Structure and function of the Bacterial metal ion effluxer SilP
Lab rotation project description
The proposed project is between the laboratory of Dr. Thomas Sorenson (Diamond Light Source), an expert in structure determination of P-APTases, and Dr. David Scott, a expert in biophysical methods (Research Complex at Harwell). This project provides a unique opportunity to work in an internationally leading facility with world class researchers. As such the student will be expected to spend at least 50 % of their time located within these facilities, although longer placements are very strongly encouraged.
Diamond/Research Centre at Harwell
Lab rotation 1, 2 & 3
Linked PhD Project Outline
Linked PhD Project Outline - snippet
Silver cations, Ag+ , are increasingly used as an antimicrobial, since they are highly toxic to eubacteria, and possess little toxicity to humans generally. However, increasing indiscriminate use of antimicrobials has seen a steady increase in bacterial silver resistance. Central to understanding bacterial silver resistance is the metal cation specific effluxer, SilP, which transports silver cations across the inner membranes of Gram negative bacteria. SilP is a P1B-ATPase, and is thought to couple conformational change with catalytic turnover of ATP and ion transport. The research question we want to answer is how does SilP discriminate between different cations and how this is coupled to conformational change and efflux.
P1B-type ATPases are a subfamily of membrane proteins that bind selectively transition metal cations (i.e., Cu+ , Ag+ , Zn2+, Cd2+, Pb2+). They undergo a well defined Post-Albers reaction that leads to a conformational change between two states –E1 and E2- which is coupled to both ATP hydrolysis and ion transport. There is a structure of a Gram positive copper P1B-type ATPase from Legionella pneumophila CopA was crystallised in the presence of Al4 - ; the structure of which is hypothesized to represent the Cu+ free E2-Pi state of the protein, and with BeF4 - which is thought to mimic the E2-P state . In addition, there is a related P1B-ATPase Zinc effluxer structure, ZntA, also crystallised in the same forms as CopA. Much of the putative copper binding and transport mechanism of these two proteins was inferred from appeals to homology arguments with the eukaryotic Type I ATPase SERCA1a, however there are significant sequence differences to the degree where there is no proton counter transport occurring in CopA or ZntA. SilP has 60 % sequence identity to CopA and 30 % to ZntA, and crucially differs from them in the regions thought to involve ion recognition and transport. The first of these the HMBD (Heavy Metal Binding Domains) is some 100 residues longer than that seen in CopA (74 residues long) and ZntA (70 residues long).
The project will be to use structural, molecular and spectroscopic techniques to reveal the molecular details of how SilP works. Results of this work are expected to be of the highest impact, and hence the aim is to publish them in the higher impact journals.