O3-6 Structural insights into the NyxA/B effector family

Abstract

Brucella spp. survival and replication inside host cells requires a type IV secretion system (T4SS  named VirB. The T4SS is a molecular nanomachine that translocates effector proteins into the host cell to subvert host cell function, metabolism and defenses. Although many VirB substrates have been identified in Brucella, few of them have been characterized functionally and structurally. NyxA and NyxB are homologous and conserved proteins found to be translocated by Brucella into the host during infection 1. The proteins were found to interact with the human Sentrin specific protein SENP3 and modify its localization in the cell. In this study we have investigated the structural properties of NyxA/B. We purified NyxA and NyxB recombinantly and solved the crystal structure of NyxB at a resolution of 2.5 Å. The protein reveals a novel dimeric fold that was confirmed by Multi-angle light scattering (MALS) and Small-Angle X-ray Scattering (SAXS) experiments. Structural analysis of the NyxB surface revealed an acidic pocket delineated by several residues strictly conserved in NyxA. In the context of the dimer, these surfaces are juxtaposed to form an extended concave negatively charged. The structure enabled the design of specific mutants at a groove formed by the dimer. A structure-function study will be presented, using site- directed mutagenesis, in vitro and in cellulointeraction assays, providing insights into NyxA/B interaction with SENP3.