Proteins at Work: A COMBINED SMALL ANGLE X-RAY SCATTERING AND THEORETICAL DETERMINATION OF THE MULTIPLE STRUCTURES INVOLVED ON THE PROTEIN KINASE FUNCTIONAL LANDSCAPE

• Published in: The Journal of biological chemistry 2010-11, Vol.285 (46), p.36121-36128
• Main Authors: Jamros, Michael A, Oliveira, Leandro C, Whitford, Paul C, Onuchic, José N, Adams, Joseph A, Blumenthal, Donald K, Jennings, Patricia A
• Format: Article
• Language: English
• Subjects: Algorithms; Crystallization; CSK Tyrosine-Protein Kinase; Energy Transfer; Escherichia coli - genetics; Models, Molecular; Molecular Dynamics Simulation; Protein Conformation; Protein Structure and Folding; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Scattering, Small Angle; src Homology Domains; src-Family Kinases; X-Ray Diffraction - methods
• ISSN: 0021-9258; 1083-351X
• DOI: 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.