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Proteins at Work

A COMBINED SMALL ANGLE X-RAY SCATTERING AND THEORETICAL DETERMINATION OF THE MULTIPLE STRUCTURES INVOLVED ON THE PROTEIN KINASE FUNCTIONAL LANDSCAPE*
Open AccessPublished:August 26, 2010DOI:https://doi.org/10.1074/jbc.M110.116947
      C-terminal Src kinase (Csk) phosphorylates and down-regulates the Src family tyrosine kinases (SFKs). Crystallographic studies of Csk found an unusual arrangement of the SH2 and SH3 regulatory domains about the kinase core, forming a compact structure. However, recent structural studies of mutant Csk in the presence of an inhibitor indicate that the enzyme accesses an expanded structure. To investigate whether wt-Csk may also access open conformations we applied small angle x-ray scattering (SAXS). We find wt-Csk frequently occupies an extended conformation where the regulatory domains are removed from the kinase core. In addition, all-atom structure-based simulations indicate Csk occupies two free energy basins. These basins correspond to ensembles of distinct global conformations of Csk: a compact structure and an extended structure. The transitions between these structures are entropically driven and accessible via thermal fluctuations that break local interactions. We further characterized the ensemble by generating theoretical scattering curves for mixed populations of conformations from both basins and compared the predicted scattering curves to the experimental profile. This population-combination analysis is more consistent with the experimental data than any rigid model. It suggests that Csk adopts a broad ensemble of conformations in solution, populating extended conformations not observed in the crystal structure that may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the ubiquitous dynamic reversible assembly processes inherent in biology.

