title: Low resolution solution structure of an enzymatic active AhpC10:AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase.
abstract: The ability of bacteria to combat oxidative stress is imperative for their survival. The Alkyl hydroperoxide Reductase (AhpR) system, composed of the AhpC and AhpF proteins, is one of the dominant antioxidant defense systems required for scavenging hydrogen peroxide and organic peroxide. Therefore, it is necessary to understand the mechanism of the AhpR ensemble formation. In previous studies, we were able to elucidate conformational flexibility of Escherichia coli AhpF during the catalytic cycle and its binding site, the N-terminal domain (NTD), to AhpC. We proposed the novel binding and release mechanism of EcAhpC-AhpF, which is mediated by the well defined redox-state linked conformational changes associated with the C-terminal tail and active site regions of EcAhpC. Here, we have proceeded further to elucidate the solution structure of E. coli AhpC and the stable ensemble formation with EcAhpF using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. The EcAhpC-AhpF complex structure with a stoichiometry of AhpC10:AhpF2 reveals that dimeric EcAhpF in its extended conformation enables the NTD disulphide centers to come in close proximity to the redox-active disulphide centers of EcAhpC, and provides an efficient electron transfer. Furthermore, the significance of the C-terminal tail of EcAhpC in ensemble formation is elucidated. SAXS data-based modeling revealed the flexible C-terminal tail of EcAhpC in solution, and its exposed nature, making it possible to contact the NTD of EcAhpF for stable complex formation.
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research (A(∗)STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Republic of Singapore.
authors.forename: Neelagandan
authors.initials: N
authors.lastname: Kamariah
authors.affiliation: Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
authors.forename: Wilson
authors.initials: W
authors.lastname: Nartey
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research (A(∗)STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Republic of Singapore.
authors.forename: Birgit
authors.initials: B
authors.lastname: Eisenhaber
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research (A(∗)STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Republic of Singapore; School of Computer Engineering, Nanyang Technological University (NTU), 50 Nanyang Drive, Singapore 637553, Republic of Singapore; Department of Biological Sciences, National University of Singapore, 8 Medical Drive, Singapore 117597, Republic of Singapore.
authors.forename: Frank
authors.initials: F
authors.lastname: Eisenhaber
authors.affiliation: Bioinformatics Institute, Agency for Science, Technology and Research (A(∗)STAR), 30 Biopolis Street, #07-01 Matrix, Singapore 138671, Republic of Singapore; Nanyang Technological University, School of Biological Sciences, 60 Nanyang Drive, Singapore 637551, Republic of Singapore. Electronic address: ggrueber@ntu.edu.sg.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology; D054464:Peroxiredoxins / Q000737:chemistry*; D011487:Protein Conformation; D014961:X-Ray Diffraction
chemical_list: D029968:Escherichia coli Proteins; D054464:Peroxiredoxins
keywords: Alkyl hydroperoxide Reductase; Bioenergetics; Oxidative stress; Reactive Oxygen Species; Redox homeostasis; Small angle X-ray scattering