Molecular Models for the Core Components of the Flagellar Type-III Secretion Complex

Abstract

We show that by using a combination of computational methods, consistent three-dimensional molecular models can be proposed for the core proteins of the type-III secretion system. We employed a variety of approaches to reconcile disparate, and sometimes inconsistent, data sources into a coherent picture that for most of the proteins indicated a unique solution to the constraints. The range of difficulty spanned from the trivial (FliQ) to the difficult (FlhA and FliP). The uncertainties encountered with FlhA were largely the result of the greater number of helix packing possibilities allowed in a large protein, however, for FliP, there remains an uncertainty in how to reconcile the large displacement predicted between its two main helical hairpins and their ability to sit together happily across the bacterial membrane. As there is still no high resolution structural information on any of these proteins, we hope our predicted models may be of some use in aiding the interpretation of electron microscope images and in rationalising mutation data and experiments.

High-resolution view of the type III secretion export apparatusin situreveals membrane remodeling and a secretion pathway

“…In addition, our in situ structure revealed that InvA forms a second cytoplasmic ring 6 nm in diameter that is located in close apposition to the bacterial membrane and in alignment with the larger ring. We hypothesize that this ring is formed by a large cytoplasmic loop (amino acids 131 to 196) predicted by secondary structure and molecular modeling within the amino terminal half of InvA [33]. Consistent with this hypothesis, removal of the large cytoplasmic domain of InvA did not affect the localization and organization of its membrane proximal ring.…”

“…The export apparatus is a central core component of T3SS that is thought to mediate the passage of type III secreted substrates through the bacterial membrane, a critical step during the protein translocation process. All the core components of the export apparatus (SpaP, SpaQ, SpaR, SpaS, and InvA in the S. Typhimurium SPI-1 T3SS) are predicted to be integral membrane proteins [33]. However, a recent cryo-EM structure of an isolated sub-complex of the flagellar-associated export apparatus made up of FliP, FliQ, and FliR (homologs of SpaP, SpaQ and SpaR) revealed that none of these proteins adopt a typical membrane protein topology and instead, they assemble into a helical structure that through fitting into a low resolution in situ structure of the injectisomes was proposed to be largely located within the bacterial periplasm [27].…”

“…However, its role in the protein secretion process is poorly understood. Secondary structure predictions and molecular modeling indicate that the amino terminal half of this protein family contains several transmembrane segments and its large carboxyterminal domain is located entirely within the bacterial cytoplasm [32,33]. It has been proposed that this protein family, alone or in association with other components of the export apparatus, may function as a proton or Na+ channel that contributes to energizing the secretion process [34,35].…”

Algorithms for matching partially labelled sequence graphs

“…In previous studies, the GREMLIN method [16] had been found to give good quality contact predictions [17, 18] and also has the benefit of generating a best structure match to the predicted contacts (based on an iterated double-dynamic programming algorithm [19]). (http://gremlin.bakerlab.org/index.php).…”

“…The accuracy of a predicted contact was evaluated as previously [18] using a ‘soft’ measure of contact based on either the separation of the -carbon atoms or the pseudo-centroids of the positions (as defined above), Both distances were inverted to give a score by a Gaussian function with an ideal separation of 10 Å for pairs of -carbon atoms or 6 Å for pseudo-centroids. (With an -carbon–centroid ‘bond’ of 2 Å, this is equivalent to the 10 Å -carbon separation).…”