      Introduction

      The Src family of tyrosine kinases (SFK)
      The abbreviations used are: SFK
      Src family of tyrosine kinases
      Csk
      C-terminal Src kinase
      SAXS
      small angle x-ray scattering
      Cbp
      Csk-binding protein.
      are modular signaling enzymes involved in the control of cellular growth and differentiation (
      • Thomas S.M.
      • Brugge J.S.
      ). The members of this family contain three important structural domains: a C-terminal tyrosine kinase domain, comprised of a small and large lobe, preceded by the noncatalytic regulatory SH2 and SH3 domains (
      • Boggon T.J.
      • Eck M.J.
      ). SFKs also contain a unique region and an N-terminal myristic acid for membrane association. The activity of c-Src, the prototype for the SFKs, is up-regulated by phosphorylation of Tyr-416 (in the activation loop of the kinase domain) and dephosphorylation of Tyr-527 (in the C-terminal tail) (Fig. 1A) (
      • Brown M.T.
      • Cooper J.A.
      ,
      • Abram C.L.
      • Courtneidge S.A.
      ). While phosphorylation of the activation loop is autocatalytic, phosphorylation of the C-terminal tail is inhibitory and requires Csk (
      • Cole P.A.
      • Shen K.
      • Qiao Y.
      • Wang D.
      ). Like the SFKs, Csk contains a C-terminal tyrosine kinase domain and N-terminal SH2 and SH3 domains (Fig. 1A) (
      • Bräuninger A.
      • Holtrich U.
      • Strebhardt K.
      • Rübsamen-Waigmann H.
      ). Unlike SFKs, Csk lacks an inhibitory C-terminal tail, is not regulated through phosphorylation, and does not possess an N-terminal sequence for membrane localization. Instead, Csk is constitutively active and increased activity is coupled to its association with membrane adaptor proteins, for example Csk-binding protein (Cbp) (
      • Kawabuchi M.
      • Satomi Y.
      • Takao T.
      • Shimonishi Y.
      • Nada S.
      • Nagai K.
      • Tarakhovsky A.
      • Okada M.
      ). Cbp localizes Csk to the membrane where phosphorylation of SFKs by Csk occurs and up-regulates Csk activity through engagement of the Csk SH2 domain (
      • Takeuchi S.
      • Takayama Y.
      • Ogawa A.
      • Tamura K.
      • Okada M.
      ). How the latter occurs is not well understood. Hydrogen-deuterium exchange studies coupled with mass spectrometric analyses indicate cross-talk between the SH2 and kinase domains within Csk. This cross-talk is activated by binding to Cbp and/or reduction of a disulfide bond distal to the active site within the kinase domain (
      • Mills J.E.
      • Whitford P.C.
      • Shaffer J.
      • Onuchic J.N.
      • Adams J.A.
      • Jennings P.A.
      ,
      • Wong L.
      • Lieser S.
      • Chie-Leon B.
      • Miyashita O.
      • Aubol B.
      • Shaffer J.
      • Onuchic J.N.
      • Jennings P.A.
      • Woods Jr., V.L.
      • Adams J.A.
      ,
      • Wong L.
      • Lieser S.A.
      • Miyashita O.
      • Miller M.
      • Tasken K.
      • Onuchic J.N.
      • Adams J.A.
      • Woods Jr., V.L.
      • Jennings P.A.
      ). In addition to this atypical mode of regulation, Csk also differs from many tyrosine kinases in its substrate specificity. Broad substrate specificity is common for tyrosine kinases, whereas Csk displays high specificity for SFKs (
      • Wang D.
      • Huang X.Y.
      • Cole P.A.
      ,
      • Nada S.
      • Okada M.
      • MacAuley A.
      • Cooper J.A.
      • Nakagawa H.
      ).
      Figure thumbnail gr1
      FIGURE 1Structural and regulatory features of c-Src and Csk. A, domain organization. c-Src contains a unique region and N-terminal fatty acylation and two activity modulating phosphorylation sites in the kinase domain at Tyr-416 (YA) and in the C-terminal tail at Tyr-527 (YT). B–D, x-ray structures of Csk and c-Src. Inactive Src is phosphorylated at YT whereas active Src is dephosphorylated at YT. The SH2, SH3, and kinase domains are colored the same as in A.
      Although c-Src and Csk share considerable sequence homology in the kinase, SH2 and SH3 domains, they differ substantially in their tertiary structures. The inactive form of c-Src (Tyr-527 phosphorylated, Tyr-416 unphosphorylated) is compact, with the SH2 and SH3 domains interacting with the kinase domain (Fig. 1B) (
      • Sicheri F.
      • Moarefi I.
      • Kuriyan J.
      ,
      • Williams J.C.
      • Weijland A.
      • Gonfloni S.
      • Thompson A.
      • Courtneidge S.A.
      • Superti-Furga G.
      • Wierenga R.K.
      ,
      • Xu W.
      • Harrison S.C.
      • Eck M.J.
      ). In c-Src, dephosphorylation of Tyr-527 results in dissociation of the C-terminal tail from the SH2 domain (Fig. 1B), which leads to the activation of c-Src (
      • Cowan-Jacob S.W.
      • Fendrich G.
      • Manley P.W.
      • Jahnke W.
      • Fabbro D.
      • Liebetanz J.
      • Meyer T.
      ). Thus, activation of c-Src involves reorientation of the regulatory domains and the loss of most interdomain contacts. For Csk, the regulatory domains adopt a different conformation relative to the kinase domain where the SH2 and SH3 domains make contacts with the small lobe of the kinase domain (Fig. 1B). This configuration is maintained by two linker segments that connect the three domains.
