Mechanistic Insights into the Metal-Dependent Activation of Zn^II-Dependent Metallochaperones

Abstract

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 II 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, Acinetobacter baumannii (AbZigA) and Staphylococcus aureus (SaZigA), in an effort to develop a model for Zn II metallochaperone activity. Zn II chelator competition experiments reveal one highaffinity (K Zn1 ≈ 10 10-10 11 M −1) metal-binding site in each GTPase, while AbZigA and SaZigA are characterized by an additional one and two (lower-affinity) metal-binding sites, respectively. Co II 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 II more effective than Co II. Both GTPases bind the product, GDP, more tightly in the apoprotein than the Zn II-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.

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII

“…15 ) in order to determine metal affinities. In view of similarity between YeiR, ZigA 22 , 23 and ZagA 24 , notably a deduced binding site for Zn II -sensor Zur in the yeiR promoter (Supplementary Fig. 16 ), occupancy with Zn II might be predicted.…”

Comparative Genomic Analysis of the DUF34 Protein Family Suggests Role as a Metal Ion Chaperone or Insertase

“…Proving metal insertion activity in vivo can be a very difficult task. For example, our group predicted that members of the COG0523 family were involved in metal insertion over 15 years ago and the experimental validation of this prediction has only been published within recent years [ 195 , 196 , 197 ]. We believe that the thorough analysis presented here should guide future experimental efforts to solve this long-standing functional enigma for one of the most conserved unknowns remaining to be confidently characterized.…”

The requirement for cobalt in vitamin B12: A paradigm for protein metalation

“…This has led to the identification of at least 15 subgroups of the COG0523 family which differ in both function and metal-selectivity [ 183 ]. These include Nha3 which activates the Fe(III)-type nitrile hydratase (NHase) [ 189 , 190 ], numerous subgroups (including YeiR, YjiA, ZigA and ZagA) connected to Zn(II) homeostasis [ 130 , [191] , [192] , [193] , [194] ], and CobW connected to vitamin B 12 [ 181 ]. An estimated 12.5% of COG0523 genes are cobW , usually located adjacent to cobN in aerobic cobalamin biosynthesis gene clusters [ 183 ].…”

“…CobW contains a CxCC sequence motif which is highly conserved in the COG0523 family [ 183 ] ( Section 5.4.2.1 ). Mutational analysis has confirmed that the CxCC sequence is necessary for metal-handling activities of various COG0523 proteins including in vivo activation of Fe(III)-type NHase by Nha3 [ 190 ], the protective effect of YeiR under conditions of Zn(II) depletion [ 191 ], and for high-affinity Zn(II) binding to ZigA [ 193 ]. It was recently discovered that, upon binding Mg(II) and GTP, CobW assembles a thiol-rich, metal-binding site that is likely localised to this same motif [ 156 ].…”