Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Backbone dynamics of the N-terminal domain of Neisserial DsbD (#237)

Stefan Nebl 1 , Roxanne P Smith 2 , Bradley C Doak 1 , Gaurav Sharma 1 , Martin L Williams 1 , Begoña Heras 2 , Biswaranjan Mohanty 1 , Martin Scanlon 1
  1. Monash Institute of Pharmaceutical Science, Parkville, VICTORIA, Australia
  2. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, Melbourne, VIC, Australia

Most Gram-negative bacteria contain a system of thiol-disulfide oxidoreductase enzymes known as disulfide bond formation proteins (Dsb). In pathogenic strains of Neisseria, one of these proteins, namely disulfide bond oxidoreductase D (DsbD) is essential for viability (1). Therefore, inhibitors of Neisserial DsbD may provide narrow-spectrum anti-Neisserial agents. This research is aimed at characterising possible structural dynamics that may be critical for the function of Neisserial DsbD.

DsbD comprises three domains; a transmembrane-domain (t-DsbD), a C-terminal domain (c-DsbD) and an N-terminal domain (n-DsbD) (2). Each domain contains two catalytic cystine residues that participate in thiol-disulfide exchange reactions (3, 4). Interaction between c-DsbD and n-DsbD is oxidation state dependant, even though the two domains have similar redox potentials. Based on published crystal structures the so-called “Phenyl-cap loop” of n-DsbD has been postulated to protect the active site from non-specific redox reactions (1, 5-7). However, the question of how unidirectional electron flow is maintained remains poorly understood.

Herein, we present backbone dynamics of n-DsbD in both oxidised and reduced states through 15N-T1, T2, 1H-15N steady-state Het-NOE, 15N and 1HN relaxation dispersion NMR experiments. Het-NOE relaxation data reveals large amplitude, subnanosecond-nanosecond motions of the Phenyl-cap loop exclusively in the oxidised state. Backbone 1HN relaxation dispersion data revealed slow motion on the microsecond-millisecond time scale in n-DsbDOx for the two residues flanking the catalytic cysteines. These motions were absent in n-DsbDRed. The lack of dynamics in the reduced state is consistent with the hypothesis that these motions modulate the interaction between n-DsbDOx and c-DsbDRed and are responsible for the unidirectional electron flow through DsbD.

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