NUCLEAR-MAGNETIC-RESONANCE ASSIGNMENT AND SECONDARY STRUCTURE OF AN ANKYRIN-LIKE REPEAT-BEARING PROTEIN - MYOTROPHIN

Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major c ause of mortality in humans. Recent cDNA cloning revealed that myotrop hin has 118 amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two- and three-dimensional heteronuclear bond correlation N MR experiments have been performed on uniformly N-15-labeled or unifor mly N-15/C-13-labeled protein to obtain the H-1, N-15, and C-13 chemic al shift assignments. The secondary structure of myotrophin has been d etermined by a combination of NOEs, NH exchange data, (3)J(HN alpha) c oupling constants, and chemical shifts of H-1(alpha), C-13(alpha), and C-13(beta). The protein has been found to consist of seven helices, a ll connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns f ollowed by a helix-turn-helix motif. A hairpin-like turn involving L32 -R36 in ANK repeat #1 exhibits slow conformational averaging on the NM R time scale and appears dynamically different from the corresponding region (D65-169) of AMC repeat #2.