Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.
Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.
Rebowski
Grzegorz
G
Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Namgoong
Suk
S
Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Boczkowska
Malgorzata
M
Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Leavis
Paul C
PC
Boston Biomedical Research Institute, Watertown, MA 02472-2899, USA.
Navaza
Jorge
J
Institut de Biologie Structurale, F-38027 Grenoble, France.
Dominguez
Roberto
R
Department of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA. Electronic address: droberto@mail.med.upenn.edu.
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011817:Rabbits; D014736:Vibrio parahaemolyticus / Q000737:chemistry
D000199:Actins; D001426:Bacterial Proteins
Acta Crystallogr D Biol Crystallogr. 2005 Apr;61(Pt 4):458-64
15805601
Biophys J. 2001 Aug;81(2):667-74
11463615
Methods Enzymol. 1982;85 Pt B:164-81
7121269
Methods Mol Biol. 2008;426:419-35
18542881
Proteins. 1998 Feb 1;30(2):144-54
9489922
Methods Enzymol. 2006;406:190-214
16472659
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11748-53
18689676
Acta Crystallogr D Biol Crystallogr. 2001 Oct;57(Pt 10):1367-72
11567147
Nature. 2009 Jan 22;457(7228):441-5
19158791
Science. 2008 Apr 11;320(5873):239-43
18403713
Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10785-90
18669664
Nat Cell Biol. 2009 Apr;11(4):451-9
19287377
Nat Rev Mol Cell Biol. 2006 Oct;7(10):713-26
16990851
J Am Chem Soc. 2006 Mar 29;128(12):3882-3
16551075
Trends Cell Biol. 2008 Oct;18(10):494-504
18774717
Cell. 2007 Oct 19;131(2):337-50
17956734
Curr Opin Struct Biol. 2010 Apr;20(2):217-25
20096561
J Biol Chem. 2007 Dec 14;282(50):36133-7
17965017
FEBS Lett. 2002 Feb 20;513(1):92-7
11911886
J Biol Chem. 2006 Oct 20;281(42):31909-19
16920713
J Biol Chem. 2002 Apr 26;277(17):14786-92
11856744
Nature. 1990 Sep 6;347(6288):44-9
2395461
Annu Rev Biophys Biomol Struct. 2007;36:451-77
17477841
Acta Crystallogr D Biol Crystallogr. 2008 Apr;64(Pt 4):454-65
18391412
Structure. 2008 May;16(5):695-704
18462674
Cell. 2003 Feb 21;112(4):453-65
12600310
Nature. 1993 Oct 28;365(6449):810-6
8413665
Cell. 2004 May 28;117(5):611-23
15163409
Structure. 2007 Feb;15(2):145-55
17292833
EMBO J. 2007 Oct 31;26(21):4597-606
17914456
Nature. 2009 Jan 22;457(7228):389-90
19158779
Proc Natl Acad Sci U S A. 2007 Oct 23;104(43):17117-22
17942696
Proc Natl Acad Sci U S A. 2005 Nov 15;102(46):16644-9
16275905
Biochemistry. 1992 Jul 14;31(27):6179-85
1627561
Trends Biochem Sci. 2004 Nov;29(11):572-8
15501675
Proc Natl Acad Sci U S A. 2005 Sep 13;102(37):13105-10
16141336
Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14527-32
16192358
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Ann N Y Acad Sci. 2007 Sep;1112:86-94
17468236
J Mol Biol. 2006 Sep 8;362(1):140-50
16893553
Cell Host Microbe. 2007 Apr 19;1(2):95-107
18005688
Mol Biol Cell. 2004 Oct;15(10):4735-48
15269284
EMBO J. 2004 Sep 15;23(18):3599-608
15329672
Ann N Y Acad Sci. 2007 Sep;1112:56-66
17468228
Nature. 2005 Feb 3;433(7025):488-94
15635372
Nature. 2005 Jan 27;433(7024):382-8
15674283
EMBO Rep. 2005 Mar;6(3):220-6
15741975
J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
19461840
Crit Rev Biochem Mol Biol. 2009 Nov-Dec;44(6):351-66
19874150