Mechanistic Insights into the Metal-Dependent Activation of Zn-Dependent Metallochaperones.

Members of the COG0523 subfamily of candidate GTPase metallochaperones function in bacterial transition-metal homeostasis, but the nature of the cognate metal, mechanism of metal transfer, and identification of target protein(s) for metal delivery remain open questions. Here, we explore the multifunctionality of members of the subfamily linked to delivering Zn to apoprotein targets under conditions of host-imposed transition-metal depletion. We examine two zinc-uptake repressor (Zur)-regulated COG0523 family members, each from a major human pathogen, (ZigA) and (ZigA), in an effort to develop a model for Zn metallochaperone activity. Zn chelator competition experiments reveal one high-affinity ( ≈ 10-10 M) metal-binding site in each GTPase, while ZigA and ZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. Co titrations reveal that both metallochaperones have similar electronic absorption characteristics that indicate the presence of two tetrahedral metal coordination sites. High-affinity metal binding at the CXCC motif activates the GTPase activity of both enzymes, with Zn more effective than Co. Both GTPases bind the product, GDP, more tightly in the apoprotein than the Zn-bound state and exhibit what is best described as a "locked" conformation around the GTP substrate. Negative thermodynamic linkage is observed between nucleotide binding and metal binding, leading to a new mechanistic model for COG0523-catalyzed metal delivery.