title: An interaction between the Walker A and D-loop motifs is critical to ATP hydrolysis and cooperativity in bacteriophage T4 Rad50.
abstract: The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site.
authors: Program for Women in Science and Engineering, Iowa State University, Ames, Iowa 50011, USA.
authors: Metzere Bierlein
authors: MB
authors: De la Rosa
authors: Scott W
authors: SW
authors: Nelson
references: FEBS Lett. 2006 Feb 13;580(4):1064-84
references: 16442101
references: Annu Rev Biochem. 2008;77:229-57
references: 18275380
references: Methods. 2000 Dec;22(4):337-54
references: 11133240
references: Nat Rev Genet. 2008 Mar;9(3):204-17
references: 18227811
references: Nature. 2010 Sep 2;467(7311):108-11
references: 20811460
references: J Biol Chem. 2011 Jan 28;286(4):2382-92
references: 21081488
references: Hum Mutat. 2010 Sep;31(9):1011-9
references: 20607857
references: Nucleic Acids Res. 1999 Feb 15;27(4):1039-46
references: 9927737
references: Microbiol Mol Biol Rev. 2008 Dec;72(4):642-71, Table of Contents
references: 19052323
references: EMBO J. 2005 Jun 1;24(11):1901-10
references: 15889153
references: Biochemistry. 1995 Jul 18;34(28):9185-92
references: 7619819
references: Methods Enzymol. 2006;408:529-39
references: 16793391
references: Cell. 2008 Sep 19;134(6):981-94
references: 18805091
references: J Bacteriol. 2000 Nov;182(21):6036-41
references: 11029422
references: Chromosome Res. 2007;15(5):551-63
references: 17674145
references: Trends Biochem Sci. 1999 Sep;24(9):364-7
references: 10470037
references: Nature. 2010 Sep 2;467(7311):112-6
references: 20811461
references: Mol Cell. 2007 Mar 9;25(5):647-61
references: 17349953
references: Microbiol Mol Biol Rev. 2002 Dec;66(4):630-70, table of contents
references: 12456786
references: Cell. 2000 Jun 23;101(7):789-800
references: 10892749
references: Biochemistry. 1988 Mar 8;27(5):1577-80
references: 3365411
references: Trends Biochem Sci. 2002 Aug;27(8):410-8
references: 12151226
references: Curr Opin Struct Biol. 2003 Apr;13(2):249-55
references: 12727520
references: J Biol Chem. 1988 Aug 15;263(23):11366-73
references: 3403532
references: Mech Ageing Dev. 2007 Jul-Aug;128(7-8):460-2
references: 17588642
references: DNA Repair (Amst). 2004 Aug-Sep;3(8-9):781-96
references: 15279764
references: Bioinformatics. 2006 Jan 15;22(2):195-201
references: 16301204
references: Trends Biochem Sci. 2009 Oct;34(10):520-31
references: 19748784
references: Nature. 2002 Aug 1;418(6897):562-6
references: 12152085
references: Oncogene. 2007 Dec 10;26(56):7780-91
references: 18066091
references: Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):37-41
references: 11120885
references: Front Biosci. 2004 May 01;9:1382-97
references: 14977554
references: DNA Repair (Amst). 2009 Sep 2;8(9):983-95
references: 19473888
references: Cell. 2008 Oct 17;135(2):250-60
references: 18957200
references: Nature. 2008 Oct 9;455(7214):770-4
references: 18806779
references: DNA Repair (Amst). 2006 Sep 8;5(9-10):1199-212
references: 16822725