Probing Src homology 2 domain ligand interactions by differential line broadening.
Few techniques for probing the role of individual amino acids in interactions of a protein with ligands are available. Chemical shift perturbations in NMR spectra provide qualitative information about the response of individual amino acids of a protein to its interactions with ligands. Line shapes derived from (15)N-HSQC spectra recorded for different steps of a ligand titration yield both kinetic constants and insight into mechanisms by which the ligand binds. Here we have analyzed line shapes for 37 signals of amino acids of the N-terminal src homology 2 domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3-kinase (PI3-K) upon binding of phosphotyrosine (ptyr)-containing peptides. Kinetic rates at individual amino acids of the SH2 varied throughout the structure. For a subset of SH2 residues, the fine structure of the NMR line shapes indicated slow motions induced by the presence of small amounts of the ligand. These complex line shapes require one or more additional conformational states on the kinetic pathway. Modeling of the observed ligand interactions suggests a quasi-allosteric initial binding step. N-SH2 mutants with altered ligand affinity or specificity were also examined. Analysis of their line shapes revealed three distinct classes of mutants with different kinetic behaviors.
Günther
Ulrich
U
Institute of Biophysical Chemistry, J. W. Goethe University, Frankfurt, Biocenter N230, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany. ugunt@bpc.uni-frankfurt.de
Mittag
Tanja
T
Schaffhausen
Brian
B
D007532:Isoleucine / Q000235:genetics; D007930:Leucine / Q000235:genetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics; D011392:Proline / Q000235:genetics; D021122:Protein Subunits; D012694:Serine / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics
D008024:Ligands; D021122:Protein Subunits; D007532:Isoleucine; D014443:Tyrosine; D012694:Serine; D011392:Proline; D019869:Phosphatidylinositol 3-Kinases; D007930:Leucine