title: Biochemical and structural studies on the high affinity of Hsp70 for ADP.
abstract: The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+)  ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i)  increased the affinity in the presence of Mg(2+)  ion, but not in its absence. Thus, P(i)  enhances the effect of the Mg(2+)  ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+)  ion. As compared with the Mg(2+)  ion-free structure, the ADP- and Mg(2+)  ion-bound NBD contains one Mg(2+)  ion, which is coordinated with the β-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+)  ion is also coordinated with one P(i)  molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+)  ion-mediated network, including the P(i)  and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+)  ion-mediated network.
authors: Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
authors: Akihiko
authors: A
authors: Arakawa
authors: Noriko
authors: N
authors: Handa
authors: Mikako
authors: M
authors: Shirouzu
authors: Shigeyuki
authors: S
authors: Yokoyama
references: Cell. 1998 Feb 6;92(3):351-66
references: 9476895
references: Cell. 2008 Jun 13;133(6):1068-79
references: 18555782
references: Mol Cell. 2005 Nov 23;20(4):513-24
references: 16307916
references: Nat Struct Mol Biol. 2008 Dec;15(12):1309-17
references: 19029896
references: J Biol Chem. 2001 Aug 31;276(35):32538-44
references: 11441021
references: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references: 9757107
references: Cell. 2006 May 5;125(3):443-51
references: 16678092
references: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references: 20383002
references: Structure. 2010 Mar 10;18(3):309-19
references: 20223214
references: Acta Crystallogr D Biol Crystallogr. 2010 Mar;66(Pt 3):223-32
references: 20179333
references: Proc Natl Acad Sci U S A. 2009 May 26;106(21):8471-6
references: 19439666
references: J Biol Chem. 1994 Apr 29;269(17):12899-907
references: 8175707
references: Biochemistry. 1995 Sep 26;34(38):12095-106
references: 7547949
references: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2184-95
references: 15572771
references: Mol Cell. 2008 Jul 25;31(2):232-43
references: 18550409
references: Methods Enzymol. 1997;276:307-26
references: 27754618
references: Acta Crystallogr D Biol Crystallogr. 2006 Jun;62(Pt 6):678-82
references: 16699196
references: Cell. 2007 Oct 5;131(1):106-20
references: 17923091
references: Mol Cell. 2010 Aug 13;39(3):321-31
references: 20705236
references: Science. 2001 Feb 23;291(5508):1553-7
references: 11222862
references: Biochemistry. 1994 Dec 6;33(48):14625-35
references: 7981225
references: Biochemistry. 1998 May 19;37(20):7456-62
references: 9585559
references: Biochemistry. 1998 Jan 13;37(2):596-607
references: 9425082
references: Biochemistry. 1998 Jan 27;37(4):1015-24
references: 9454592
references: Mol Cell. 2005 Feb 4;17(3):367-79
references: 15694338
references: Nature. 1993 Oct 14;365(6447):664-6
references: 8413631
references: Mol Cell. 2010 Apr 9;38(1):89-100
references: 20385092
references: J Biol Chem. 1996 Jul 5;271(27):15874-8
references: 8663302
references: Nat Rev Mol Cell Biol. 2004 Oct;5(10):781-91
references: 15459659
references: EMBO Rep. 2004 Jun;5(6):567-71
references: 15170475
references: J Biol Chem. 1993 Nov 15;268(32):24323-9
references: 8226982
references: J Biol Chem. 1995 Feb 3;270(5):2247-50
references: 7836457
references: Nat Struct Biol. 2001 May;8(5):427-32
references: 11323718
references: J Biol Chem. 2008 May 30;283(22):15502-11
references: 18400763
references: Mol Cell. 2007 Nov 9;28(3):422-33
references: 17996706
references: Science. 1997 Apr 18;276(5311):431-5
references: 9103205
references: EMBO J. 1997 Oct 15;16(20):6209-16
references: 9321400
references: Biochemistry. 1999 Aug 17;38(33):10823-30
references: 10451379