pK(a) coupling at the intein active site: implications for the coordination mechanism of protein splicing with a conserved aspartate.

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
Read More Add to Saved list
  • Author(s): Du Z;Du Z; Zheng Y; Patterson M; Liu Y; Wang C
  • Source:
    Journal of the American Chemical Society [J Am Chem Soc] 2011 Jul 06; Vol. 133 (26), pp. 10275-82. Date of Electronic Publication: 2011 Jun 09.
  • Publication Type:
    Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: American Chemical Society Country of Publication: United States NLM ID: 7503056 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-5126 (Electronic) Linking ISSN: 00027863 NLM ISO Abbreviation: J Am Chem Soc Subsets: MEDLINE
    • Publication Information:
      Publication: Washington, DC : American Chemical Society
      Original Publication: Easton, Pa. [etc.]
    • Subject Terms:
      Aspartic Acid* ; Catalytic Domain* ; Conserved Sequence* ; Inteins* ; Protein Splicing*; Rec A Recombinases/*chemistry ; Rec A Recombinases/*metabolism; Hydrogen Bonding ; Models, Molecular ; Mutation ; Mycobacterium tuberculosis/enzymology ; Protons ; Rec A Recombinases/genetics ; Solutions
    • Abstract:
      Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pK(a) of 6.1, two units above the normal pK(a) of aspartate. The elevated pK(a) of D422 is coupled to the depressed pK(a) of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pK(a) to normal, while a D422G mutation increases the C1 pK(a) from 7.5 to 8.5. The pK(a) coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing.
    • References:
      Biotechnol Prog. 2000 Nov-Dec;16(6):1055-63. (PMID: 11101334)
      Nat Struct Biol. 1998 Jan;5(1):31-6. (PMID: 9437427)
      J Synchrotron Radiat. 2004 Jan 1;11(Pt 1):109-12. (PMID: 14646148)
      J Mol Biol. 2010 Jul 23;400(4):755-67. (PMID: 20562025)
      Chem Rec. 2006;6(4):183-93. (PMID: 16900466)
      Methods Enzymol. 2000;328:478-96. (PMID: 11075362)
      Biotechniques. 1999 Jul;27(1):110-4, 116, 118-20. (PMID: 10407673)
      Nat Struct Biol. 1996 Nov;3(11):946-50. (PMID: 8901873)
      J Am Chem Soc. 2009 Aug 19;131(32):11581-9. (PMID: 19630416)
      Nat Methods. 2008 Apr;5(4):303-5. (PMID: 18272963)
      J Mol Biol. 2005 Nov 11;353(5):1093-105. (PMID: 16219320)
      J Biol Chem. 2003 Oct 3;278(40):39133-42. (PMID: 12878593)
      J Biomol NMR. 1999 Mar;13(3):289-302. (PMID: 10212987)
      J Mol Biol. 2002 Mar 1;316(4):919-29. (PMID: 11884132)
      Science. 1990 Nov 2;250(4981):651-7. (PMID: 2146742)
      J Inorg Biochem. 2004 Jan;98(1):161-6. (PMID: 14659645)
      J Am Chem Soc. 2008 Jun 18;130(24):7536-7. (PMID: 18498162)
      Nat Chem Biol. 2010 Jul;6(7):527-33. (PMID: 20495572)
      J Biomol NMR. 2001 Dec;21(4):377-82. (PMID: 11824758)
      Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11466-71. (PMID: 9326633)
      J Mol Biol. 2011 Feb 25;406(3):430-42. (PMID: 21185311)
      J Mol Biol. 2007 Mar 16;367(1):162-73. (PMID: 17254599)
      J Biol Chem. 1990 Apr 25;265(12):6726-33. (PMID: 2139027)
      Nat Protoc. 2008;3(4):679-90. (PMID: 18388951)
      Genetics. 1997 Sep;147(1):73-85. (PMID: 9286669)
      EMBO J. 1996 Oct 1;15(19):5146-53. (PMID: 8895558)
      Biomol NMR Assign. 2008 Dec;2(2):111-3. (PMID: 19636882)
      J Bacteriol. 1997 Oct;179(20):6378-82. (PMID: 9335286)
      Arch Biochem Biophys. 2000 Mar 1;375(1):138-44. (PMID: 10683259)
      J Mol Biol. 2009 Nov 13;393(5):1106-17. (PMID: 19744499)
      Biochemistry. 2001 Oct 2;40(39):11698-705. (PMID: 11570870)
      J Magn Reson. 1997 Jan;124(1):274-8. (PMID: 9424317)
      Biochemistry. 1995 Dec 12;34(49):16186-93. (PMID: 8519776)
      Nat Biotechnol. 1999 Sep;17(9):889-92. (PMID: 10471931)
      Annu Rev Biochem. 2000;69:447-96. (PMID: 10966466)
      J Am Chem Soc. 2003 Sep 3;125(35):10561-9. (PMID: 12940738)
      J Mol Biol. 2005 Dec 9;354(4):916-26. (PMID: 16288917)
      J Magn Reson. 2003 Jan;160(1):65-73. (PMID: 12565051)
      Biochemistry. 2008 Jul 15;47(28):7430-40. (PMID: 18570440)
      Proc Natl Acad Sci U S A. 2009 Jul 7;106(27):11005-10. (PMID: 19541659)
      Proteins. 2002 Aug 1;48(2):388-403. (PMID: 12112705)
      Nucleic Acids Res. 2002 Jan 1;30(1):383-4. (PMID: 11752343)
      Proteins. 2007 Mar 1;66(4):778-95. (PMID: 17186527)
    • Grant Information:
      R01 GM081408-03S1 United States GM NIGMS NIH HHS; R01GM44844 United States GM NIGMS NIH HHS; R01 GM081408-04 United States GM NIGMS NIH HHS; R01 GM081408-03 United States GM NIGMS NIH HHS; R01 GM081408 United States GM NIGMS NIH HHS; R01GM81408 United States GM NIGMS NIH HHS; R01 GM039422 United States GM NIGMS NIH HHS; R01 GM044844 United States GM NIGMS NIH HHS
    • Accession Number:
      0 (Protons)
      0 (Solutions)
      30KYC7MIAI (Aspartic Acid)
      EC 2.7.7.- (Rec A Recombinases)
    • Publication Date:
      Date Created: 20110525 Date Completed: 20120820 Latest Revision: 20211020
    • Publication Date:
      20231215
    • Accession Number:
      PMC3126871
    • Accession Number:
      10.1021/ja203209f
    • Accession Number:
      21604815