PDBsum entry 2wkp

Transferase, cell adhesion

PDB id: 2wkp

Contents

Protein chain

320 a.a. *

Ligands

GTP

FMN

Metals

_CA

_MG

Waters ×313

* Residue conservation analysis

PDB id:

2wkp

Name:

Transferase, cell adhesion

Title:

Structure of a photoactivatable rac1 containing lov2 wildtype

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: rac1_human, 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.

Resolution:

1.90Å R-factor: 0.167 R-free: 0.195

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

Date:

16-Jun-09 Release date: 18-Aug-09

Protein chain

O49003  (O49003_AVESA) -  non-specific serine/threonine protein kinase from Avena sativa

Seq: 923 a.a.

Struc: 320 a.a.*

Protein chain

P63000  (RAC1_HUMAN) -  Ras-related C3 botulinum toxin substrate 1 from Homo sapiens

Seq: 192 a.a.

Struc: 320 a.a.*

* PDB and UniProt seqs differ at 173 residue positions (black crosses)

Enzyme reactions

• Enzyme class 1: E.C.2.7.11.1 - non-specific serine/threonine protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

L-seryl-[protein] (Bound ligand (Het Group name = GTP) corresponds exactly) + ATP = O-phospho-L-seryl-[protein] + ADP + H(+)

• Enzyme class 2: E.C.3.6.5.2 - small monomeric GTPase.
• Reaction: GTP + H2O = GDP + phosphate + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

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.

ABSTRACT

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.