Our goal is to understand the mechanism and regulation of two classes of macromolecular machines: the Clp/Hsp100 family of protein unfolding enzymes and the proteins that catalyze DNA transposition. These biological catalysts are being studied using biochemistry, structural biology, molecular biology, and genetics.
Protein-catalyzed protein remodeling
Cells need to be able to unfold stable proteins to take apart protein complexes and aggregates, to transport proteins between cellular compartments and to degrade unstable or damaged proteins. Members of the Clp/Hsp100 protein family (a AAA+ ATPase subfamily) are powerful protein-unfolding enzymes. Clp/Hsp100 proteins are present in bacteria, plants, and animals. We initially identified the E.coli ClpX protein based on its ability to destabilize an exceedingly stable protein-DNA complex. ClpX also forms a complex with a peptidase complex known as ClpP to generate the ClpXP ATP-dependent protease.
Using model protein substrates we demonstrated that Clp/Hsp100 enzymes have the capacity to completely unfold their substrate proteins. This unfolding reaction is essential for Clp-mediated protein degradation, as the entry pore to the ClpP protease chamber is ~10Å, and therefore too small to allow passage of anything other than an unfolded polypeptide. Some of our current studies are focused on understanding the mechanism of protein unfolding, and elucidating how the protein processing reactions that result in complete unfolding for proteolysis, differ from those that are involved in destabilizing protein complexes.
We are also very interested in the strategies used by Clp/Hsp100 proteins to recognize proteins as substrates and in how this recognition is regulated. We recently completed a large-scale proteomic screen for new cellular substrates for ClpXP. This analysis identified about 60 new substrate proteins. These proteins included transcription factors, metabolic enzymes and proteins involved in the starvation and oxidative stress responses. An additional 30 proteins were identified in cells experiencing DNA damage. This analysis reveals that ClpXP plays a large role in resculpting the bacterial proteome, essentially as cells cope with changing environmental conditions.
Substrate proteins carry specific peptide sequences in exposed regions, often near either the N- or C-terminus of the protein, that are used as substrate-recognition tags. For ClpX, we have identified and characterized five classes of these recognition motifs. These peptide sequences can be recognized directly by the Clp/Hsp100 enzyme, or recognition can be further controlled by the action of additional proteins that serve as delivery factors. One such factor is the E. coli SspB protein. We have recently solved the structure of this protein with its bound peptide. This analysis, coupled with biochemical experiments, has provided insight into the mechanism used to regulate protein-recognition within the cell.
DNA Transposition: Transposable elements appear to have successfully invaded all forms of life, promoting their movement from one DNA site to another by a type of genetic recombination called transposition. The impact of transposition on genome stability and human health is immense. Many transposable elements can insert into essentially any DNA sequence and are thus a common source of mutations and genome rearrangements. The rapid spread of antibiotic resistance genes is largely a result of transposable elements moving throughout bacterial populations. Furthermore, retroviruses, including HIV, integrate into the host chromosome via a mechanism nearly identical to transposition. Although transposition of most elements is rare, the bacterial virus Mu can achieve 100 rounds of transposition in one hour; thus Mu transposition is well suited for mechanistic analysis.
Transposition of many elements occurs using a common set of DNA cleavage and joining reactions. This similarity in the mechanism is reflected in the proteins that catalyze the reactions. Structural and functional studies of the Mu transposase have contributed to the understanding of this important protein family. We have found and characterized regions of Mu transposase that are homologous to regions in other transposases and retroviral integrases. Structural studies of the central domain of Mu transposase and two retroviral integrases confirmed this relationship by demonstrating that the core domains of these proteins have a nearly identical fold. Analysis of the arrangement of subunits in the active tetramer of the Mu transposase also has provided mechanistic insight into how pairing of DNA molecules is coupled to catalysis of recombination.
Transposons are among the simplest genetic entities, yet they often exhibit sophisticated means of interacting with their host cells and responding to changing cellular environments. Illuminating the regulation of transposition is intricately intertwined with understanding the mechanism. Mu transposition is subject to at least two types of control: (1) regulation of DNA target site choice; and (2) control of the choice between using a non-replicative or a replicative transposition mechanism. In each of the examples of regulation of the Mu transposition pathway, the process is either known or proposed to involve direct protein-protein contacts between the transpososome and other proteins. We are currently focusing on defining interactions between Mu transposase and its regulatory proteins, and probing how these interactions provide precision and flexibility to the recombination mechanism.
Wang KH, Sauer RT and Baker TA (2007) ClpS modulates but is not essential for bacterial N-end rule degradation. Genes Dev. 21:403-8.
Neher SB, Villén J, Oakes EC, Bakalarski CE, Sauer RT, Gygi SP and Baker TA (2006) Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon. Mol Cell 22:193–204.
Levchenko I, Grant RA, Flynn JM, Sauer RT and Baker TA (2005) Versatile modes of peptide recognition by the AAA+ adaptor protein SspB. Nat Struct Mol Biol. 12:520-525.
Kenniston JA, Baker TA, Fernandez JM and Sauer RT (2003) Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of an AAA+ degradation machine. Cell 114: 511-20.
Neher SB, Flynn JM, Sauer RT and Baker TA (2003) Latent ClpX-recognition signals ensure LexA destruction after DNA damage. Genes and Development 17: 1084-1089.
Flynn JM, Neher SB, Kim YI, Sauer RT and Baker TA (2003) Proteomic discovery of ClpXP substrates reveals five classes of ClpX-recognition signals. Mol Cell.11: 671-83.