Conservation of Helical Bundle Structure between the Exocyst Subunits Nicole J. Croteau1, Melonnie L. M. Furgason 1 , Damien Devos2, Mary Munson 1 * 1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2 EMBL, Heidelberg, Germany Abstract Background: The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies. Methodology/Principal Findings: Using advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60% a-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners. Conclusions/Significance: Although all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folded domain of Sec10p. Citation: Croteau NJ, Furgason MLM, Devos D, Munson M (2009) Conservation of Helical Bundle Structure between the Exocyst Subunits. PLoS ONE 4(2): e4443. doi:10.1371/journal.pone.0004443 Editor: Dafydd Jones, Cardiff University, United Kingdom Received November 24, 2008; Accepted January 2, 2009; Published February 13, 2009 Copyright: 2009 Croteau et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the EU grant 3D-Repertoire (LSHG-CT-2005-5120828) to D.D., and by the US National Institutes of Health grant GM068803 to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mary.munson@umassmed.edu Introduction The exocyst is a large, eight protein complex localized to sites of polarized secretion that is required for exocytosis and cytokinesis in eukaryotes ([1–3]; and references therein). Its specific function(s) is unclear, but it interacts with small Ras superfamily GTPases on secretory vesicles and the plasma membrane, where it is hypothesized to tether vesicles to the plasma membrane prior to membrane fusion [4]. The complex also interacts with the regulatory protein, Sec1p [5], and the plasma membrane SNARE (soluble N-ethylmaleimide sensitive protein receptor) protein Sec9p [6]. These interactions indicate a role for the exocyst and its subunits in the quality control of exocytic trafficking, as well as in facilitating SNARE complex assembly and vesicle fusion at the plasma membrane. Elucidation of the exocyst’s function, and that of other related tethering complexes [7–10], requires biochemical and structural analyses of the individual subunits, as well as various protein-protein interactions within the complex. Each of the exocyst subunits is predicted to be a-helical. They possess less than 10% sequence identity with each other, although limited sequence similarity has been detected using PSI-BLAST analyses [7,11]. They also show little similarity to other proteins or domains, except for short regions of predicted coiled coils [1,7]. Several recent crystal structures of domains from individual subunits have been determined: nearly full-length yeast and human Exo70 [12–14], and the C-terminal domains of yeast Exo84p [12], yeast Sec6p [15] and Drosophila Sec15 [16]. They show similar structures containing multiple helical bundles, yielding an overall similar shape (Figure 1A). Specific details of the bundles differ, especially the surface residues, but the helical bundle topologies are identical, suggesting divergent evolution from an ancient exocyst ancestor protein for these four exocyst components [3,15]. Progress for the other subunits has been hindered by lack of soluble protein. Insolubility can occur for many different reasons: recombinant proteins may not fold correctly when overexpressed in Escherichia coli cells, they may not have the correct post- translational modifications, or they may be insoluble in the absence of co-factors or binding partners. Much effort has been spent to develop methods to address these issues ([17–21]; and references therein), including the use of different strains, selective growth conditions, fusion tags, co-expression with binding partners, and expression of independently folded structural domains. Often, the latter strategy is approached by using secondary structure predictions to deduce a domain, making numerous constructs with slight variations at the N- and/or C- termini, or by using limited proteolytic digestions to cleave unstructured or floppy regions, thereby defining a core domain. Limited proteolysis proved critical for most of the current exocyst structures; however, this strategy relies on availability of at least PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4443