The process of translocating a large passenger domain by a much smaller beta domain is not currently understood. Described below are three models that have been proposed to explain passenger domain translocation. In one model, the C-terminus of the passenger domain is folded into the β-domain pore in the periplasm in a post-translocation conformation. The prefolded β-domain is then inserted into the OM and the passenger domain is transported across the OM by a concerted mechanism that possibly involves Omp85, an essential protein that promotes OM protein integration and assembly. An advantage of this model is that it circumvents the need for one or more passenger domains to be translocated through a relatively small barrel pore in the absence of an external energy source. A second translocation model focuses on the unusual architecture of passenger domains, which all appear to contain β-solenoid motifs. These motifs could supply the energy needed for translocation by folding on the extracellular side of the OM once a small portion has reached the cell surface. In this model, a short hairpin comprising the C-terminus of the passenger domain is positioned inside the barrel pore with its tip protruding into the extracellular space. Folding at the tip of the hairpin would then pull the rest of the passenger domain through the pore. A third model is based on the observation that the β-domain of IgA protease forms multimeric ring-like structures when the protein is produced in E. coli. The central cavity is about 20 in diameter, and was postulated to transport multiple passenger domains. A major focus of this project is EspP, a classical autotransporter associated with diarrheagenic strains of E. coli. It belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) family of autotransporters, whose passengers encode serine proteases that cleave various mammalian proteins. Biochemical studies have indicated that EspP is a monomer like NalP. Once the EspP passenger domain is translocated across the OM, it is cleaved from the membrane embedded β-domain between two asparagine residues (N1023/N1024) and released from the cell surface. The Asn/Asn cleavage site defines the boundary of the EspP passenger domain (residues 56-1023) and β-domain (residues 1024 1300). Although the passenger domain contains a serine protease motif located at residues 261-264, this motif is not used to cleave the two domains. Our goals for this project are to solve crystal structures of the pre- and post-cleavage forms of one or more autotransporters and to design experiments to probe substrate translocation across the outer membrane. Structure determination of a bacterial autotransporter To learn what happens to the β-domain after cleavage and release of the passenger domain, we determined the crystal structure of the native β-domain of EspP at 2.7 resolution in 2007. This is the first structure of an autotransporter β-domain post-cleavage, and it consists of a monomeric 12-stranded β-barrel with its N-terminal 15 residues inserted into the barrel lumen from the periplasmic side. In agreement with a recently proposed autocatalytic cleavage mechanism, residues implicated in cleavage are located deep inside the β-barrel, in a region of EspP that would be embedded in the OM. Interestingly, the structure suggests that two discrete conformational changes occur after cleavage and release of the passenger domain, one that confers increased stability on the β-domain and another that restricts access to the barrel pore. Our structure does not support an oligomeric translocation model, but rather a model in which a single β-barrel facilitates the translocation of a single passenger domain to the extracellular surface. Progress for 2012: Since solving the post-cleavage structure of EspP in 2007, we have been working on pre-cleavage structures by mutating active site residues so that secretion occurs but cleavage is prevented. We solved three new crystal structures, performed pulse-chase experiments using site-directed mutants, and did molecular dynamics simulations to deduce the reaction mechanism. Analysis of the residues and electrostatic surfaces surrounding the active site asparagine reveal how its side chain is constrained to rotamers favourable for cleavage, with the carboxyamide group correctly positioned over the main chain carbonyl carbon. We located potential catalytic water molecules that could be activated by three conserved acidic residues to deprotonate the active site asparagine. By comparing pre-and post-cleavage structures of EspP we conclude that a glutamate located at the active site loses a proton during or after cleavage that gets transferred to the new N-terminus, helping to overcome a significant energy barrier in the cleavage pathway. This manuscript was published in J. Mol. Biol. in 2012. To learn more about protein translocation, we initiated a new autotransporter project which focuses on proteins related to EspP but having a different gene organization. Whether these proteins have the same structure and function the same way, is currently unclear. This past year we solved structures of the (outer membrane) beta domains for intimin and invasin using state-of-the-art techniques for membrane protein crystallization. We began with intimin and eventually found conditions for detergent-based crystallization (the most common method for membrane protein crystallization here detergent mimics the lipid bilayer). These crystals diffracted X-rays to 3.2 resolution. We then explored crystallization using DMPC/CHAPSO bicelles (lipid discs capped by detergent) and monoolein lipidic cubic phase. Both of these methods envelop the membrane protein in lipids rather than detergent molecules, with consequent changes in crystal packing and crystal order. In both lipid systems the intimin crystals were significantly better ordered, diffracting X-rays to 1.8 resolution, and we solved thte intimin structure using selenomethionine MAD phasing on lipidic cubic phase crystals. This is the first ever example of a lipidic cubic phase structure determined by using experimental phases (there are fewer than 20 lipidic cubic phase structures solved to date, all using molecular replacement except for ours). We also recently solved the structure of invasin from lipidic cubic phase at 2.5 resolution. In addition to learning about protein translocation for these proteins (which differ structurally from EspP), we plan to write a methods paper comparing native data sets for intimin crystallized by detergent, bicelle, or lipidic cubic phase to compare spacegroups, packing, and crystal resolution. This work was recently published in Structure. A third focus of this proposal is a structure/function study of how Gram Negative bacteria fold and insert newly synthesized outer membrane proteins into the outer membrane. This essential function is accomplished by the BAM complex, consisting of BamA, an integral outer membrane protein, and four lipoproteins BamB, C, D, and E. We solved the structure of BamB (J. Mol. Biol. 2012), contributed to a structure/function study on BamC (J. Mol. Bio. 2012), and are currently working on structures of the other components and complexes. This work will impact drug and vaccine design against all Gram negative bacterial pathogens because this is the only completely conserved and essential complex for all of these bacteria.

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2012
Total Cost
$691,347
Indirect Cost
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State
Country
Zip Code
Bakelar, Jeremy; Buchanan, Susan K; Noinaj, Nicholas (2016) The structure of the β-barrel assembly machinery complex. Science 351:180-6
Kuszak, Adam J; Jacobs, Daniel; Gurnev, Philip A et al. (2015) Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40. J Biol Chem 290:26204-17
Noinaj, Nicholas; Kuszak, Adam J; Buchanan, Susan K (2015) Heat Modifiability of Outer Membrane Proteins from Gram-Negative Bacteria. Methods Mol Biol 1329:51-6
Noinaj, Nicholas; Buchanan, Susan K (2015) Summary and Future Directions. Methods Mol Biol 1329:279-80
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Kuszak, Adam J; Noinaj, Nicholas; Buchanan, Susan K (2015) Methods to Characterize Folding and Function of BamA Cross-Link Mutants. Methods Mol Biol 1329:137-47
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Noinaj, Nicholas; Kuszak, Adam J; Balusek, Curtis et al. (2014) Lateral opening and exit pore formation are required for BamA function. Structure 22:1055-62
Noinaj, Nicholas; Kuszak, Adam J; Gumbart, James C et al. (2013) Structural insight into the biogenesis of β-barrel membrane proteins. Nature 501:385-90
Madden, Jeremy T; Toth, Scott J; Dettmar, Christopher M et al. (2013) Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline. J Synchrotron Radiat 20:531-40

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