Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship.
Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.
Yuan
Chunhua
C
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA. cyuan@ccic.ohio-state.edu
Li
Junan
J
Mahajan
Anjali
A
Poi
Ming Jye
MJ
Byeon
In-Ja L
IJ
Tsai
Ming-Daw
MD
D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D015513:Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry
D015513:Oncogene Proteins; C403591:PSMD10 protein, human; D011518:Proto-Oncogene Proteins; D013912:Threonine; D006639:Histidine; D046988:Proteasome Endopeptidase Complex