Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jul 22;286(29):26093-106.
doi: 10.1074/jbc.M110.197053. Epub 2011 May 24.

Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs)

Hui Wang  1 , Prasanta K Hota, Yufeng Tong, Buren Li, Limin Shen, Lyudmila Nedyalkova, Susmita Borthakur, Soonjeung Kim, Wolfram Tempel, Matthias Buck, Hee-Won Park
Affiliations

Structural basis of Rnd1 binding to plexin Rho GTPase binding domains (RBDs)

Hui Wang et al. J Biol Chem. .

Abstract

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Structures of the complex of plexin-B1 RBD and Rnd1. A, ribbon diagram of the plexin-B1 RBD·Rnd1 complex homodimer/heterotetramer. The asymmetric unit consists of plexin-B1 RBD chains A (cyan) and C (green) (residues 1746–1852), forming a homodimer, and Rnd1 chains B (violet) (residues 13–188) and D (hot pink) (residues 7–188), bound on opposite sides to the RBD dimer. The homodimer interface is stabilized by a π-cation interaction between Trp1830 and Arg1832 (21). B, one unit of the plexin-B1 RBD (cyan) and Rnd1 (violet) complex. Several of the β strands (β1–β7) and two α-helices (α1 and α2) are labeled in plexin-B1 RBD. C, superimposition of the RBDs of the plexin-B1 RBD·Rnd1 complex (cyan) with the free plexin-B1 RBD (green) illustrates conformational changes that take place in the β1-β2 loop, the β3-β4 loop, and the η1-η2 region upon complex formation with Rnd1.
FIGURE 2.
FIGURE 2.
Interface of the plexin-B1 RBD·Rnd1 complex. A, transparent surface representation of the plexin-B1 RBD·Rnd1 complex. The plexin-B1 RBD and Rnd1 are shown in cyan and violet, respectively. The β3 and β4 strands and helix α2 of the plexin-B1 RBD are located in the interface, as are the β2 and β3 strands, the 310-helices η1 and η2, and one α-helix of Rnd1. The residues in the interface are represented by a stick model. The close-up view of the interface is shown on the right, where the hydrophobic core is highlighted by a gray transparent circle. B, amino acid interactions at the interface are shown in a LIGPLOT diagram (53). Hydrogen bonds are presented as dashed lines; interatomic distances are in angstroms. “Radiating” curves indicate hydrophobic contacts between the corresponding atoms or residues and the surrounding residues. C, two areas of the interface contact are shown, illustrating that the three residues of Rnd1, Phe47, Glu48, and Tyr114, are flipped toward plexin-B1 (shown in cyan), as seen in the superimposition of the bound (magenta for complex with plexin-B1) and the free (blue) structures of Rnd1.
FIGURE 3.
FIGURE 3.
Interface of the plexin-A2 RBD·Rnd1 complex. In part similar to Fig. 2A, a transparent surface representation of plexin-A2 RBD·Rnd1 complex is shown, with the interface displayed in an expanded view. B, LIGPLOT diagram of contacts at the interface. Rnd1 residues Trp66 and Tyr114 are omitted for clarity (but are shown in D). C, superimposition of the RBDs of the plexin-A2 RBD·Rnd1 complex (gold) and free plexin-B1 (green) illustrates conformational changes that take place. D, superposition of two regions in contact showing perturbations similar to those of the Rnd1 side-chain structure upon association with plexin-A2 RBD as seen in the case of plexin-B1 RBD binding. Gold, RBD residues; blue, free Rnd1; magenta, bound Rnd1 residues.
FIGURE 4.
FIGURE 4.
Crystal structure of plexin-C1 and -D1 RBD. A, ribbon diagram of plexin-C1 RBD. B, superimposition of the plexin-C1 RBD (green) and the free (blue) and bound (cyan) forms of the plexin-B1 RBD. C, plexin-D1 RBD. D, superimposition of the plexin-D1 RBD (gold) and the free (blue) and bound (cyan) forms of the plexin-B1 RBD in a ribbon diagram.
FIGURE 5.
FIGURE 5.
Structure-based sequence alignments. Structure-based sequence alignment of human plexin RBDs (A) and Rnd1 to -3 and Rac1 to -3 Rho GTPase (B). Alignments were generated using ESPript 2.1 (54). Sequence similarities are highlighted in yellow, and sequence identities are shown as white letters on a red background. Secondary structural elements (arrows for β-strands and coils for α-helices) are indicated at the top. Blue and green triangles indicate residues at the plexin-A2 and -B1 RBD·Rnd1 complex interface, respectively, as discussed under “Results.”
FIGURE 6.
FIGURE 6.
Analytical gel filtration assay for binding of Rnd1 to GTPases (A) and plexin-A2 RBD dimerization (B). A, the blue star indicates the peak corresponding to the plexin-A2 RBD·Rnd1 complex. The data show that there is no binding between plexin-C1 or plexin-D1 and Rnd1. The inset shows the elution volumes of standard proteins (158, 44, 17, and 1.4 kDa) on a Superdex 75 10/300 GL column (GE Healthcare), plotted against log molecular masses, which were used to estimate the molecular masses of samples. B, plexin-A2 RBD dimerization measured under similar conditions as above. The data show that the RBD dimerizes, albeit at a slightly higher concentration than observed for plexin-B1 RBD (40).
FIGURE 7.
FIGURE 7.
Representative ITC data showing binding of Rnd1 to plexin RBDs. A, ITC raw data (base line-corrected) and fitted data for the interaction of Rnd1 with the RBD of plexin-A2. No interaction is detected between RBD of plexin-C1 and Rnd1 (B), whereas a modest binding is generated by the T1264V/R1265V mutation in RBD of plexin-C1 toward Rnd1 (C).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tamagnone L., Artigiani S., Chen H., He Z., Ming G. I., Song H., Chedotal A., Winberg M. L., Goodman C. S., Poo M., Tessier-Lavigne M., Comoglio P. M. (1999) Cell 99, 71–80 - PubMed
    1. Negishi M., Oinuma I., Katoh H. (2005) Cell Mol. Life Sci. 62, 1363–1371 - PMC - PubMed
    1. Patel B. N., Van Vactor D. L. (2002) Curr. Opin. Cell Biol. 14, 221–229 - PubMed
    1. Gitler A. D., Lu M. M., Epstein J. A. (2004) Dev. Cell 7, 107–116 - PubMed
    1. Serini G., Valdembri D., Zanivan S., Morterra G., Burkhardt C., Caccavari F., Zammataro L., Primo L., Tamagnone L., Logan M., Tessier-Lavigne M., Taniguchi M., Püschel A. W., Bussolino F. (2003) Nature 424, 391–397 - PubMed

Publication types

LinkOut - more resources