James W Coulton

Biography

Co-chair of CREATE-CDMC Member of the Scientific Committee

Research Interest

The Coulton research group studies membrane proteins (MPs) that are required for transport of iron, an essential nutrient, across the bacterial cell envelope. Our on-going research collaborations with colleagues world-wide emphasize structural determinants of MPs required for transport, including solving their 3-D structures by X-ray crystallography. For import of iron-siderophore complexes, seven proteins in the cell envelope of Escherichia coli are essential. We use FhuA from E. coli as a model bacterial outer MP. TonB, partner protein of FhuA, is one of three proteins from the energy-transducing complex TonB–ExbB–ExbD that is embedded in the cytoplasmic membrane. Initial studies of the TonB interactome began with our X-ray structure 2GRX for the co-crystal of TonB–FhuA. We recently adopted complementary strategies to generate models for the 3-D organization of the TonB interactome. Having isolated abundant (mg) quantities of purified ExbB–ExbD complexes and substituted amphipols (APol) for detergents, we collected low-resolution data: small angle X-ray scattering (SAXS) and small angle neutron scattering (SANS) (Sverzhinsky et al., Journal of Membrane Biology 2014). Medium resolution electron microscopy resolved particles (10 nm diameter) by negative staining (Sverzhinsky et al., Structure 2014; Journal of Bacteriology 2015). Remarkably, the stoichiometry of ExbB4–ExbD2 changed on addition of TonB: ExbD4–ExbD1–TonB1. The accompanying video shows a scale model for interaction of known proteins of E. coli that participate in transport of iron-siderophores across the cell envelope. Video credit: A. Sverzhinsky. Our steepest challenge is to grow 3-D crystals, a partnership with I. Moraes at the Membrane Protein Laboratory, Diamond Light Source, Oxford; S. Iwata, Director. Growing MP crystals requires novel strategies and leading infrastructure: lipidic cubic phase and screening hundreds of conditions with nano-robotics. Crystals must be of sufficient quality that they diffract; to date we observe diffraction to 2.9 angstroms at the microfocus beamline I24, Diamond Light Source. Unlike crystals that are grown for soluble proteins (usually to 100 µm in size), the MP crystals of the ExbB–ExbD complex that we reproducibly grow in lipidic cubic phase are “showers”, only 5 to 10 µm in all dimensions. Our 2015 visits to world-unique beam lines, X-ray free electron laser XFEL, at Stanford University, and at SPring-8/SACLA in Japan provided opportunities for data collection on the ExbB-ExbD complex. When we fully understand the structure and function of TonB, ExbB, and ExbD, then we will know a critical mechanism whereby Gram-negative bacteria acquire iron. Knowledge advanced by outcomes from our research will enable the design of antibacterial compounds that block iron import, thus markedly slowing bacterial growth.