PDBsum entry 2wkq

Go to PDB code: 
protein ligands metals links
Transferase, cell adhesion PDB id
Protein chain
317 a.a.
EDO ×2
_CL ×3
Waters ×397
PDB id:
Name: Transferase, cell adhesion
Title: Structure of a photoactivatable rac1 containing the lov2 c450a mutant
Structure: Nph1-1, ras-related c3 botulinum toxin substrate 1. Chain: a. Fragment: nph1-1 residues 404-546 and p21-rac1, residues 4-180. Synonym: p21-rac1, ras-like protein tc25, cell migration-inducing gene 5 protein,. Engineered: yes. Mutation: yes
Source: Avena sativa, homo sapiens. Oat, human. Organism_taxid: 4498, 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.
1.60Å     R-factor:   0.169     R-free:   0.186
Authors: Y.I.Wu,D.Frey,O.I.Lungu,A.Jaehrig,I.Schlichting,B.Kuhlman, K.M.Hahn
Key ref:
Y.I.Wu et al. (2009). A genetically encoded photoactivatable Rac controls the motility of living cells. Nature, 461, 104-108. PubMed id: 19693014 DOI: 10.1038/nature08241
16-Jun-09     Release date:   18-Aug-09    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
O49003  (O49003_AVESA) -  NPH1-1
923 a.a.
317 a.a.*
Protein chain
Pfam   ArchSchema ?
P63000  (RAC1_HUMAN) -  Ras-related C3 botulinum toxin substrate 1
192 a.a.
317 a.a.*
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 173 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     intracellular   19 terms 
  Biological process     epithelial cell morphogenesis   76 terms 
  Biochemical function     nucleotide binding     12 terms  


DOI no: 10.1038/nature08241 Nature 461:104-108 (2009)
PubMed id: 19693014  
A genetically encoded photoactivatable Rac controls the motility of living cells.
Y.I.Wu, D.Frey, O.I.Lungu, A.Jaehrig, I.Schlichting, B.Kuhlman, K.M.Hahn.
The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.
  Selected figure(s)  
Figure 2.
Figure 2: Localized activation or inactivation of PA-Rac1 induces myosin-dependent migration. a, Protrusion/retraction map after a single pulse of activating illumination. MEFs expressing PA-Rac1 (left) generated protrusions at the site of irradiation (red) and retraction at the opposite side of the cell (blue) (in all 50 cells studied). Irradiation of the dominant-negative T17N mutant of PA-Rac1 (right) produced retraction near the point of irradiation, with protrusion in area(s) other than the site of irradiation (in all 25 cells studied). b, Repeated activation of PA-Rac1 at the cell edge induces directional migration. (MEF, 2-min intervals, average 0.8 m movement per pulse, n = 6.) c, Localized activation of PA-Rac1 in the presence of ML-7 (MLCK inhibitor, 1 M), blebbistatin (myosin II ATPase inhibitor, 1 M) or Y-27632 (ROCK inhibitor, 10 M). Protrusions analysed as in panel a. d, Effect of myosin or ROCK inhibition on the ability of Rac1 to specify the direction of movement. The cosine of the angle between two lines (from the irradiation spot to the cell centroid at time 0, from the centroid at time 0 to the centroid at the end of the experiment) indicated how much the cell deviates from the direction specified by local irradiation. For c, d, n > 25; means 95% confidence intervals; throughout Fig. 3 irradiation at 458 nm, spot diameter = 10 m; time shown is in minutes and seconds.
Figure 4.
