Refined solution structure and backbone dynamics of 15N-labeled C12A-p8MTCP1 studied by NMR relaxation.
MTCP1 (for Mature-T-Cell Proliferation) was the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8MTCP1 protein encoded by the MTCP1 oncogene has been previously determined by homonuclear proton two-dimensional NMR methods at 600 MHz: it consists of an original scaffold comprising three alpha-helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an alpha-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 the details of this new scaffold, we uniformly labeled with nitrogen-15 a mutant of this protein (C12A-p8MTCP1) in which the unbound cysteine at position 12 has been replaced by an alanine residue, thus allowing reproducibly high yields of recombinant protein.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 15N-edited 3D experiments, and from a nearly complete set of phi angular restraints allowing the estimation of the helical content of the structured part of the protein. Moreover, measurements of 15N spin relaxation times and heteronuclear 15N¿1H¿NOEs provided additional insights into the dynamics of the protein backbone. The analysis of the linear correlation between J(0) and J(omega) was used to interpret relaxation parameters. It appears that the apparent relative disorder seen in helix III is not simply due to a lack of experimental constraints, but associated with substantial contributions of sub-nanosecond motions in this segment.
Barthe
P
P
Centre de Biochimie Structurale, CNRS-UMR 9955, INSERM-U414, Université de Montpellier I, Faculté de Pharmacíe, France.
Chiche
L
L
Declerck
N
N
Delsuc
M A
MA
Lefèvre
J F
JF
Malliavin
T
T
Mispelter
J
J
Stern
M H
MH
Lhoste
J M
JM
Roumestand
C
C
D004268:DNA-Binding Proteins; D004058:Diffusion; D006801:Humans; D007938:Leukemia / Q000235:genetics; D008432:Mathematical Computing; D008958:Models, Molecular; D009038:Motion; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D012996:Solutions; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D015513:Oncogene Proteins; D012996:Solutions; C118783:TCL1B protein, human; D014157:Transcription Factors