¹Department of Microbiology and Biochemistry, Michigan State University, East Lansing, MI 48824
²Department of Chemistry, Michigan State University, East Lansing, MI 48824
³Department of Chemistry and Institute for Biomolecular Science, University of South Florida, Tampa, FL 33620
⁴Center for Metalloenzyme Studies, Department of Chemistry and Biochemistry & Molecular Biology, University of
    Georgia, Athens, GA 30602
 
 
Identification of Metal-Binding Residues in the Klebsiella aerogenes Urease Nickel Metallochaperon, UreE
Biochemistry 1999, 38, 4078-4088 (abstract 20)

Colpas, G. J.;¹ Brayman, T. G.;¹ Ming, L.-J.;²  Hausinger, R. P.*¹

¹Department of Microbiology and Biochemistry, Michigan State University, East Lansing, MI 48824
²Department of Chemistry and Institute for Biomolecular Science, University of South Florida, Tampa, FL 33620
 

     Metallochaperones are intracellular metal-binding proteins that protect the cell from reactivity of free metal ions while delivering specific cations to target metalloproteins.  Perhaps the best characterized metallochaperones are those involved in cellular trafficking of copper (1).  For example, yeast uses the Ctr1 protein to transport Cu(I) across the plasma membrane and then transfers the metal ion to one of at least three small, soluble, Cu chaperone proteins.  Atx1 (or the human homologue, HAH1 (2)) specifically delivers Cu to Ccc2, an intracellular membrane-associated protein involved in activation of the multicopper oxidase Fet3 that is needed for Fe transport (3).  Similarly, Lys7 (or CCS in humans) uniquely ferries Cu to the copper/zinc superoxide dismutase (4).  Finally, Cox17 is a Cu-binding protein that selectively provides the metal to cytochrome c oxidase (5).  The precise mechanisms for Cu delivery by these Cu-binding proteins are poorly defined, and auxiliary proteins may be required for loading of the metal ion into these metallochaperones or for achieving proper target specificity. Numerous other enzymes are known to require accessory proteins for metallocenter assembly (reviewed in (6)), and it is likely that metallochaperones participate in specific delivery of many of the essential transition metals to a variety of additional apoproteins.

     Nickel is specifically incorporated into five enzymes (hydrogenase, carbon monoxide dehydrogenase/acetyl-S-coenzyme A synthase, methyl-S-coenzyme M reductase, one form of superoxide dismutase, and urease) (reviewed in (7)), and several distinct Ni-specific metallochaperone proteins appear to exist.  For example, HypB is essential for Ni insertion into hydrogenase by a process that requires GTP hydrolysis (8, 9).  The amino-terminal region of HypB in some, but not all, microorganisms contains a histidine-rich region that has been shown to bind Ni (10, 11).  Experimental evidence obtained with cells synthesizing mutant protein lacking the His-rich region is consistent with HypB serving both in Ni delivery to hydrogenase as well as in Ni storage (12).  Activation of CO dehydrogenase similarly involves a protein containing a His-rich region, CooJ, where in this case the potential metal-binding motif is located at the carboxyl terminus (13).  Analogous to the case of hypB mutants (14), cooJ mutants can be phenotypically suppressed by increasing the Ni concentration in the medium (13).  Purified CooJ from Rhodospirillum rubrum has been shown to bind four Ni per monomer, and to competitively bind several other divalent metals (15).  In addition, an interaction with several other proteins, including CO dehydrogenase, was observed during purification of this protein.  Extensive studies have revealed many aspects of the elaborate metallocenter assembly pathway for synthesis of the Ni-containing coenzyme F430 found in methyl-S-coenzyme M reductase (16), but it remains unclear whether a metallochaperone participates in this process.  Similarly, no evidence has been reported that a chaperone aids in synthesis of Ni-containing superoxide dismutase; however, studies of this most recent Ni enzyme are in their infancy and such a role cannot be excluded.  If a metallochaperone is utilized for superoxide dismutase activation, a counterpart must be available in Escherichia coli as shown by the ability to obtain active recombinant enzyme upon expression of truncated Streptomyces coelicolor sodN (17).  The work described in this manuscript focuses on characterization of a possible Ni metallochaperone associated with urease.  As detailed below, the UreE accessory protein, shown to be associated with urease activation (18), is a Ni-binding protein (19).

     Purified Klebsiella aerogenes UreE reversibly binds 5-6 Ni(II) ions per 34 kD homodimer (average Kd ~10 mM) (19).  Spectroscopic experiments with this protein indicate the presence of pseudo-octahedral Ni(II) coordinated by an average of 3-5 imidazole ligands, implicating a role for the carboxyl terminus where 10 of the last 15 residues are histidine (20).  Despite its distinctive sequence, however, the His-rich region found in the K. aerogenes protein is not conserved in all UreE homologues (Figure 1).  Furthermore, a truncated form of K. aerogenes UreE lacking the His-rich region, termed H144*UreE,1 is competent for activating urease in vivo (21).  Thus, internal Ni-binding sites, not the histidine residues at the carboxyl terminus, are necessary for UreE to assist in K. aerogenes urease activation.  Equilibrium dialysis measurements with H144*UreE indicate that two Ni(II) per dimer are cooperatively bound, with an average Kd of ~8 mM.  Competition experiments with Cu, Zn, Co, and Cd indicate that these divalent metal ions compete (to varying degrees) with Ni binding to H144*UreE in vitro (21).  Spectroscopic studies of Ni, Cu, and Co binding to H144*UreE reveal the two binding sites per homodimer to be distinct in their properties, consistent with an observed sequential binding pattern for the two sites (22).  In addition, the coordination environments differ for each type of metal.  Thus, the two Cu sites have tetragonal coordination environments with two histidine donors each, but a cysteine donor coordinates only the second metal ion that is bound to the dimer.  Co is bound exclusively by N/O donors including at least three histidines in the two pseudo-octahedral sites.  Ni is chelated in a manner similar, but not identical, to Co with at least three histidines in two approximately six-coordinate N/O sites.  The differences in coordination environments observed for each type of metal ion are proposed to facilitate in vivo Ni selection for urease activation (22).

     Here, we have used site directed mutagenesis methods to create variants of H144*UreE in order to identify the metal-binding ligands.  The variants include substitution of alanine for each of two cysteine residues (positions 79 and 89) and all five histidines (residues 91, 96, 109, 110, and 112) in the truncated protein (Figure 1).  In addition, the tyrosine at position 90 (associated with a CYH motif) and an aspartate at position 111 (in a HHDH motif) were changed to phenylalanine and alanine, respectively.  We examined the effects of these nine changes on the ability of the metallochaperone to form active urease in vivo, and we characterized the interactions between purified H144*UreE variants and selected metal ions by equilibrium dialysis and spectroscopic methods.  Based on results from these studies, in combination with those from prior spectroscopic analyses (22), we propose a model in which two distinct metal-binding sites are present at the dimer interface and we identify several ligands at each metal-binding site.  Furthermore, we show that only one of the metal sites (involving the highly conserved His96 and Asp111 residues) appears to be important for in vivo activation of urease.
 

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