BPS2026 – Role of the identical nucleotide binding domains in the E. coli ABC transporter MetNI-Q

Membrane transporters contribute to the unique selective permeability of the plasma membrane, which is integral for cellular homeostasis. Found in every kingdom of life, ATP-binding cassette (ABC) transporters are a superfamily of ATP-powered unidirectional membrane transporters that transport a variety of substrates against chemical gradients. ABC transporters are thought to follow the widely accepted alternating access model. In this model, ATP binding at the interface of the highly conserved nucleotide binding domains (NBDs) causes a conformational change in the transmembrane domains, opening a pathway for substrate to cross the membrane. However, variations in the number of functional NBDs across the family raises questions regarding the role of ATP hydrolysis, specifically how ATP hydrolysis influences rates of transport, how it serves to reset the transporter for the next cycle, and how it affects the binding affinity of its cognate substrate binding proteins. To understand the mechanism of transport, we are studying the E . coli MetNI-Q transport system, which transports methionine into the cell. The MetNI transporter is a homodimer, and the structure of MetNI-Q reveals two identical NBDs that dimerize upon binding two ATP molecules in a head-to-tail orientation. To understand whether both NBDs are necessary for optimal function, we expressed and purified MetNI heterodimers with inactivating mutations in one or both NBDs. Preliminary in vitro kinetic data of ATP hydrolysis suggests that ATP hydrolysis is impaired if one NBD is not able to bind ATP. However, if two molecules of ATP are able to bind but only one NBD can hydrolyze ATP, this combination is sufficient to achieve wild type levels of substrate turnover. Given the highly conserved nature of the NBDs, a detailed understanding of their interdependence may be broadly applicable to the ABC transporter family.