Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux.

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism.

P(II) signal transduction proteins are ATPases whose activity is regulated by 2-oxoglutarate.

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. In all these organisms, P(II) proteins coordinate many facets of nitrogen metabolism by interacting with and regulating the activities of enzymes, transcription factors, and membrane transport proteins. The primary mode of signal perception by P(II) proteins derives from their ability to bind the effector molecules 2-oxoglutarate (2-OG) and ATP or ADP. The role of 2-OG as an indicator of cellular nitrogen status is well understood, but the function of ATP/ADP binding has remained unresolved. We have now shown that the Escherichia coli P(II) protein, GlnK, has an ATPase activity that is inhibited by 2-OG. Hence, when a drop in the cellular 2-OG pool signals nitrogen sufficiency, 2-OG depletion of GlnK causes bound ATP to be hydrolyzed to ADP, leading to a conformational change in the protein. We propose that the role of ATP/ADP binding in E. coli GlnK is to effect a 2-OG-dependent molecular switch that drives a conformational change in the T loops of the P(II) protein. We have further shown that two other P(II) proteins, Azospirillum brasilense GlnZ and Arabidopsis thaliana P(II), have a similar ATPase activity, and we therefore suggest that this switch mechanism is likely to be a general property of most members of the P(II) protein family.

Control of AmtB-GlnK complex formation by intracellular levels of ATP, ADP, and 2-oxoglutarate.

P(II) proteins are one of the most widespread families of signal transduction proteins in nature, being ubiquitous throughout bacteria, archaea, and plants. They play a major role in coordinating nitrogen metabolism by interacting with, and regulating the activities of, a variety of enzymes, transcription factors, and membrane transport proteins. The regulatory properties of P(II) proteins derive from their ability to bind three effectors: ATP, ADP, and 2-oxoglutarate. However, a clear model to integrate physiological changes with the consequential structural changes that mediate P(II) interaction with a target protein has so far not been developed. In this study, we analyzed the fluctuations in intracellular effector pools in Escherichia coli during association and dissociation of the P(II) protein GlnK with the ammonia channel AmtB. We determined that key features promoting AmtB-GlnK complex formation are the rapid drop in the 2-oxoglutarate pool upon ammonium influx and a simultaneous, but transient, change in the ATP/ADP ratio. We were also able to replicate AmtB-GlnK interactions in vitro using the same effector combinations that we observed in vivo. This comprehensive data set allows us to propose a model that explains the way in which interactions between GlnK and its effectors influence the conformation of GlnK and thereby regulate its interaction with AmtB.