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Dr. Aaron Smith

Northwestern University

April 5th, 2016 – Spring Seminar

Time and location: 10:45 – 11:45 AM, Commons 331

The ABCs of P1Bs: Membrane Metallotransporters at the Forefront of Metal Homeostasis

Host: Dr. Elsa Garcin

The P1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. Prior studies have focused chiefly on the involvement of these enzymes in copper and zinc transport. However, recent bioinformatic data reveal that these enzymes may be involved in the maintenance of nearly every biologically-relevant transition metal. Moreover, these data demonstrate that diversity exists in the nature and scope of the soluble P1B-ATPase N- and C-terminal metal-binding domains (MBDs), which are thought to modulate the enzyme allosterically. Biochemical, structural, and functional characterization of one such novel MBD from the Cupriavidus metallidurans P1B-ATPase CzcP demonstrates that this domain binds two metal ions in distinct and unique sites, and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Activity assays with the full-length CzcP and a truncated CzcP lacking the MBD indicate that this domain is necessary for maximal activity, consistent with an allosteric function. Homology modeling data suggest that this MBD may communicate to a metal-binding site (MBS) buried in the transmembrane (TM) region. Characterization using site-directed mutagenesis, ATPase assays, and electron paramagnetic resonance (EPR) techniques reveals the amino acids necessary for metal binding and transport in the TM region. Furthermore, binding of metal within this site is capable of labilizing metal in the MBD, suggesting cross-talk between the two domains. Mutations of key amino acids in the TM region affect the growth of E. coli cells overexpressing key CzcP variants in the presence of cadmium, demonstrating a correlation between in vitro activity assays and in vivo function. Given that CzcP likely evolved from an ancestral P1B-ATPase, these data suggest that an evolutionarily-adapted flexibility in the TM region likely afforded CzcP the ability to transport cadmium and zinc in addition to cobalt.

Abstract for his talk: Presentation Abstract