      Crystallographic analysis of Csk and c-Src suggest that tyrosine kinases can position regulatory domains about a central kinase core in multiple configurations (
      • Sicheri F.
      • Moarefi I.
      • Kuriyan J.
      ,
      • Williams J.C.
      • Weijland A.
      • Gonfloni S.
      • Thompson A.
      • Courtneidge S.A.
      • Superti-Furga G.
      • Wierenga R.K.
      ,
      • Xu W.
      • Harrison S.C.
      • Eck M.J.
      ,
      • Cowan-Jacob S.W.
      • Fendrich G.
      • Manley P.W.
      • Jahnke W.
      • Fabbro D.
      • Liebetanz J.
      • Meyer T.
      ,
      • Ogawa A.
      • Takayama Y.
      • Sakai H.
      • Chong K.T.
      • Takeuchi S.
      • Nakagawa A.
      • Nada S.
      • Okada M.
      • Tsukihara T.
      ). These configurations are important for controlling catalytic activity of the kinase domain. For Csk, interactions between the SH2 domain and small lobe of the kinase domain, which are facilitated by the SH2-kinase linker, are necessary for efficient catalysis (
      • Wong L.
      • Lieser S.
      • Chie-Leon B.
      • Miyashita O.
      • Aubol B.
      • Shaffer J.
      • Onuchic J.N.
      • Jennings P.A.
      • Woods Jr., V.L.
      • Adams J.A.
      ,
      • Lin X.
      • Wang Y.
      • Ahmadibeni Y.
      • Parang K.
      • Sun G.
      ). When the regulatory domains are removed activity decreases by about two orders of magnitude (
      • Sun G.
      • Budde R.J.
      ,
      • Sondhi D.
      • Cole P.A.
      ). In contrast, the SH2 and SH3 domains play a repressive role in controlling the tyrosine kinase activity of c-Src through interaction with the C-terminal tail and the SH2 kinase linker (
      • Williams J.C.
      • Weijland A.
      • Gonfloni S.
      • Thompson A.
      • Courtneidge S.A.
      • Superti-Furga G.
      • Wierenga R.K.
      ). These studies indicate that for tyrosine kinases configurations of the regulatory domains relative to the kinase domain is of functional importance. Whereas the crystallographic data may lead one to believe that domain movement in c-Src is highly cooperative and operates in a switch-like manner, recent studies demonstrate that the regulatory mechanism is more complex. For example, whereas SAXS studies reveal that activation of c-Src is coupled to an increase in the radius of gyration, modeling studies indicate that an open form akin to that in the crystal structure represents only a minor fraction (15%) of the overall population of molecules (
      • Bernadó P.
      • Pérez Y.
      • Svergun D.I.
      • Pons M.
      ). Therefore, the data are consistent with the possibility that c-Src may adopt a broad ensemble of solution conformations upon activation, where the catalytically active open conformation is only one of these forms. In the case of Csk, the different arrangement of domains raises the question of whether this unique form is the major Csk conformation or whether other species are populated under solution conditions.
      Here, we employed SAXS measurements to describe the solution structure of Csk. Interestingly, we find that Csk samples an extended conformation relative to the crystal structure. Rigid body modeling indicates that the SH2 and SH3 domains adopt positions where they are not tightly associated with the kinase core. Recent studies have shown the viability of molecular dynamics simulations to provide a structural interpretation of SAXS data (
      • Yang S.
      • Park S.
      • Makowski L.
      • Roux B.
      ,
      • Kim S.J.
      • Dumont C.
      • Gruebele M.
      ,
      • Pelikan M.
      • Hura G.L.
      • Hammel M.
      ). In this study, molecular dynamics simulations that employ an all-atom structure-based model (
      • Whitford P.C.
      • Noel J.K.
      • Gosavi S.
      • Schug A.
      • Sanbonmatsu K.Y.
      • Onuchic J.N.
      ) are applied in two ways to characterize the ensemble of CSK conformations present in solution: (a) comparing the structures obtained from the simulation with the experimental SAXS data and (b) reconstructing the distribution of states in solution using a system constrained mainly by the entropy. These studies taken together with solution studies of Src (
      • Bernadó P.
      • Pérez Y.
      • Svergun D.I.
      • Pons M.
      ) suggest that divergent configurations resulting from global motions of multi-domain protein kinases may be common and that solution approaches are necessary to identify and characterize these conformations.

      Acknowledgments

      We thank Hiro Tsuruta for assistance in the experiments run at SSRL. Initial x-ray scattering data were collected at the University of Utah using facilities that are supported by the United States Dept. of Energy, Grant No. DE-FG02-05ER64026 (to Jill Trewhella). X-ray scattering data for publication were collected at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U. S. Dept. of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Dept. of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. We acknowledge Sulyman Barkho and Jeff Noel for insightful discussions.

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