Figure 4: Crystallization and structural modelling of PA-Rac1. a, Dark state crystal structure of PA-Rac1. Blue, LOV domain; red, J helix; green, Rac1. b, Interacting residues at the LOV–Rac interface (arrow in panel a), including Trp 56. c, Mutating Cdc42 to include the Trp involved in stabilizing the LOV2–Rac1 interaction substantially improved LOV inhibition of Cdc42. Lane 1, PA-Cdc42; linking LOV to Cdc42 using the same truncations that produced good inhibition for Rac does not inhibit Cdc42–PAK binding. Lane 2, PA-Cdc42–CRIB; covalently linking the CRIB domain of PAK to PA-Cdc42 blocks PAK binding. Lane 3, PA-Cdc42(F56W) (PA-Cdc42W); introduction of the tryptophan substantially improves LOV inhibition of Cdc42 binding to PAK. Lane 4, lit state mutant of PA-Cdc42(F56W) (PA-Cdc42W(I539E), showing that Cdc42 inhibition is sensitive to the lit/dark state of the LOV domain. Supplementary Movie 16 and Supplementary Fig. 14 demonstrate the ability of PA-Cdc42(F56W) to produce filopodia and protrusions in living cells.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2009, 461, 104-108) copyright 2009.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
23325214 Y.Sasai (2013).
Cytosystems dynamics in self-organization of tissue architecture.
  Nature, 493, 318-326.  
22596318 A.L.Slusarczyk, A.Lin, and R.Weiss (2012).
Foundations for the design and implementation of synthetic genetic circuits.
  Nat Rev Genet, 13, 406-420.  
22750946 M.Galic, S.Jeong, F.C.Tsai, L.M.Joubert, Y.I.Wu, K.M.Hahn, Y.Cui, and T.Meyer (2012).
External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane.
  Nat Cell Biol, 14, 874-881.  
22842228 M.Krauthammer, Y.Kong, B.H.Ha, P.Evans, A.Bacchiocchi, J.P.McCusker, E.Cheng, M.J.Davis, G.Goh, M.Choi, S.Ariyan, D.Narayan, K.Dutton-Regester, A.Capatana, E.C.Holman, M.Bosenberg, M.Sznol, H.M.Kluger, D.E.Brash, D.F.Stern, M.A.Materin, R.S.Lo, S.Mane, S.Ma, K.K.Kidd, N.K.Hayward, R.P.Lifton, J.Schlessinger, T.J.Boggon, and R.Halaban (2012).
Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma.
  Nat Genet, 44, 1006-1014.
PDB codes: 3sbd 3sbe 3th5
21352235 A.Losi, and W.Gärtner (2011).
Old chromophores, new photoactivation paradigms, trendy applications: flavins in blue light-sensing photoreceptors.
  Photochem Photobiol, 87, 491-510.  
21076938 B.Chen, Y.Gao, T.Jiang, J.Ding, Y.Zeng, R.Xu, and X.Jiang (2011).
Inhibition of tumor cell migration and invasion through knockdown of rac1 expression in medulloblastoma cells.
  Cell Mol Neurobiol, 31, 251-257.  
22016058 C.M.Welch, H.Elliott, G.Danuser, and K.M.Hahn (2011).
Imaging the coordination of multiple signalling activities in living cells.
  Nat Rev Mol Cell Biol, 12, 749-756.  
21499630 C.Wang, Z.Zhu, Y.Song, H.Lin, C.J.Yang, and W.Tan (2011).
Caged molecular beacons: controlling nucleic acid hybridization with light.
  Chem Commun (Camb), 47, 5708-5710.  
21437309 E.D'Este, G.Baj, P.Beuzer, E.Ferrari, G.Pinato, E.Tongiorgi, and D.Cojoc (2011).
Use of optical tweezers technology for long-term, focal stimulation of specific subcellular neuronal compartments.
  Integr Biol (Camb), 3, 568-577.  
21621038 H.Shadpour, J.S.Zawistowski, A.Herman, K.Hahn, and N.L.Allbritton (2011).
Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors.
  Anal Chim Acta, 696, 101-107.  
21261629 I.H.van Stokkum, M.Gauden, S.Crosson, R.van Grondelle, K.Moffat, and J.T.Kennis (2011).
The primary photophysics of the Avena sativa phototropin 1 LOV2 domain observed with time-resolved emission spectroscopy.
  Photochem Photobiol, 87, 534-541.  
21191370 J.E.Toettcher, C.A.Voigt, O.D.Weiner, and W.A.Lim (2011).
The promise of optogenetics in cell biology: interrogating molecular circuits in space and time.
  Nat Methods, 8, 35-38.  
21822294 J.Herrou, and S.Crosson (2011).
Function, structure and mechanism of bacterial photosensory LOV proteins.
