Refined solution structure and backbone dynamics of N-15-labeled C12A-p8(MTCP1) studied by NMR relaxation

MTCP1 (for Mature-T-Cell Proliferation) was the first gene unequivocally id entified in the group of uncommon leukemias with a mature phenotype. The th ree-dimensional solution structure of the human p8(MTCP1) protein encoded b y the MTCP1 oncogene has been previously determined by homonuclear proton t wo-dimensional NMR methods at 600 MHz: it consists of an original scaffold comprising three alpha-helices, associated with a new cysteine motif. Two o f the helices are covalently paired by two disulfide bridges, forming an al pha-hairpin which resembles an antiparallel coiled-coil. The third helix is orientated roughly parallel to the plane defined by the alpha-antiparallel motif and appears less well defined. In order to gain more insight into th e details of this new scaffold, we uniformly labeled with nitrogen-15 a mut ant of this protein (C12A-p8(MTCP1)) in which the unbound cysteine at posit ion 12 has been replaced by an alanine residue, thus allowing reproducibly high yields of recombinant protein. The refined structure benefits from 211 additional NOEs, extracted from N-15-edited 3D experiments, and from a nea rly complete set of phi angular restraints allowing the estimation of the h elical content of the structured part of the protein. Moreover, measurement s of N-15 spin relaxation times and heteronuclear N-15{H-1}NOEs provided ad ditional insights into the dynamics of the protein backbone. The analysis o f the linear correlation between J(0) and J(omega) was used to interpret re laxation parameters. It appears that the apparent relative disorder seen in helix III is not simply due to a lack of experimental constraints, but ass ociated with substantial contributions of sub-nanosecond motions in this se gment.