NMR structure determination of the tetramerization domain of the Mnt repressor: An asymmetric alpha-helical assembly in slow exchange

The structure and dynamics of the chymotryptic tetramerization domain of th e Mnt repressor of Salmonella bacteriophage P22 have been studied by NMR sp ectroscopy. Two sets of resonances (A and B) were found, representing the a symmetry within the homotetramer. Triple-resonance techniques were used to obtain unambiguous assignments of the A and B resonances. Intra-monomeric N OEs, which were distinguished from the inter-monomeric NOEs by exploiting C -13/N-15-filtered NOE experiments, demonstrated a continuous alpha-helix of approximately seven turns for both the A and B monomers. The asymmetry fac ilitated the interpretation of inter-subunit NOEs, whereas the antiparallel alignment of the subunits allowed further discrimination of inter-monomeri c NOEs. The three-dimensional structure revealed an unusual asymmetric pack ing of a dimer of two antiparallel right-handed intertwined coiled alpha-he lices. The A and B forms exchange on a timescale of seconds by a mechanism that probably involves a relative sliding of the two coiled coils. The amid e proton solvent exchange rates demonstrate a stable tetrameric structure. The essential role of Tyr 78 in oligomerization of Mnt, found by previous m utagenesis studies, can be explained by the many hydrophobic and hydrogen b onding interactions that this residue participates in with adjacent monomer s.