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