Conformational change in the vinculin C-terminal depends on a critical histidine residue (His-906).
A phospholipid-controlled interaction between the N-terminal and C-terminal domains of vinculin is thought to be a major mechanism that regulates binding activities of the protein. To probe the mechanisms underlying these interactions we used chemical modification and site-directed mutagenesis directed at histidine residues. Diethylpyrocarbonate (DEPC) modification of the C-terminal, but not the N-terminal, domain greatly decreased affinity of the N-terminal-C-terminal binding, implicating histidine residues in the C-terminal. Mutation of either or both C-terminal histidines (at positions 906 and 1026), however, did not affect N-C binding at neutral pH. The H906A mutation did prevent DEPC effects and also prevented the normal decrease in binding affinity for the N-terminal at lower pH. We found that the wild type C-terminal domain, but not the H906A mutant, underwent a conformational change at pH 6.5, reflected in an altered circular dichroism spectrum and apparent oligomerization. Phospholipid also induced conformational changes in the wild type C-terminal domain but not in the H906A mutant, even though the mutant protein did bind to the phospholipid. Finally, the sensitivity of the N-C interaction to phospholipid was much reduced by the H906A mutation. These results show that H906 plays a key role in the conformational dynamics of the C-terminal domain and thus the regulation of vinculin.
Miller
G J
GJ
Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Ball
E H
EH
D000409:Alanine; D019943:Amino Acid Substitution; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D010743:Phospholipids / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D016596:Vinculin / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins; D016596:Vinculin; D006639:Histidine; D004047:Diethyl Pyrocarbonate; D000409:Alanine