Crystal structures of actin-related protein 2/3 complex with bound ATP or ADP.

Actin-related protein (Arp) 2/3 complex stimulates formation of actin filaments at the leading edge of motile cells. Nucleation of filaments depends on hydrolysis of ATP bound to Arp2. Here we report crystal structures of Arp2/3 complex with bound ATP or ADP. The nucleotides are immobilized on the face of subdomains 3 and 4 of Arp2, whereas subdomains 1 and 2 are flexible and absent from the electron density maps. This flexibility may explain why Arp2 does not hydrolyze ATP until the complex is activated. ATP stabilizes a relatively closed conformation of Arp3 with the gamma-phosphate bridging loops from opposite sides of the cleft. ADP binds Arp3 in a unique conformation that favors an open cleft, revealing a conformational change that may occur in actin and Arps when ATP is hydrolyzed and phosphate dissociates. These structures provide the an opportunity to compare all nucleotide-binding states in an actin-related protein and give insights into the function of both the Arp2/3 complex and actin.

[1]  H. Mannherz,et al.  Structure of gelsolin segment 1-actin complex and the mechanism of filament severing , 1993, Nature.

[2]  W. Kabsch,et al.  Atomic structure of the actin: DNase I complex , 1990, Nature.

[3]  S. Brenner,et al.  The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization. , 1980, The Journal of biological chemistry.

[4]  M. Carlier,et al.  Control of Actin Dynamics in Cell Motility , 1999, The Journal of Biological Chemistry.

[5]  V. Luzzati,et al.  Traitement statistique des erreurs dans la determination des structures cristallines , 1952 .

[6]  M. Carlier,et al.  Activation of Arp2/3 Complex by Wiskott-Aldrich Syndrome Protein Is Linked to Enhanced Binding of ATP to Arp2* , 2001, The Journal of Biological Chemistry.

[7]  T. Pollard,et al.  Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins , 2000, Nature.

[8]  T D Pollard,et al.  Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins. , 2001, Annual review of biochemistry.

[9]  T. Pollard,et al.  Hydrolysis of ATP by polymerized actin depends on the bound divalent cation but not profilin. , 2002, Biochemistry.

[10]  T. Pollard,et al.  Structure of Arp2/3 Complex in Its Activated State and in Actin Filament Branch Junctions , 2001, Science.

[11]  J. E. Estes,et al.  Nucleotide binding to actin. Cation dependence of nucleotide dissociation and exchange rates. , 1993, The Journal of biological chemistry.

[12]  Edward H. Egelman,et al.  How does ATP hydrolysis control actin's associations? , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[14]  C. Schutt,et al.  The structure of an open state of beta-actin at 2.65 A resolution. , 1996, Journal of molecular biology.

[15]  D. Safer,et al.  Beta thymosins as actin binding peptides , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[16]  P. Rubenstein,et al.  Biochemical Consequences of the Cardiofunk (R177H) Mutation in Yeast Actin* , 2003, Journal of Biological Chemistry.

[17]  T D Pollard,et al.  Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments , 1986, The Journal of cell biology.

[18]  Roberto Dominguez,et al.  Crystal Structure of Monomeric Actin in the ATP State , 2003, Journal of Biological Chemistry.

[19]  R. Mullins,et al.  Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Ivan Rayment,et al.  Trisoxazole macrolide toxins mimic the binding of actin-capping proteins to actin , 2003, Nature Structural Biology.

[21]  T D Pollard,et al.  Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. , 2000, Annual review of biophysics and biomolecular structure.

[22]  N. Swamy,et al.  Crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution. , 2002, Biochemistry.

[23]  S. Almo,et al.  The structure of nonvertebrate actin: Implications for the ATP hydrolytic mechanism , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Thomas D. Pollard,et al.  Interaction of WASP/Scar proteins with actin and vertebrate Arp2/3 complex , 2000, Nature Cell Biology.

[25]  C. Schutt,et al.  The structure of crystalline profilin–β-actin , 1993, Nature.

[26]  T. Pollard,et al.  Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization. , 1999, Biochemistry.

[27]  Thomas D. Pollard,et al.  Activation by Cdc42 and Pip2 of Wiskott-Aldrich Syndrome Protein (Wasp) Stimulates Actin Nucleation by Arp2/3 Complex , 2000, The Journal of cell biology.

[28]  Roberto Dominguez,et al.  Crystal structures of the vitamin D-binding protein and its complex with actin: Structural basis of the actin-scavenger system , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[29]  T. Pollard,et al.  The Arp2/3 complex nucleates actin filament branches from the sides of pre-existing filaments , 2001, Nature Cell Biology.

[30]  Marie-France Carlier,et al.  The β-Thymosin/WH2 Domain Structural Basis for the Switch from Inhibition to Promotion of Actin Assembly , 2004, Cell.

[31]  T D Pollard,et al.  The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Wei Yang,et al.  Structural analysis, identification, and design of calcium‐binding sites in proteins , 2002, Proteins.

[33]  T. Pollard,et al.  Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose , 1994, The Journal of cell biology.

[34]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[35]  T. Pollard,et al.  Interaction of Actin Monomers with AcanthamoebaActophorin (ADF/Cofilin) and Profilin* , 1998, The Journal of Biological Chemistry.

[36]  D. ben-Avraham,et al.  Normal mode analysis of G-actin. , 1993, Journal of molecular biology.

[37]  M. Carlier,et al.  ATP hydrolysis on actin-related protein 2/3 complex causes debranching of dendritic actin arrays , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[38]  L. Otterbein,et al.  The Crystal Structure of Uncomplexed Actin in the ADP State , 2001, Science.

[39]  A. DePace,et al.  The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly , 1997, The Journal of cell biology.

[40]  T. Pollard,et al.  Crystal Structure of Arp2/3 Complex , 2001, Science.

[41]  H. Berendsen,et al.  Systematic analysis of domain motions in proteins from conformational change: New results on citrate synthase and T4 lysozyme , 1998, Proteins.

[42]  T. Pollard,et al.  Identification of functionally important residues of Arp2/3 complex by analysis of homology models from diverse species. , 2004, Journal of molecular biology.

[43]  R. Mullins,et al.  Activation of Arp2/3 Complex: Addition of the First Subunit of the New Filament by a WASP Protein Triggers Rapid ATP Hydrolysis on Arp2 , 2004, PLoS biology.