Structural characterization of the natively unfolded N-terminal domain of human c-Src kinase: insights into the role of phosphorylation of the unique domain.

Institute for Research in Biomedicine, Parc Científic de Barcelona, Baldiri Reixac 10, Barcelona, Spain.
Journal of Molecular Biology (Impact Factor: 3.91). 07/2009; 391(1):136-48. DOI: 10.1016/j.jmb.2009.06.018
Source: PubMed

ABSTRACT The N-terminal regions of the members of Src family of non-receptor protein tyrosine kinases are intrinsically unfolded and contain the maximum sequence divergence among them. In this study, we have addressed the structural characterization by nuclear magnetic resonance of this region of 84 residues that encompasses the SH4 and the unique domains (USrc) of the human c-Src. With this aim, the backbone assignment was performed using (13)C-detected experiments that overcome the spectral resolution problems and the large number of prolines that are typical for intrinsically unfolded proteins. The analysis of the residual dipolar couplings measured for the USrc indicates the presence of a low populated helical structure in the 60-75 region. No long-range contacts between remote fragments of the chain were detected with paramagnetic relaxation enhancement experiments. The structural characterization was extended to two different phosphorylation states of USrc that encompassed three different phosphorylated sites, Ser17, Thr37, and Ser75. The structural and conformational changes upon phosphorylation were monitored through chemical shift perturbations and residual dipolar couplings, indicating that modifications occur at local level and no global rearrangements were apparent. These results suggest a scenario where phosphorylation induces a global electrostatic perturbation that could be involved in the membrane unbinding of c-Src and that could be related with the localization of the enzyme. These observations suggest the unique domain of Src kinases as a source of selectivity and reinforce the relevant role of intrinsically disordered proteins in biological processes.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Intrinsically disordered proteins are found extensively in cell signaling pathways where they often are targets of posttranslational modifications e.g. phosphorylation. Such modifications can sometimes induce or disrupt secondary structure elements present in the modified protein. CD79a and CD79b are membrane-spanning, signal-transducing components of the B-cell receptor. The cytosolic domains of these proteins are intrinsically disordered and each has an immunoreceptor tyrosine-based activation motif (ITAM). When an antigen binds to the receptor, conserved tyrosines located in the ITAMs are phosphorylated which initiate further downstream signaling. Here we use NMR spectroscopy to examine the secondary structure propensity of the cytosolic domains of CD79a and CD79b in vitro before and after phosphorylation. The phosphorylation patterns are identified through analysis of changes of backbone chemical shifts found for the affected tyrosines and neighboring residues. The number of the phosphorylated sites is confirmed by mass spectrometry. The secondary structure propensities are calculated using the method of intrinsic referencing, where the reference random coil chemical shifts are measured for the same protein under denaturing conditions. Our analysis revealed that CD79a and CD79b both have an overall propensity for α-helical structure that is greatest in the C-terminal region of the ITAM. Phosphorylation of CD79a caused a decrease in helical propensity in the C-terminal ITAM region. For CD79b, the opposite was observed and phosphorylation resulted in an increase of helical propensity in the C-terminal part.
    PLoS ONE 01/2014; 9(4):e96199. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Intrinsically disordered proteins (IDPs) are characterized by highly flexible solvent exposed backbones and can sample many different conformations. These properties confer them functional advantages, complementary to those of folded proteins, which need to be characterized to expand our view of how protein structural and dynamic features affect function beyond the static picture of a single well defined 3D structure that has influenced so much our way of thinking. NMR spectroscopy provides a unique tool for the atomic resolution characterization of highly flexible macromolecules in general and of IDPs in particular. The peculiar properties of IDPs however have profound effects on spectroscopic parameters. It is thus worth thinking about these aspects to make the best use of the great potential of NMR spectroscopy to contribute to this fascinating field of research. In particular, after many years of dealing with exclusively heteronuclear NMR experiments based on (13)C direct detection, we would like here to address their relevance when studying IDPs.
    Journal of Magnetic Resonance 04/2014; 241:115-25. · 2.30 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Defining the structural, dynamic, and energetic properties of the unfolded state of proteins is critical for an in-depth understanding of protein folding, protein thermodynamics, and protein aggregation. Here we analyze long-range contacts and compactness in two apparently fully unfolded ensembles of the same protein: the acid unfolded state of the C-terminal domain of the ribosomal protein L9 (CTL9) in the absence of high concentrations of urea as well as the urea unfolded state at low pH. SAXS reveals that the two states are expanded with values of Rg differing by less than 7 %. Paramagnetic relaxation enhancement (PRE) NMR studies, however, reveal that the acid unfolded state samples conformations that facilitate contacts between residues that are distant in sequence while the urea unfolded state ensemble does not. The experimental PRE profiles for the acid unfolded state differ significantly from these predicted using an excluded volume (EV) limit ensemble, but these long-range contacts are largely eliminated by the addition of 8 M urea. The work shows that expanded unfolded states can sample very different distributions of long-range contacts yet still have similar radii of gyration. The implications for protein folding and for the characterization of unfolded states are discussed.
    Biochemistry 11/2013; · 3.38 Impact Factor


Available from
May 20, 2014