The zinc coordination site of the bacteriophage Mu translational activator protein, Com.
The bacteriophage Mu Com protein is a small "zinc finger-like" protein that binds a specific site in com-mom operon mRNA and activates translation of the mom open-reading-frame. Com contains six cysteine and five histidine residues that have the potential to form several alternative zinc-finger-like motifs. We have used oligonucleotide site-directed mutagenesis to individually alter each of these amino acids (Cys to Ser, and His to Asn or Gln) and tested the various forms of Com for their ability to function in vivo. We observed that mutation of any one of the four N-terminal cysteine residues (Cys-6, 9, 26 or 29) resulted in loss of Com activity. The Com protein requires zinc in order to fold into its functional tertiary structure, as demonstrated by characteristic 1H nuclear magnetic resonance (NMR) chemical shifts. 1H chemical shifts revert to random coil values in the presence of the metal chelator EDTA. The metal-binding specificity and thermal stability of Com also has been investigated using 1H NMR. We report the use of 113Cd NMR, 1H-113Cd heteronuclear spin-echo difference spectroscopy HSED and Zn extended X-ray absorption fine structure spectroscopy EXAFS to determine the zinc/protein stoichiometry as 1:1 and the ligand environment as tetrathiolate. Comparative NMR spectra of Com mutants C6S and C39S suggest position 6 is involved in zinc coordination, while position 39 is not metal-liganded. These studies indicate that the metal coordination, site of Com is a four-cysteine complex, involving residues 6, 9, 26 and 29.
Witkowski
R T
RT
Department of Chemistry, University of Rochester, NY 14627, USA.
Hattman
S
S
Newman
L
L
Clark
K
K
Tierney
D L
DL
Penner-Hahn
J
J
McLendon
G
G
D000595:Amino Acid Sequence; D010583:Bacteriophage mu / Q000737:chemistry*; D001483:Base Sequence; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics; D016335:Zinc Fingers
C068482:Com protein, Enterobacteria phage Mu; D016601:RNA-Binding Proteins; D014764:Viral Proteins