title: How the ankyrin and SOCS box protein, ASB9, binds to creatine kinase.
abstract: The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. The ASB proteins function as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that specifically transfer ubiquitin to cellular proteins targeting them for degradation by the proteasome. ASB9 binds to creatine kinase (CK) and targets it for degradation; however, the way in which ASB9 interacts with CK is not yet known. We present a complete characterization of the binding of ASB9 to CK. One ASB9 molecule binds to a dimer of CK. The binding affinity of ASB9(1-252) was extremely tight, and no dissociation could be observed. Deletion of the 34 N-terminal amino acids forming ASB9(35-252) resulted in weakening of the binding, so that a binding affinity of 2.6 nM could be measured. Amide hydrogen-deuterium exchange (HDXMS) experiments showed that both ASB9(1-252) and ASB9(35-252) protected the same region of CK, residues 182-203, which forms one side of the active site. The HDXMS experiments indicated that the N-terminal disordered region and first ankyrin repeat of ASB9 are protected from exchange in the complex. Molecular docking yielded a structural model consistent with all of the data that suggested the N-terminal residues of ASB9(1-252) may lie in one CK active site. This model was corroborated by enzymatic activity assays and mutational analysis.
authors: Department of Chemistry and Biochemistry, University of California, San Diego , 9500 Gilman Drive, La Jolla, California 92093-0378, United States.
authors: Deepa
authors: D
authors: Balasubramaniam
authors: Jamie
authors: J
authors: Schiffer
authors: Jonathan
authors: J
authors: Parnell
authors: Stephan P
authors: SP
authors: Mir
authors: Rommie E
authors: RE
authors: Amaro
authors: Elizabeth A
authors: EA
authors: Komives
mesh_terms: D000595:Amino Acid Sequence; D003402:Creatine Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D006801:Humans; D062105:Molecular Docking Simulation; D011485:Protein Binding; D059748:Proteolysis; D017384:Sequence Deletion; D050826:Suppressor of Cytokine Signaling Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C540562:ASB9 protein, human; D050826:Suppressor of Cytokine Signaling Proteins; D003402:Creatine Kinase
references: J Protein Chem. 2000 Jan;19(1):59-66
references: 10882173
references: Breast Cancer Res. 2008;10(3):R40
references: 18460216
references: Protein Sci. 1999 Nov;8(11):2258-69
references: 10595529
references: Biochemistry. 2010 Mar 2;49(8):1560-7
references: 20055496
references: Annu Rev Biochem. 1998;67:425-79
references: 9759494
references: Nature. 2002 Apr 18;416(6882):703-9
references: 11961546
references: Biochemistry. 2013 Aug 6;52(31):5236-46
references: 23837592
references: Proteins. 2006 Nov 15;65(3):712-25
references: 16981200
references: J Chem Theory Comput. 2013 Sep 10;9(9):3878-88
references: 26592383
references: J Mol Biol. 2006 Jul 7;360(2):421-34
references: 16756995
references: Nat Rev Mol Cell Biol. 2005 Jan;6(1):9-20
references: 15688063
references: Protein J. 2012 Apr;31(4):275-84
references: 22418839
references: Nat Rev Mol Cell Biol. 2009 May;10(5):319-31
references: 19352404
references: J Biol Chem. 1959 Dec;234:3201-4
references: 13836864
references: J Mol Biol. 2013 Sep 9;425(17):3166-77
references: 23806657
references: Biochemistry. 2001 Oct 2;40(39):11698-705
references: 11570870
references: Protein Pept Lett. 2009;16(3):333-5
references: 19275750
references: Proc Natl Acad Sci U S A. 2014 Jan 7;111(1):225-30
references: 24367071
references: FEBS Lett. 2005 Dec 19;579(30):6796-802
references: 16325183
references: Bioinformatics. 2014 Jun 15;30(12):1771-3
references: 24532726
references: Structure. 2014 Apr 8;22(4):572-81
references: 24613485
references: Biochemistry. 2007 Sep 18;46(37):10629-42
references: 17718514
references: Cell. 2008 Sep 19;134(6):995-1006
references: 18805092
references: Curr Opin Struct Biol. 2010 Dec;20(6):714-21
references: 20880695
references: Int J Cancer. 2014 May 1;134(9):2189-98
references: 24174293
references: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14705-10
references: 9843953
references: Phys Chem Chem Phys. 2014 Apr 14;16(14):6480-5
references: 24605363
references: J Biol Chem. 2007 Feb 16;282(7):4728-37
references: 17148442
references: Int J Oncol. 2010 Nov;37(5):1105-11
references: 20878058
references: BMC Biol. 2010;8:23
references: 20302626
references: J Chem Theory Comput. 2008 Mar;4(3):435-47
references: 26620784