title: Pb2+ as modulator of protein-membrane interactions.
abstract: Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.
authors: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
authors: Krystal A
authors: KA
authors: Morales
authors: Mauricio
authors: M
authors: Lasagna
authors: Alexey V
authors: AV
authors: Gribenko
authors: Youngdae
authors: Y
authors: Yoon
authors: Gregory D
authors: GD
authors: Reinhart
authors: James C
authors: JC
authors: Lee
authors: Wonhwa
authors: W
authors: Cho
authors: Pingwei
authors: P
authors: Li
authors: Tatyana I
authors: TI
authors: Igumenova
references: J Biol Chem. 1998 Jul 10;273(28):17544-52
references: 9651347
references: J Mol Biol. 2002 Jul 5;320(2):277-91
references: 12079385
references: Chem Rev. 2001 Aug;101(8):2353-64
references: 11749377
references: Neurotoxicol Teratol. 2004 May-Jun;26(3):359-71
references: 15113598
references: Neurotoxicology. 2004 Jun;25(4):599-604
references: 15183013
references: J Biomol NMR. 1995 Nov;6(3):277-93
references: 8520220
references: Curr Opin Chem Biol. 2001 Apr;5(2):223-7
references: 11282351
references: J Phys Chem B. 2010 Mar 25;114(11):4005-9
references: 20192256
references: Methods Mol Biol. 2003;233:105-13
references: 12840502
references: Biochemistry. 2002 Sep 24;41(38):11411-24
references: 12234184
references: Biochemistry. 2003 Feb 11;42(5):1254-65
references: 12564928
references: Biochemistry. 2004 Sep 21;43(37):11727-39
references: 15362857
references: Nature. 1988 Jul 7;334(6177):71-3
references: 3386747
references: Acta Crystallogr D Biol Crystallogr. 2000 Apr;56(Pt 4):421-30
references: 10739915
references: J Am Chem Soc. 2005 Jul 6;127(26):9495-505
references: 15984876
references: EMBO J. 1999 Nov 15;18(22):6329-38
references: 10562545
references: Proc Natl Acad Sci U S A. 2009 Apr 21;106(16):6603-7
references: 19346474
references: Angew Chem Int Ed Engl. 2007;46(4):553-6
references: 17152108
references: Angew Chem Int Ed Engl. 2010 Oct 25;49(44):8177-80
references: 20859984
references: Biochemistry. 2001 Apr 17;40(15):4672-8
references: 11294634
references: J Biol Chem. 1994 Jan 14;269(2):834-7
references: 8288636
references: Biochemistry. 2001 Nov 20;40(46):13898-905
references: 11705379
references: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 Aug 1;63(Pt 8):653-6
references: 17671360
references: Trends Biochem Sci. 1998 Jun;23(6):217-21
references: 9644976
references: Nature. 1979 Oct 25;281(5733):690-2
references: 551288
references: Biochim Biophys Acta. 2006 Aug;1761(8):838-49
references: 16945584
references: Toxicology. 1999 Jan 1;132(1):19-32
references: 10199578
references: Biochemistry. 1982 Mar 2;21(5):1055-61
references: 7074048
references: J Biol Chem. 2003 Apr 4;278(14):12452-60
references: 12531893
references: J Mol Biol. 2001 Apr 6;307(4):1023-34
references: 11286553
references: Biochemistry. 1986 Sep 23;25(19):5819-25
references: 3778883
references: Mol Cell. 2002 Jan;9(1):145-54
references: 11804593
references: J Biol Chem. 2001 Jan 12;276(2):1531-7
references: 11032836
references: Science. 1996 Jul 12;273(5272):248-51
references: 8662510
references: J Neurochem. 2001 Mar;76(6):1724-35
references: 11259490
references: Structure. 1999 Jul 15;7(7):865-75
references: 10425687
references: J Biol Chem. 2008 Sep 19;283(38):26047-58
references: 18621733
references: J Biol Chem. 2005 Oct 28;280(43):36452-63
references: 16079140
references: Nat Rev Mol Cell Biol. 2010 Feb;11(2):103-12
references: 20094051
references: Proc Natl Acad Sci U S A. 2010 May 25;107(21):9608-13
references: 20453199
references: J Inorg Biochem. 2008 Oct;102(10):1901-9
references: 18684507
references: N Engl J Med. 2003 Apr 17;348(16):1517-26
references: 12700371
references: J Biol Chem. 1990 Mar 15;265(8):4223-6
references: 2106517
references: J Mol Biol. 2007 Aug 17;371(3):608-21
references: 17586528
references: J Neurochem. 1995 Jun;64(6):2667-73
references: 7760046
references: Methods Mol Biol. 2002;172:295-303
references: 11833355
references: Protein Expr Purif. 2005 Sep;43(1):1-9
references: 16084395
references: Methods Enzymol. 2001;339:204-38
references: 11462813
references: Biophys J. 1995 Mar;68(3):1009-18
references: 7756521
references: J Biol Chem. 2006 Jul 21;281(29):20521-9
references: 16672222
references: Toxicol Appl Pharmacol. 1999 Apr 1;156(1):40-5
references: 10101097
references: Acta Crystallogr D Biol Crystallogr. 1998 May 1;54(Pt 3):438-40
references: 9761921
references: Physiol Rev. 2008 Oct;88(4):1341-78
references: 18923184
references: Biochemistry. 1998 Nov 17;37(46):16106-15
references: 9819203
references: J Mol Biol. 2010 Sep 17;402(2):301-10
references: 20659476