title: Proteins at work: a combined small angle X-RAY scattering and theoretical determination of the multiple structures involved on the protein kinase functional landscape.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, La Jolla, California 92093, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Jamros
authors.forename: Leandro C
authors.initials: LC
authors.lastname: Oliveira
authors.forename: Paul C
authors.initials: PC
authors.lastname: Whitford
authors.forename: José N
authors.initials: JN
authors.lastname: Onuchic
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Adams
authors.forename: Donald K
authors.initials: DK
authors.lastname: Blumenthal
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Jennings
mesh_terms: D000465:Algorithms; D000081247:CSK Tyrosine-Protein Kinase; D003460:Crystallization; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction / Q000379:methods*; D018909:src Homology Domains; D019061:src-Family Kinases
chemical_list: D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases
references.citation: Chem Rev. 2006 Aug;106(8):3055-79
references.pmid: 16895318
references.citation: Annu Rev Cell Dev Biol. 1997;13:513-609
references.pmid: 9442882
references.citation: J Phys Chem B. 2009 Feb 5;113(5):1517-21
references.pmid: 19140753
references.citation: Curr Opin Struct Biol. 2004 Feb;14(1):70-5
references.pmid: 15102452
references.citation: Proteins. 2009 May 1;75(2):430-41
references.pmid: 18837035
references.citation: Proteins. 1995 Mar;21(3):167-95
references.pmid: 7784423
references.citation: J Mol Biol. 2006 Apr 7;357(4):1263-73
references.pmid: 16483606
references.citation: Gen Physiol Biophys. 2009 Jun;28(2):174-89
references.pmid: 19592714
references.citation: J Biol Chem. 2006 Mar 24;281(12):8183-9
references.pmid: 16439366
references.citation: Nature. 1997 Feb 13;385(6617):595-602
references.pmid: 9024657
references.citation: J Am Chem Soc. 2004 Jul 14;126(27):8426-32
references.pmid: 15237999
references.citation: J Struct Biol. 2010 Jan;169(1):95-105
references.pmid: 19800974
references.citation: J Mol Biol. 2005 Aug 5;351(1):131-43
references.pmid: 16002086
references.citation: Nature. 2000 Apr 27;404(6781):999-1003
references.pmid: 10801129
references.citation: Chem Rev. 2001 Jun;101(6):1763-78
references.pmid: 11709998
references.citation: HFSP J. 2008 Dec;2(6):307-13
references.pmid: 19436496
references.citation: J Comput Chem. 2005 Dec;26(16):1701-18
references.pmid: 16211538
references.citation: J Mol Biol. 2007 Feb 2;365(5):1460-8
references.pmid: 17137590
references.citation: Biochemistry. 2001 Feb 20;40(7):2004-10
references.pmid: 11329267
references.citation: Structure. 2005 Jun;13(6):861-71
references.pmid: 15939018
references.citation: Exp Cell Res. 2000 Jan 10;254(1):1-13
references.pmid: 10623460
references.citation: J Phys Chem A. 2006 Jan 19;110(2):671-6
references.pmid: 16405339
references.citation: J Mol Biol. 2008 Feb 15;376(2):492-505
references.pmid: 18164031
references.citation: Phys Chem Chem Phys. 2008 Jan 28;10(4):471-81
references.pmid: 18183310
references.citation: J Biol Chem. 2006 Dec 8;281(49):38004-12
references.pmid: 17018524
references.citation: J Mol Biol. 2005 Apr 29;348(2):335-47
references.pmid: 15811372
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 29;106(52):22124-9
references.pmid: 20018738
references.citation: J Phys Chem B. 2008 May 15;112(19):6131-6
references.pmid: 18251535
references.citation: Oncogene. 2004 Oct 18;23(48):7918-27
references.pmid: 15489910
references.citation: J Mol Biol. 2000 May 19;298(5):937-53
references.pmid: 10801360
references.citation: EMBO J. 1996 Sep 16;15(18):4909-18
references.pmid: 8890164
references.citation: EMBO J. 2003 Sep 15;22(18):4616-24
references.pmid: 12970174
references.citation: J Mol Biol. 2007 Mar 9;366(5):1661-71
references.pmid: 17217965
references.citation: Curr Opin Chem Biol. 2003 Oct;7(5):580-5
references.pmid: 14580561
references.citation: J Biol Chem. 2000 Sep 22;275(38):29183-6
references.pmid: 10918051
references.citation: Nature. 1991 May 2;351(6321):69-72
references.pmid: 1709258
references.citation: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W657-61
references.pmid: 20525782
references.citation: J Mol Biol. 2004 Jul 30;341(1):93-106
references.pmid: 15312765
references.citation: Biophys J. 2008 Jun;94(12):4924-31
references.pmid: 18326645
references.citation: Proteins. 2009 Dec;77(4):881-91
references.pmid: 19626713
references.citation: Cell. 2008 Jul 11;134(1):124-34
references.pmid: 18614016
references.citation: Oncogene. 1993 May;8(5):1241-7
references.pmid: 7683130
references.citation: J Biol Chem. 2003 Feb 7;278(6):3664-70
references.pmid: 12446738
references.citation: Nature. 1997 Feb 13;385(6617):602-9
references.pmid: 9024658
references.citation: J Mol Biol. 1997 Dec 19;274(5):757-75
references.pmid: 9405157
references.citation: J Mol Biol. 2008 Oct 17;382(4):1089-106
references.pmid: 18694757
references.citation: PLoS One. 2009 Nov 04;4(11):e7683
references.pmid: 19888460
references.citation: Oncogene. 2006 Sep 7;25(40):5495-506
references.pmid: 16636672
references.citation: Biophys J. 2009 Jun 3;96(11):4449-63
references.pmid: 19486669
references.citation: Arch Biochem Biophys. 1999 Jul 15;367(2):167-72
references.pmid: 10395732
references.citation: Biochemistry. 1999 Aug 24;38(34):11147-55
references.pmid: 10460171
references.citation: Biochim Biophys Acta. 1996 Jun 7;1287(2-3):121-49
references.pmid: 8672527
references.citation: Gene. 1992 Jan 15;110(2):205-11
references.pmid: 1371489
references.citation: J Biol Chem. 2003 Jun 27;278(26):24072-7
references.pmid: 12686554
references.citation: J Biol Chem. 2002 Apr 26;277(17):14351-4
references.pmid: 11884384
references.citation: Biophys J. 2005 Aug;89(2):1237-50
references.pmid: 15923225