Structural variability of EspG chaperones from mycobacterial ESX-1, ESX-3 and ESX-5 type VII secretion systems

Abstract

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 EspG 1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG 1 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 EspG 3 chaperones from four different crystal forms. Comparison of available EspG 3 structures reveals several conformations of the putative PE-PPE binding site. Analysis of EspG 1 , EspG 3 and EspG 5 chaperones using smallangle X-ray scattering (SAXS) reveals that EspG 1 and EspG 3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific PE5-PPE4-EspG 3 complex based on the SAXS analysis.. CC-BY-NC-ND4.0 International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/250803 doi: bioRxiv preprint first

The ESX-1 substrate PPE68 has a key function in the ESX-1 mediated secretion inMycobacterium marinum

“…Based on available structural data on PE:PPE:EspG complexes and observed molecular weight of the major cytosolic complex, we postulate that the MMAR_2894:PPE68:EspG 1 complex forms a dimer of trimers. Dimers of EspG 3 from M. tuberculosis and M. smegmatis have been crystalized, suggesting that dimerization could occur via the chaperone (12, 67).…”

PE5-PPE4-EspG3 trimer structure from mycobacterial ESX-3 secretion system gives insight into cognate substrate recognition by ESX systems

“…Our structure is the first of EspG3 solved in complex with a cognate PE-PPE dimer, and thus we wanted to compare it to the previously solved unbound EspG3 structures. In total, there are six available EspG3 structures, four of EspG3ms (PDB codes: 4L4W, 4RCL, 5SXL, and 4W4J (13,16)), one EspG3mt (4W4I (13)), and one EspG3mm (5DLB (16)). These six structures can be classified into two different forms, an 'open' and a 'closed' form.…”

“…PE5ms-PPE4ms-EspG3ms trimer SAXS data (SASDDX2 (16)) was compared to a single copy of the mixed PE5mt-PPE4mt-EspG3mm trimer structure (PDB code: 6UUJ) using CRYSOL (17). Ab initio reconstruction of the envelope was completed using GASBOR (33).…”