Protein modeling combined with spectroscopic techniques: an attractive quick alternative to obtain structural information.
Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth beta-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.
Kubala
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M
Department of Protein Structure, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. kubala@biomed.cas.cz
Obsil
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Obsilová
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Lánský
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Amler
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D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000502:physiology; D008958:Models, Molecular; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000502:physiology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013912:Threonine / Q000502:physiology
D048948:14-3-3 Proteins; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D013912:Threonine; D000255:Adenosine Triphosphate; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D000254:Sodium-Potassium-Exchanging ATPase