title: Insights into the importance of hydrogen bonding in the gamma-phosphate binding pocket of myosin: structural and functional studies of serine 236.
abstract: The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during gamma-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP.vanadate have been determined. In contrast to the previous structure of wild-type S1dC, the S236A.MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Jeremiah J
authors.initials: JJ
authors.lastname: Frye
authors.forename: Vadim A
authors.initials: VA
authors.lastname: Klenchin
authors.forename: Clive R
authors.initials: CR
authors.lastname: Bagshaw
authors.forename: Ivan
authors.initials: I
authors.lastname: Rayment
mesh_terms: D000199:Actins / Q000737:chemistry / Q000502:physiology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000502:physiology; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry* / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004023:Dictyostelium; D006860:Hydrogen Bonding; D024462:Myosin Type II / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship
chemical_list: D000199:Actins; D000266:Adenylyl Imidodiphosphate; D012694:Serine; D000255:Adenosine Triphosphate; D024462:Myosin Type II; D009218:Myosins
references.citation: Acta Crystallogr D Biol Crystallogr. 2000 Dec;56(Pt 12):1622-4
references.pmid: 11092928
references.citation: Philos Trans R Soc Lond B Biol Sci. 2004 Dec 29;359(1452):1819-28
references.pmid: 15647158
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Biochemistry. 1997 Sep 30;36(39):11619-28
references.pmid: 9305951
references.citation: Nat Struct Mol Biol. 2005 Apr;12(4):378-9
references.pmid: 15750603
references.citation: Structure. 2007 May;15(5):553-64
references.pmid: 17502101
references.citation: Biochemistry. 1995 Jul 18;34(28):8960-72
references.pmid: 7619795
references.citation: Biochemistry. 1978 Aug 22;17(17):3432-42
references.pmid: 150856
references.citation: Nature. 2003 Sep 25;425(6956):419-23
references.pmid: 14508494
references.citation: Gene. 1995 Aug 30;162(1):129-34
references.pmid: 7557400
references.citation: Biochem J. 1988 Feb 1;249(3):735-43
references.pmid: 3355494
references.citation: Biol Chem. 1999 Jul-Aug;380(7-8):1017-23
references.pmid: 10494855
references.citation: Annu Rev Biochem. 1999;68:687-728
references.pmid: 10872464
references.citation: J Biol Chem. 1998 Oct 16;273(42):27404-11
references.pmid: 9765269
references.citation: Biochemistry. 1996 Apr 30;35(17):5404-17
references.pmid: 8611530
references.citation: J Muscle Res Cell Motil. 1998 Jun;19(5):491-504
references.pmid: 9682136
references.citation: J Muscle Res Cell Motil. 1995 Jun;16(3):325-32
references.pmid: 7560005
references.citation: Biochemistry. 1997 Jan 14;36(2):317-23
references.pmid: 9003183
references.citation: Curr Opin Cell Biol. 2004 Feb;16(1):61-7
references.pmid: 15037306
references.citation: Science. 1993 Jul 2;261(5117):50-8
references.pmid: 8316857
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: J Biol Chem. 2000 Dec 8;275(49):38494-9
references.pmid: 10954715
references.citation: Biochemistry. 2000 Dec 26;39(51):16135-46
references.pmid: 11123942
references.citation: Anal Biochem. 1989 Feb 1;176(2):313-8
references.pmid: 2500867
references.citation: J Biol Chem. 2004 Aug 20;279(34):35557-63
references.pmid: 15205456
references.citation: Biochemistry. 1997 Nov 18;36(46):14037-43
references.pmid: 9369475
references.citation: Biochemistry. 1995 Jul 18;34(28):8973-81
references.pmid: 7619796
references.citation: Nature. 2005 Jun 9;435(7043):779-85
references.pmid: 15944696
references.citation: J Biol Chem. 2002 Aug 9;277(32):28459-67
references.pmid: 11971905
references.citation: Biochem J. 1974 Aug;141(2):351-64
references.pmid: 4281654
references.citation: EMBO J. 2004 Nov 24;23(23):4527-37
references.pmid: 15510214
references.citation: Philos Trans R Soc Lond B Biol Sci. 2004 Dec 29;359(1452):1843-55
references.pmid: 15647160
references.citation: Biophys J. 2009 May 6;96(9):3673-81
references.pmid: 19413972
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Biochemistry. 2001 Oct 23;40(42):12727-37
references.pmid: 11601998
references.citation: Protein Expr Purif. 2007 Sep;55(1):53-68
references.pmid: 17543538
references.citation: J Muscle Res Cell Motil. 2002;23(7-8):673-83
references.pmid: 12952066
references.citation: Cell. 1998 Sep 4;94(5):559-71
references.pmid: 9741621