  Nat Rev Microbiol, 9, 713-723.  
21191368 K.Deisseroth (2011).
  Nat Methods, 8, 26-29.  
21250783 M.E.Auldridge, and K.T.Forest (2011).
Bacterial phytochromes: more than meets the light.
  Crit Rev Biochem Mol Biol, 46, 67-88.  
21271630 M.V.Golynskiy, M.S.Koay, J.L.Vinkenborg, and M.Merkx (2011).
Engineering protein switches: sensors, regulators, and spare parts for biology and biotechnology.
  Chembiochem, 12, 353-361.  
21482721 M.Vicente-Manzanares, K.Newell-Litwa, A.I.Bachir, L.A.Whitmore, and A.R.Horwitz (2011).
Myosin IIA/IIB restrict adhesive and protrusive signaling to generate front-back polarity in migrating cells.
  J Cell Biol, 193, 381-396.  
21191371 P.Hegemann, and A.Möglich (2011).
Channelrhodopsin engineering and exploration of new optogenetic tools.
  Nat Methods, 8, 39-42.  
21305623 U.Krauss, T.Drepper, and K.E.Jaeger (2011).
Enlightened enzymes: strategies to create novel photoresponsive proteins.
  Chemistry, 17, 2552-2560.  
21517885 W.Gärtner, and P.Hegemann (2011).
Introduction to the Symposium-in Print: Blue light effects.
  Photochem Photobiol, 87, 489-490.  
21212476 Y.Chen, S.Guzik, J.P.Sumner, J.Moreland, and A.P.Koretsky (2011).
Magnetic manipulation of actin orientation, polymerization, and gliding on myosin using superparamagnetic iron oxide particles.
  Nanotechnology, 22, 065101.  
19911402 A.Deiters (2010).
Principles and applications of the photochemical control of cellular processes.
  Chembiochem, 11, 47-53.  
20835487 A.Möglich, and K.Moffat (2010).
Engineered photoreceptors as novel optogenetic tools.
  Photochem Photobiol Sci, 9, 1286-1300.  
20192744 A.Möglich, X.Yang, R.A.Ayers, and K.Moffat (2010).
Structure and function of plant photoreceptors.
  Annu Rev Plant Biol, 61, 21-47.  
20850295 A.Rana, and R.E.Dolmetsch (2010).
Using light to control signaling cascades in live neurons.
  Curr Opin Neurobiol, 20, 617-622.  
20354536 B.N.Manz, and J.T.Groves (2010).
Spatial organization and signal transduction at intercellular junctions.
  Nat Rev Mol Cell Biol, 11, 342-352.  
20945451 B.P.Callahan, M.J.Stanger, and M.Belfort (2010).
Protease activation of split green fluorescent protein.
  Chembiochem, 11, 2259-2263.  
20543835 D.Fan, Z.Yin, R.Cheong, F.Q.Zhu, R.C.Cammarata, C.L.Chien, and A.Levchenko (2010).
Subcellular-resolution delivery of a cytokine through precisely manipulated nanowires.
  Nat Nanotechnol, 5, 545-551.  
20535203 D.G.Spiller, C.D.Wood, D.A.Rand, and M.R.White (2010).
Measurement of single-cell dynamics.
  Nature, 465, 736-745.  
20187057 D.K.Sinha, P.Neveu, N.Gagey, I.Aujard, C.Benbrahim-Bouzidi, T.Le Saux, C.Rampon, C.Gauron, B.Goetz, S.Dubruille, M.Baaden, M.Volovitch, D.Bensimon, S.Vriz, and L.Jullien (2010).
Photocontrol of protein activity in cultured cells and zebrafish with one- and two-photon illumination.
  Chembiochem, 11, 653-663.  
20562867 D.Strickland, X.Yao, G.Gawlak, M.K.Rosen, K.H.Gardner, and T.R.Sosnick (2010).
Rationally improving LOV domain-based photoswitches.
  Nat Methods, 7, 623-626.  
20717145 E.Papusheva, and C.P.Heisenberg (2010).
Spatial organization of adhesion: force-dependent regulation and function in tissue morphogenesis.
