PDBsum entry 2wkq

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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   18 terms 
  Biological process     epithelial cell morphogenesis   70 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

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PDB codes: 3sbd 3sbe 3th5
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Cell adhesion: integrating cytoskeletal dynamics and cellular tension.
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Programming cells: towards an automated 'Genetic Compiler'.
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Sensing and controlling protein dynamics.
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Hold me tightly LOV.
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Pdro, a protein associated with late endosomes and lysosomes and implicated in cellular cholesterol homeostasis.
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20566876 P.J.Hanley, Y.Xu, M.Kronlage, K.Grobe, P.Schön, J.Song, L.Sorokin, A.Schwab, and M.Bähler (2010).
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LOV conquers (sm)All GTPases.
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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.
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20485291 W.A.Lim (2010).
Designing customized cell signalling circuits.
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20665617 W.Gärtner (2010).
Lights on: a switchable fluorescent biliprotein.
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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.