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PNAS 106 (37): 15610-15615

Copyright © 2009 by the National Academy of Sciences.

Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

Huawei Hea, Taehong Yangb, Jonathan R. Termana,b, and Xuewu Zhanga,c,1

Departments of aPharmacology, bNeuroscience, and cBiochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041


Figure 1
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Fig. 1.. Overall structure of the plexin A3 intracellular region. (A) Schematic diagram of the plexin A3 intracellular region. The two catalytically critical arginines are shown. (B) Structure of the plexin A3 intracellular region. The color scheme is the same as that in A. (C) Superimposition of the GAP domains of plexin A3 and p120 Ras GAP (PDB ID: 1WQ1) suggesting an autoinhibited conformation of the plexin A3 GAP domain. Parts with significant differences between the two structures are highlighted with red arrows. (D) Cross-section view of the docking of Ras onto the plexin A3 GAP, based on the structure of the p120Ras GAP/Ras complex. Steric clashes between the GAP domain and Ras are highlighted with red arrows.

 

Figure 2
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Fig. 2.. The dimer of the plexin B1 RBD core reported previously (2R2O) mimics an intra-molecular interaction between the RBD core and the C-terminal linker in plexin A3.

 

Figure 3
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Fig. 3.. Interaction between the RBD and GAP domain. (A) Interface between the RBD and the GAP domain. The N-terminal linker (N-linker) and C-terminal linker (C-linker) are colored in green and cyan, respectively. (B) COS-7 cell collapse assays showing that mutations in the RBD/GAP interface abolish plexin-induced cell collapse. The percentages of collapse are average of three experiments. Error bars: standard deviations. Representative images of cells are shown and collapsed cells are marked with arrowheads. (C) In vivo axon guidance assay of the L1524G mutant in Drosophila plexin A. Increased expression of wild type (WT) plexin A induces axons to abnormally cross the midline of the central nervous system (arrowheads). Both truncation of the cytoplasmic domain ({Delta}Cyto) and the L1524G mutation eliminate this effect. Genotypes: (Control) ELAV-GAL4,UAS:HA Plexin A/+; (WT) ELAV-GAL4,UAS:HA Plexin A/UAS:HA Plexin A; ({Delta}Cyto) ELAV-GAL4, UAS:HA Plexin A/UAS: HA Plexin A{Delta}Cyto; (L1524G) ELAV-GAL4, UAS:HA Plexin A/UAS:HA Plexin A L1524G. ***P < 0.0005 by two-tailed Student's t test.

 

Figure 4
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Fig. 4.. Rnd1 alone does not induce conformational changes in plexin. (A) Superimposition of the plexin A3 intracellular domain, the plexin B1 intracellular domain (PDB ID: 3HM6), the plexin B1 RBD core (PDB ID: 2R2O), and the plexin B1 RBD core/Rnd1 complex (PDB ID: 2REX). (B) Detailed view of the Rho GTPase binding site in the structures of plexin A3 and the plexin B1 RBD/Rnd1 complex. (C) Sequence alignment of the Rho GTPase-binding region in plexins. Residues involved in Rho GTPase binding are highlighted with circles. The two brown circles indicate the two residues that are conserved in class A and B plexins, but different in class C and D plexins. The secondary structure elements are based on the plexin A3 structure. m, Murine; d, Drosophila.

 

Figure 5
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Fig. 5.. Interaction between the N-terminal segment and the GAP domain. (A) Sequence alignment of the N-terminal segment of plexins. Residues making contacts with the GAP domain are highlighted with black circles. m, Murine; d, Drosophila. (B) Interactions between the GAP domain and the N-terminal segment. The three right panels are exploded views of the correspondingly numbered boxes in the left panel. Changes of view angles are indicated by the rotation axis on the top of each panel. (C) Mutational analyses of the interface between the N-terminal segment and the GAP domain in the COS-7 collapse assay. (D) Mutational analyses of the corresponding interface residues in Drosophila plexin A using the vivo axon guidance assay. Genotypes: (M1320R) ELAV-GAL4, UAS:HA Plexin A/UAS:HA Plexin A M1320R; (L1338R) ELAV-GAL4, UAS:HA Plexin A/UAS:HA Plexin A L1338R; and as in Fig. 3C. *P < 0.05; ***P < 0.001 by two-tailed Student's t test.

 


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