Structural investigation of Mycobacterial protein complexes involved in the stationary phase stress response

Abstract

36Large protein complexes play key roles in mediating biological processes in the cell. Little 37 structural information is known on the protein complex mediators governing persistence in the 38 host for Mycobacterium tuberculosis (Mtb). We applied the 'shotgun EM' method for the 39 structural characterisation of protein complexes produced after exposure to stationary phase 40 stress for the model Mycobacterium, M smegmatis (Msm). We identified glutamine synthetase 41 I, essential for Mtb virulence, in addition to bacterioferritin, critical for Mtb iron regulation, 42 and encapsulin, which produces a cage-like structure to enclose target proteins. Further 43 investigation found that encapsulin carries dye-decolourising peroxidase (DyP), a potent 44 protein antioxidant, as the primary cargo during stationary phase stress. Our 'proof-of-concept' 45 application of this method offers insight into identifying potential key-mediators in Mtb 46 persistence. 47 immune response (1). Although some studies have shown potential key mediators in Mtb 53 survival in the host (2-4) there is a lack of structural information to guide drug discovery or 54 elucidate biological function. 55 56Structural data provides compelling evidence for the existence of protein complexes which are 57 physiologically relevant to the cell. In addition, this data offers valuable information on subunit 58 composition and mechanism of interaction, which yields biological insight governing its 59 potential actions (5). Single-particle transmission electron microscopy (TEM) is a powerful 60 3 method to reconstruct large protein complexes. The method has been successfully used to solve 61 the structures of protein complexes in a range of organisms from homogenous (6) as well as 62 heterogenous (7-10) samples. Combining 3D reconstruction of protein complexes from 63negative stain EM images with information obtained from mass spectrometry data ('shotgun 64 EM') (10) allows for a potentially high-throughput approach to finding complexes which could 65 play a key role in Mtb pathogenesis. 66 67 Although the 'shotgun EM' approach offers a faster method for solving the structures of a range 68 of protein complexes without the need for extensive purification (11), the approach still needs 69 to be tested and adapted for the organism of application (9,10,12). Here, we present our 70 'shotgun EM' methodology for the purification and TEM 3D reconstruction of Mycobacterial 71protein complexes from the model organism, M smegmatis (Msm), after exposure to stationary 72 phase stress which is known to induce a protective effect against subsequent exposure to 73 oxidative stress (13). We identified three protein complexes (glutamine synthetase I (GSI) (E.C 74 6.3.1.2), bacterioferritin (BrfA) (E.C 1.16.3.1), and encapsulin); the initial identity assigned by 75 mass spectrometry, as well as the 3D model and subunit composition, was validated by fitting 76 of homologous crystallographic structures into the low-resolution density. We demonstrate that 77 encapsulin encloses...