  EMBO J, 29, 2753-2768.  
21081920 E.Peter, B.Dick, and S.A.Baeurle (2010).
Mechanism of signal transduction of the LOV2-Jα photosensor from Avena sativa.
  Nat Commun, 1, 122.  
20409272 J.Chory (2010).
Light signal transduction: an infinite spectrum of possibilities.
  Plant J, 61, 982-991.  
20729930 J.T.Parsons, A.R.Horwitz, and M.A.Schwartz (2010).
Cell adhesion: integrating cytoskeletal dynamics and cellular tension.
  Nat Rev Mol Cell Biol, 11, 633-643.  
20592249 K.B.Walters, J.M.Green, J.C.Surfus, S.K.Yoo, and A.Huttenlocher (2010).
Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome.
  Blood, 116, 2803-2811.  
20702081 K.Clancy, and C.A.Voigt (2010).
Programming cells: towards an automated 'Genetic Compiler'.
  Curr Opin Biotechnol, 21, 572-581.  
20861876 K.H.Wrighton (2010).
Sensing and controlling protein dynamics.
  Nat Rev Mol Cell Biol, 11, 680-681.  
20676078 K.M.Hahn, and B.Kuhlman (2010).
Hold me tightly LOV.
  Nat Methods, 7, 595, 597.  
20667710 N.A.Frost, J.M.Kerr, H.E.Lu, and T.A.Blanpied (2010).
A network of networks: cytoskeletal control of compartmentalized function within dendritic spines.
  Curr Opin Neurobiol, 20, 578-587.  
20544018 P.Guillaumot, C.Luquain, M.Malek, A.L.Huber, S.Brugière, J.Garin, D.Grunwald, D.Régnier, V.Pétrilli, E.Lefai, and S.N.Manié (2010).
Pdro, a protein associated with late endosomes and lysosomes and implicated in cellular cholesterol homeostasis.
  PLoS One, 5, e10977.  
20566876 P.J.Hanley, Y.Xu, M.Kronlage, K.Grobe, P.Schön, J.Song, L.Sorokin, A.Schwab, and M.Bähler (2010).
Motorized RhoGAP myosin IXb (Myo9b) controls cell shape and motility.
  Proc Natl Acad Sci U S A, 107, 12145-12150.  
  20948805 R.Kosoff, and J.Chernoff (2010).
LOV conquers (sm)All GTPases.
  F1000 Biol Rep, 2, 0.  
20159593 S.K.Yoo, Q.Deng, P.J.Cavnar, Y.I.Wu, K.M.Hahn, and A.Huttenlocher (2010).
Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish.
  Dev Cell, 18, 226-236.  
20303157 V.Gradinaru, F.Zhang, C.Ramakrishnan, J.Mattis, R.Prakash, I.Diester, I.Goshen, K.R.Thompson, and K.Deisseroth (2010).
Molecular and cellular approaches for diversifying and extending optogenetics.
  Cell, 141, 154-165.  
20485291 W.A.Lim (2010).
Designing customized cell signalling circuits.
  Nat Rev Mol Cell Biol, 11, 393-403.  
20665617 W.Gärtner (2010).
Lights on: a switchable fluorescent biliprotein.
  Chembiochem, 11, 1649-1652.  
20691580 W.Weber, and M.Fussenegger (2010).
Synthetic gene networks in mammalian cells.
  Curr Opin Biotechnol, 21, 690-696.  
20473296 X.Wang, L.He, Y.I.Wu, K.M.Hahn, and D.J.Montell (2010).
Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo.
  Nat Cell Biol, 12, 591-597.  
20008333 X.Yan, Y.Shen, and X.Zhu (2010).
Live show of Rho GTPases in cell migration.
  J Mol Cell Biol, 2, 68-69.  
19857985 A.Deiters (2009).
Light activation as a method of regulating and studying gene expression.
  Curr Opin Chem Biol, 13, 678-686.  
19965465 J.D.Scott, and T.Pawson (2009).
Cell Signaling in Space and Time: Where Proteins Come Together and When They're Apart.
  Science, 326, 1220-1224.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.