Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase.
Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His-X2-His. This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging. In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition. We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity. Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate. The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme. The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry. The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity.
Miller
W T
WT
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Hill
K A
KA
Schimmel
P
P
D000412:Alanine-tRNA Ligase / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013329:Structure-Activity Relationship; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D010446:Peptide Fragments; D006639:Histidine; D012313:RNA; D000412:Alanine-tRNA Ligase; D015032:Zinc; D003545:Cysteine