Transposable elements 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 genomic architecture and human health is immense. Many transposable elements can insert into any DNA sequence and are thus a common source of mutations. The 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. A related recombination reaction is also responsible for assembly of the immunoglobulin and T cell receptor genes during development of the immune system.
Our work on DNA transposition is centered on studies of bacteriophage Mu, which transposes extraordinarily frequently, making it an excellent system for analyzing the mechanism of transposition. Our interest in proteins that catalyze the disassembly and unfolding of other proteins arouse directly from studies of Mu transposition. The ClpX chaperone, a member of the Clp/Hsp100 ATPase family (a subfamily of the AAA+ ATPases), is required to disassemble the transposition machinery after recombination is complete. Clp/Hsp100 proteins are present in bacteria, plants, and animals, where they are involved in intracellular degradation, protein transport into organelles, solubilization of protein aggregates, and the dismantling of supramolecular assemblies.
Mu Transposase: A Member of the Transposase/Integrase Superfamily
Transposition of many elements occurs using a common set of DNA cleavage and joining reactions that take place on the two ends of the mobile element's DNA. This similarity in the mechanism is reflected in the proteins that catalyze the reactions. Mu transposase (MuA) carries a core domain containing residues that contribute to the enzyme's active site. This domain is highly conserved among many transposases and the retroviral integrases. We have determined how the conserved catalytic domains are organized in the active tetramer of Mu transposase. These experiments led to two important conclusions: (1) the same catalytic domain donates the active site for both DNA cleavage and the subsequent DNA joining on one end of the genome, and (2) the subunit donating this catalytic domain to one end of the genome is bound to the other DNA end. This interwoven subunit arrangement explains the precision with which recombination occurs using a pair of DNA signals and provides an example of how the architecture of a protein-DNA complex can define the reaction products.
Regulation of Transposition
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. An understanding of the regulation of transposition is intricately intertwined with an understanding of 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 nonreplicative or a replicative transposition mechanism. Regulation appears to involve direct protein-protein contacts between the Mu transposase and other proteins. We are focusing on defining interactions between Mu transposase and its regulatory proteins, and probing how these interactions provide precision and flexibility to the recombination mechanism. (A grant from the National Institutes of Health provides support for these projects on DNA transposition.)
Protein-Catalyzed Disassembly of Protein-DNA Complexes and Chaperone-Assisted Degradation
Timely disassembly of higher order protein-DNA complexes is essential for the proper control of biological processes. We became interested in protein disassembly when we found that the ClpX protein, a member of the Clp/Hsp100 family, promotes a protein-remodeling reaction that helps release the transposase from the DNA following recombination. This work contributed to the conclusion that Clp/Hsp100 proteins are chaperones specifically suited for disassembling or disaggregating other proteins. ClpX also functions as an essential component of the ClpXP protease. The ClpP component is a cylindrical protein chamber that provides the peptidase-active sites to degrade proteins that are chosen and delivered by ClpX. To increase our understanding of Clp/Hsp100 protein function, we are focusing on four research areas.
Protein processing during disassembly and degradation. Several groups studying Clp/Hsp100 proteins demonstrated that these enzymes have the capacity to unfold their substrate proteins completely. My group completed two studies of this type with ClpXP. We found that the rate of unfolding of the stable model substrate was accelerated by 7 orders of magnitude by ClpX. Furthermore, experiments with proteins of various intrinsic stabilities strongly supported the conclusion that unfolding occurs via an active mechanism, rather than by passive trapping of the transiently unfolded state. Unfolding is essential for ClpXP-mediated protein degradation, as the entry pore to the ClpP protease chamber is ~10 Å, too small to allow passage of anything other than an unfolded polypeptide.
With the knowledge of the ClpX mechanism based on degradation studies, we returned to studying the mechanism used by ClpX to destabilize the Mu transposase–DNA complex. Several lines of experiments reveal that protein unfolding by ClpX is involved in disassembly, but that only some of the subunits in the complex are unfolded. Thus, we conclude that the ability of ClpX to unfold proteins is sufficient to explain its role in both protein destabilization and ATP-dependent proteolysis.
Substrate recognition. Our work has helped formulate the conclusion that Clp/Hsp100 proteins recognize short regions of peptide sequence exposed on a folded protein. A short peptide from the carboxyl-terminal domain of MuA is required for its recognition by ClpX and is sufficient to convert a heterologous protein into a ClpX substrate. The ssrA degradation tag, an 11-residue peptide added cotransitionally to proteins as a result of ribosome stalling, is also sufficient to convert essentially any linked protein into a ClpX substrate. Analysis of proteins carrying these tags revealed that the tag peptides are unstructured, do not themselves destabilize proteins, and directly bind to ClpX. Using a proteomic screen to identify new substrates, we have identified more than 60 proteins that interact with ClpX (see below). Analysis of these proteins revealed the presence of five classes of ClpX-recognition motifs; one of these motifs is ssrA-like and one is MuA-like. These results represent the first description of general rules governing substrate recognition by an AAA+ ATPase.
SspB: a specificity-enhancing factor for ClpX. In addition to direct recognition, regulatory proteins enhance specificity and allow regulation of substrate recognition by ClpX/Hsp100 proteins. We identified and solved the crystal structure of SspB (stringent starvation protein B), a regulatory protein that binds to the ssrA tag, enhances binding of tagged proteins to ClpX, and stimulates degradation of these substrates by ClpXP. The structure reveals a well-defined peptide-binding cleft and suggests how the SspB-substrate complex may dock on ClpX to enhance recognition.
Impact of Clp-mediated degradation of the bacterial proteome. We have developed a method to trap proteins in vivo that are substrates for ClpXP or the related enzyme ClpAP. This allowed the identification of more than 60 new ClpXP substrates in cells growing under nonstressed conditions; numerous additional proteins were found when cells were exposed to a DNA-damaging agent. The proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. We are continuing to use this approach to understand the global role of proteolysis in resculpting the proteome in response to changing environmental conditions.
As of March 23, 2016