Structural and biophysical characterisation of the interaction between OpcA from Neisseria meningitidis and the actin-binding domain of human alpha-actinin

Abstract

The outer membrane protein OpcA is a ten-stranded transmembrane protein that facilitates adhesion and invasion of Neisseria meningitidis into human epithelial and endothelial cells. Recently, the human cytoskeletal protein alpha-actinin was identified as a target of OpcA, and this interaction was shown to take place within the host cytoplasm after bacterial invasion. We have developed and optimised OpcA refolding and purification methods in order to improve solubility and prevent aggregation, and have investigated binding between OpcA and the actin-binding domain of alpha-actinin isoform 4 (hAct-4) using structural and biophysical techniques. Using NMR chemical shift perturbation, we have demonstrated that the two proteins bind directly to each other in vitro. Binding was also confirmed by surface plasmon resonance, which provided binding affinity and kinetics constants of this interaction. The OpcA/hAct-4 complex was crystallised, yielding crystals that diffracted to a resolution limit of 3.9A, which SDS-PAGE analysis indicated were composed of both proteins at approximately equal concentrations. Our work has not only provided evidence at the structural level that this interaction occurs directly, but has also developed a method of solubilising OpcA into a mixed detergent micelle, enabling production of high quality NMR spectra.

Date of Award	3 Jan 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeremy Derrick (Supervisor)