Our lab uses molecular biology, protein chemistry, and X-ray crystallography to investigate the structure and mechanism of the proteins and protein complexes involved in targeting, translocation and catalysis at the cellular membrane surface. We are particularly interested in membrane bound proteases.
Below we briefly descibe some of the proteins and protein complexes currently being investigated in the lab.
Shown in the figure above are the components of the Sec-dependent protein targeting and translocation system. Each protein whose structure has been solved is shown in ribbon diagram, rendered to scale. Each protein whose structure has not yet been solved is shown as a blue block. Those structures that were solved in the Paetzel lab are shown in blue ribbon (for more information see the publication and structure sections of the Paetzel Lab website).
The majority of protein translocation in bacteria occurs post translationally via the Sec-system. The Sec-system is made up of the proteins SecA, SecB, SecD, SecE, SecF, SecG, SecY, YajC, YidC, SRP (Ffh), SRP receptor (FtsY) type I signal peptidase (SPase I), and signal peptide peptidase (SppA) (See Figure above). Typically, the homotetrameric molecular chaperone SecB (or in some cases the homodimeric chaperone CsaA) interacts with the newly synthesized preprotein in the cytoplasm and targets the protein to the SecA-SecYEG translocase at the membrane surface. The homodimer SecA, in an energy dependant event (ATP), aids in the partial translocation of the presumably unfolded protein across the membrane. The pore through which the protein passes is thought to be formed by the integral membrane proteins SecY, SecE and SecG. The role of three additional membrane bound proteins, SecD, SecF (both of which are predicted to have large periplasmic domains) and YajC is less clear, but are thought to improve translocation efficiency and plug the pores opening between translocation events. After the targeting and translocation steps have occurred the secretory pre-protein is then released from the SecYEG pore and is tethered to the membrane via its signal peptide. Type I signal peptidase (SPase), an essential membrane-bound endopeptidase, functions to cleave off the signal peptide releasing the secretory protein to its final destination. The remnant signal peptides, left in the membrane following preprotein cleavage by SPase, are then cleaved by signal peptide peptidase (SppA) which allows for removal of signal peptides from the membrane. Polytopic membrane proteins are targeted to the membrane via the signal recognition particle (SRP; Ffh and a 4.5 S RNA) and are assisted in the membrane assembly by the SRP receptor (FtsY) and the recently described protein YidC.
The analysis of the Sec-system at the atomic level will provide a wealth of new and complementary information for the understanding of how proteins are secreted from the cell or from one compartment in the cell to another.
This work will likely provide important insights into the structural reasons for many hereditary and autoimmune diseases that result from mutations in the genes involved in protein trafficing and translocation.
Bacteria are used to produce many pharmaceutically and biotechnologically important proteins at industrial scale by expressing and secreting the proteins of interest directly into the growth media via the bacterial general secretory pathway. Improving the efficiency of the bacterial Sec-system may lead to greater yields, profits and potential new products.
The bacterial Sec-dependent protein translocation system and signal peptidase in particular are being pursued as targets for the development of a novel class of antibiotics.
Signal peptidase (SPase) is the membrane bound serine endoprotease responsible for cleaving off the N-terminal signal (or leader) peptide extensions from secretory proteins. A major focus of our research involves studying the structure and catalytic mechanism of signal peptidases. The first crystal structure of E. coli signal peptidase revealed a novel proteolytic mechanism that utilizes a serine nucleophile, lysine general base and an unusual oxyanion-hole made up of a serine side chain hydroxyl group and a main chain amide group to stabilize the oxyanion intermediate. It also revealed a shallow hydrophobic binding pocket adjacent to the active-site which explained its substrate specificity (small, neutral residues at the -1 and -3 position relative to the cleavage-site) (Paetzel et al, 1998, 2002, 2004). We are now working to solve crystal structures of SPases from other species as well as inhibitor-enzyme and substrate-enzymes complexes. In addition, we are pursuing the structural and mechanistic analysis of other enzymes that utilize the unusual Ser/Lys catalytic mechanism. These include the VP4 protease from birnavirus and the bacterial signal peptide peptidase (sppA).