A protein pre-organized to trap the nucleotide moiety of coenzyme B-12: Refined solution structure of the B-12-binding subunit of glutamate mutase from Clostridium tetanomorphum

Uniformly C-13,N-15-labeled MutS, the coenzyme B-12-binding subunit of glut amate mutase from Clostridium tetanomorphum, was prepared by overexpression from an Escherichia coli strain. Multidimensional heteronuclear NMR spectr oscopic experiments with aqueous solutions of C-13, N-15-labeled MutS provi ded signal assignments for roughly 90% of the 1025 hydrogen, 651 carbon, an d 173 nitrogen atoms and resulted in about 1800 experimental restraints. Ba sed on the information from the NMR experiments, the structure of MutS was calculated, confirming the earlier less detailed structure obtained with N- 15-labeled MutS. The refined analysis allowed a precise determination of th e secondary and tertiary structure including several crucial side chain int eractions. The structures of (the apoprotein) MutS in solution and of the B -12-binding subunit in the crystal of the corresponding homologous holoenzy me from Clostridium cochlearium differ only in a section that forms the wel l-structured helix alpha1 in the crystal structure and that also comprises the cobalt-coordinating histidine residue. In the apoprotein MutS, this par t of the B-12-binding subunit is dynamic. The carboxy-terminal end of this section is conformationally flexible and has significant propensity for,an a-helical structure ("nascent helix"). This dynamic section in MutS is a de cisive element for the binding of the nucleotide moiety or coenzyme B-12 an d appears to be stabilized as a helix (alpha1) upon trapping of the nucleot ide of the B-12 cofactor.