Raghavendar Reddy Sanganna Gari

Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species

We have established a reconstitution system for the translocon SecYEG in proteoliposomes in which 55% of the accessible translocons are active. This level corresponds to the fraction of translocons that are active in vitro when assessed in their native environment of cytoplasmic membrane vesicles. Assays using these robust reconstituted proteoliposomes and cytoplasmic membrane vesicles have revealed that the number of SecYEG units involved in an active translocase depends on the precursor undergoing transfer. The active translocase for the precursor of periplasmic galactose-binding protein contains twice the number of heterotrimeric units of SecYEG as does that for the precursor of outer membrane protein A.

Transient Collagen Triple Helix Binding to a Key Metalloproteinase in Invasion and Development

Skeletal development and invasion by tumor cells depends on proteolysis of collagen by the pericellular metalloproteinase MT1-MMP. Its hemopexin-like (HPX) domain binds to collagen substrates to facilitate their digestion. Spin labeling and paramagnetic nuclear magnetic resonance (NMR) detection have revealed how the HPX domain docks to collagen I-derived triple helix. Mutations impairing triple-helical peptidase activity corroborate the interface. Saturation transfer difference NMR suggests rotational averaging around the longitudinal axis of the triple-helical peptide. Part of the interface emerges as unique and potentially targetable for selective inhibition. The triple helix crosses the junction of blades I and II at a 45° angle to the symmetry axis of the HPX domain, placing the scissile Gly∼Ile bond near the HPX domain and shifted ∼25 Å from MMP-1 complexes. This raises the question of the MT1-MMP catalytic domain folding over the triple helix during catalysis, a possibility accommodated by the flexibility between domains suggested by atomic force microscopy images.

Dynamic Structure of the Translocon SecYEG in Membrane DIRECT SINGLE MOLECULE OBSERVATIONS

Background: Numerous proteins are exported across membranes by the translocon SecYEG, a highly conserved complex. Results: Multiple structural conformations and oligomeric states of SecYEG observed in lipid bilayers. Conclusion: Cytoplasmic membrane-external segments of SecYEG that orchestrate translocon function are highly dynamic. Significance: Direct visualization of disordered, flexible structures and oligomeric states in lipid bilayers provides a near-native vista of the translocon. Purified SecYEG was reconstituted into liposomes and studied in near-native conditions using atomic force microscopy. These SecYEG proteoliposomes were active in translocation assays. Changes in the structure of SecYEG as a function of time were directly visualized. The dynamics observed were significant in magnitude (∼1–10 Å) and were attributed to the two large loops of SecY linking transmembrane helices 6–7 and 8–9. In addition, we identified a distribution between monomers and dimers of SecYEG as well as a smaller population of higher order oligomers. This work provides a new vista of the flexible and dynamic structure of SecYEG, an intricate and vital membrane protein.

Glass is a Viable Substrate for Precision Force Microscopy of Membrane Proteins

Though ubiquitous in optical microscopy, glass has long been overlooked as a specimen supporting surface for high resolution atomic force microscopy (AFM) investigations due to its roughness. Using bacteriorhodopsin from Halobacterium salinarum and the translocon SecYEG from Escherichia coli, we demonstrate that faithful images of 2D crystalline and non-crystalline membrane proteins in lipid bilayers can be obtained on microscope cover glass following a straight-forward cleaning procedure. Direct comparison between AFM data obtained on glass and on mica substrates show no major differences in image fidelity. Repeated association of the ATPase SecA with the cytoplasmic protrusion of SecYEG demonstrates that the translocon remains competent for binding after tens of minutes of continuous AFM imaging. This opens the door for precision long-timescale investigations of the active translocase in near-native conditions and, more generally, for integration of high resolution biological AFM with many powerful optical techniques that require non-birefringent substrates.

Heparinoids Activate a Protease, Secreted by Mucosa and Tumors, via Tethering Supplemented by Allostery

Activation by glycosaminoglycans (GAGs) is an emerging trend among extracellular proteases important in disease. ProMMP-7, the zymogen of a matrix metalloproteinase secreted by mucosal epithelial and tumor cells, is activated at their surfaces by sulfated GAGs, but how? ProMMP-7 is activated in trans by representative heparin oligosaccharides in a length-dependent manner, with a large jump in activation at lengths of 16 monosaccharides. Imaging by atomic force microscopy visualized small complexes of proMMP-7 molecules linked by 8-mer lengths of heparinoids and extended assembles formed with 16-mer lengths of heparin. Complexes of proMMP-7 with polydisperse heparin or heparan sulfate were more diverse. Heparinoids evidently accelerate activation by tethering multiple proMMP-7 molecules together for proteolytic attack among neighbors. Removal of either the prodomain or C-terminal peptide sequence of KRSNSRKK from MMP-7 prevents formation of the long arrays induced by heparin 16-mers or heparan sulfate. The role of the C-terminus in activation assays suggests it contributes to remote, allosteric binding of GAGs. Enhancement of proteolytic velocity of MMP-by GAGs indicates them to be effectors of V-type allostery. GAGs from proteoglycans appear to assemble proMMP-7 molecules for activation, an event preceding its tumorigenic or antibacterial proteolytic activities at cell surfaces.

Single Molecule Studies of the General Secretory System

In bacteria and archaea many proteins use the protein conducting channel SecYEG either to transport across the membrane bilayer or to integrate into the bilayer. Further, it is known that the ATPase SecA binds SecYEG to perform translocation. Crystal structures of detergent-solubilized SecYEG and SecA bound to SecYEG have been reported [Nature 427, 36 (2004); Nature 455, 936 (2008)]. In recent years atomic force microscopy (AFM) has emerged as an important complementary tool to study membrane proteins at the single molecule level in near native conditions. In this work we study two central components of the bacterial secretory system (SecYEG and SecA) in membrane via AFM. We have obtained images of proteoliposomes containing just SecYEG, and SecYEG proteoliposomes assembled in the presence of SecA (SecY·A). All samples were adsorbed on mica surfaces and imaged in aqueous buffer solution. We collected several hundred images of each sample to provide statistics. Heights of SecYEG and SecY·A protruding above the lipid bilayer are in close agreement with crystal structure data and the topological asymmetry of SecYEG allows orientation determination. From volume calculations we are able to differentiate SecYEG monomers from dimers and higher order oligomeric states. Images of SecA bound to lipid (i.e, in the absence of SecYEG) were also obtained. In this case, the heights of SecA bound to the lipid are significantly different than the heights of SecY·A suggesting distinct binding modes of SecA to lipid compared to SecA to SecYEG. Further experiments and analysis will be required to conclusively determine the oligomeric state of active SecYEG during translocation.