cost of unfolding occurs because protein substrates that resist denaturation are released from ClpX, resulting in a clutch-like mechanism that prevents stalling of the AAA+ motor when substrates cannot be unfolded.

Translocation of polypeptides is the essential mechanical function of AAA+ enzymes like ClpX, ClpA, and HslU. Translocation of the degradation tag drives protein unfolding and the unfolded polypeptide is then translocated into the peptidase for degradation. Remarkably, ClpX translocates polypeptides either in the C-terminal or N-terminal direction and can translocate two or three polypeptides at once. We are currently trying to identify the molecular determinants in the translocating chain that allow recognition by ClpX or HslU and how interactions between the translocating polypeptide and these ATPases help coordinate degradation. We are also probing the mechanisms by which communication between the AAA+ ATPases and peptidases contribute to efficient substrate degradation.

Although ClpX and HslU are both hexamers assembled from identical subunits, positive and negative allosteric interactions partition the six potential nucleotide-binding sites of each enzyme into three classes with asymmetric properties. Certain sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free even under conditions of ATP saturation. Binding to the degradation tag of protein substrates requires ATP occupancy of more than one set of sites. In ClpX, the second set of sites can be also be occupied by ADP. During the normal ATPase cycle, ClpX and HslU probably never pass through all-ADP states, allowing these enzyme to maintain interactions with protein substrates and with their partner peptidases.

By linking wild type and inactive mutant ClpX subunits to form “covalent” hexamers, we have found that ATP hydrolysis in a single subunit can drive the translocation of protein substrates into ClpP. Moreover, enzymes with just two active subunits unfold native substrates and use ATP as efficiently as wild type for substrate translocation. These and related studies provide evidence for directional communication between ClpX subunits, for additive subunit contributions to overall hexamer activity, and for a probabilistic sequence of ATP hydrolysis in different subunits of the hexamer. Molecular machines with highly diverse quaternary architectures and molecular functions (including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes) could share common operating principles based on similar asymmetric mechanisms.

DegS and periplasmic stress: Trans-membrane signaling between intracellular compartments is often controlled by regulated proteolysis. E. coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sE-dependent transcription of stress genes in the cytoplasm. This process requires a proteolytic cascade initiated by the DegS protease, which destroys a trans-membrane protein (RseA) that normally binds to and inhibits sE. We have shown that peptides ending with OMP C-terminal sequences bind the DegS PDZ domain, activate DegS cleavage of RseA, and induce sE-dependent transcription, suggesting that DegS acts as a sensor of envelope stress by binding unassembled OMPs. We are currently investigating the peptide-dependent activation of DegS, which involves relief of inhibitory interactions between its PDZ and protease domains, probing the determinants of DegS recognition of RseA, studying how DegS cleavage of RseA regulates the next steps in the proteolytic cascade, and determining how the RseB protein binds to RseA and inhibits its degradation by DegS.

The tmRNA tagging and ribosome-rescue system: To maintain protein-quality control, cells must rid themselves of incomplete or damaged proteins produced by errors during translation. In bacteria, this process is mediated by tmRNA (ssrA), a molecule with tRNA and mRNA-like properties. When translation stalls, tmRNA rescues the ribosome and mediates addition of a degradation tag to the nascent polypeptide. These ssrA-tagged proteins are subsequently degraded by cellular proteases, including ClpXP and ClpAP. We are studying the mechanisms that lead to translational stalling as well as the structure and function of tmRNA and proteins involved in its tagging and rescue activities.

We have found that tmRNA can act either at the 3’ end of mRNAs lacking stop codons or at internal mRNA sites, including rare codons and stop codons, where ribosomes appear to pause or stall, leaving an unoccupied A-site. In some instances, mRNA cleavage at this unoccupied A-site precedes tmRNA action and tagging at internal sites. Proteomic studies have shown that many full-length proteins are subject to tagging and preliminary studies suggest that tagging of this type may be connected to the protein-folding status of the nascent polypeptide. Using mutant tmRNAs encoding peptide sequences that do not lead to degradation of the tagged protein, we find that roughly 1 in 250 translation events in E. coli terminate in ribosome rescue and tagging.

Selected Publications
Martin, A., Baker, T.A. & Sauer, R.T. Rebuilt AAA+ motors reveal operating principles for ATP-fueled machines. Nature 437, 1115-1120. (2005)

Hersch, G.L., Burton, R.E., Bolon, D.N., Baker, T.A. & Sauer, R.T. Asymmetric interactions of ATP with the AAA+ ClpX6 unfoldase: allosteric control of a protein machine. Cell 121, 1017-1027. (2005)

Bolon, D.N., Grant, R.A., Baker, T.A. & Sauer, R.T. Specificity versus stability in computational protein design. Proc. Natl. Acad. Sci. USA 102, 12724-12729. (2005)

Tabtiang, R.K., Cezairliyan, B.O., Grant, R.A, Cochrane, J.C. & Sauer, R.T. Consolidating critical binding determinants by non-cyclic rearrangement of protein secondary structure. Proc. Natl. Acad. Sci. USA 102, 2305-2309. (2005)

Burton, R.E., Baker, T.A. & Sauer, R.T. Nucleotide-dependent substrate recognition by the AAA+ HslUV protease. Nat. Struct. & Mol. Biol. 12, 245-251. (2005)

Kenniston, J.A., Baker, T.A. & Sauer, R.T. Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing. Proc. Natl. Acad. Sci. USA 102, 1390-1395. (2005)

McGinness, K.E., Baker, T.A., & Sauer, R.T. Engineering controllable protein degradation. Mol. Cell 22, 701-707. (2006)

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