TY - JOUR T1 - Structural variability of EspG chaperones from mycobacterial ESX-1, ESX-3 and ESX-5 type VII secretion systems JF - bioRxiv DO - 10.1101/250803 SP - 250803 AU - Anne T. Tuukkanen AU - Diana Freire AU - Sum Chan AU - Mark A. Arbing AU - Robert W. Reed AU - Timothy J. Evans AU - Grasilda Zenkeviciutė AU - Jennifer Kim AU - Sara Kahng AU - Michael R. Sawaya AU - Catherine T. Chaton AU - Matthias Wilmanns AU - David Eisenberg AU - Annabel H. A. Parret AU - Konstantin V. Korotkov Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/10/13/250803.abstract N2 - Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. Additionally, we describe the structures of EspG3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG3 structures. Analysis of EspG1, EspG3 and EspG5 chaperones using small-angle X-ray scattering (SAXS) reveals that EspG1 and EspG3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG3-PE5-PPE4 complex based on the SAXS analysis.HighlightsThe crystal structure of EspG1 reveals the common architecture of the type VII secretion system chaperonesStructures of EspG3 chaperones display a number of conformations that could reflect alternative substrate binding modesEspG3 chaperones dimerize in solutionA model of EspG3 in complex with its substrate PE-PPE dimer is proposed based on SAXS dataAbbreviations usedEOMensemble optimization methodMDmolecular dynamicsRMSDroot-mean-square deviationNSDnormalized spatial discrepancyRMSFroot-mean-square-fluctuationsSAXSsmall-angle X-ray scatteringSECsize-exclusion chromatographySeMetselenomethionineTEVtobacco etch virusTCEPtris(2-carboxyethyl)phosphine ER -