Supplementary Figures for Tong et al.: Structure and function of the intracellular region of the plexin-b1 transmembrane receptor

Transcription

1 Supplementary Figures for Tong et al.: Structure and function of the intracellular region of the plexin-b1 transmembrane receptor

2 Figure S1. Plexin-B1 GAP segments are homologous to RasGAPs. Sequence alignment of plexin-b1, SYNGAP and p120gap (also known as RASA1) and R-RasGAP (RASA3), using the color scheme of Fig 1a. The sequence homology is moderately high; for example plexin-b1 has 19.7% identity (38.2% similarity) with the N- and 21% identity (37.8% similarity) with the C-terminal parts of the p120gap sequence. The location of the catalytic Arginine finger and supporting Arginine are indicated (first and second triangles, respectively). Highly conserved residues are shown in yellow, completely conserved in red. Regions of the polypeptide chain not observed in the plexin-b1 crystal structure are shaded grey.

3 Figure S2. Fo - Fc omit map of the coupling loop. A simulated annealing Fo - Fc omit map was generated using the Phenix program (ref S1) by omitting the coupling loop (residues 1879 to 1890). The resulting electron density map is contoured at 3 sigma, and superimposed with the final coupling loop model, a) shown in same orientation as Fig. 2, b) same as a) but rotated by ~ 40 o about a vertical axis in the page to provide a different view.

4 Figure S3. Overview of the structure in crystal lattice. Shown are two possible orientations for crystallographic symmetry-related molecules. The color scheme is similar to Fig.1a (RBD- yellow, N-terminal helix red, coupling loop green, GAP domains- cyan/magenta or grey). PISA analysis (ref. S2) indicates that neither of these are real interfaces but instead originate from crystal packing. Loops missing from the structure are not long enough to allow a swap to make a dimer between structures in the crystal lattice. Similarly the number of residues that are missing due to lack of electron density are too few to connect the RBD and C-terminal GAP segment to the coupling loop of a neighboring molecule (27 residues N- and 23 C-terminal to the coupling loop). Thus, it is unlikely that the loop could come from a neighboring molecule and, therefore, the structure is that of a monomer.

5 (I) (II) Abs 280nm (iv) (v) (iii) (ii) Ovalbumin (i) BSA (ii) Human IgG (iii) Ferritin (iv) Thyroglobulin (v) Elution Volume (ml) (i) K av Calibration plot for Superdex 200 PC3.2/30 Ovalbumin (43 kd) BSA (68 kd) Human IgG (150 kd) 0.2 Ferritin (440 kd) 0.1 Thyroglobulin (669 kd) Log (Mr) (III) (IV) 40 wtplexinb1 (i) Rnd1(ii) wtplexinb1+rnd1(iii) monomer kd, V e = 1.61 ml complex kd, V e = 1.56 ml 40 wtplexinb1 (i) Rac1 (ii) wtplexinb1+rac1(iii) monomer kd, V e = 1.61 ml complex kd, V e = 1.55 ml Abs 280nm 20 (iii) (i) Abs 280nm 20 (iii) (i) 0 (ii) 0 (ii) Elution volume/ ml Elution volume/ ml Figure S4. Gel filtration data for the intracellular plexin-b1 protein and its complex formation with Rnd1 and Rac1. Plexin s molecular weight in solution and the binding with GTPases were determined by analytical grade column chromatography, (Superdex 200 PC3.2/30cm gel filtration column; GE Healthcare). Panels I and II. The gel filtration column was calibrated using 5 standard molecular weight proteins in 20 mm Tris buffer (ph 7.5, 1mM TCEP, 50 mm sodium chloride, flow rate 30 µl/min at 4 o C). The plot of the gel phase coefficient (K av ) vs. log (M r ) is linear between 669 kd (Thyroglobulin) to 43 kd (Ovalbumin), where K av = (V e -V o )/(V t -V o ), V e = elution volume, V o = column void volume, V t = total column bed volume (2.4 ml), M r = molecular weight. Blue Dextran 2000 (M r 2000 kd) shows the column void volume (V o = 1.14 ml). Panels III and IV. 50 µl of a 20 µm sample of plexin-b1 was applied with/without Rac1.Q61L or Rnd1 GTPase in a ratio of 1:2. Panel IV also shows that the plexin is prone to forming higher order oligomers in presence of Rac1.Q61L.

6 Figure S5. Superposition of RBD from free dimer [PDB: 2R2O] (green/magenta) and Rnd1-bound state [2REX] (pink/cyan) a) showing minimal perturbation of the RBD structure and b) & c) a modest twisting of dimer bridge in response to Rnd1 binding or different crystal contacts.

7 Figure S6. Alignment of 9 human plexins (first part of sequence). Regions of interest are indicated as follows: Blue *, residue sidechains involved in RBD-coupling loop-gap interactions. Red *, residues involved in plexin:h-/r-ras interactions in the docked models. For comparison, the Green line delineates regions of interaction between p120gap and Ras (34).

8 Figure S6 continued.

9 Figure S7. Structures of the GAP active sites of four complexes during the simulations. a) p120gap : H-Ras, b) p120gap : R-Ras c) plexin-b1 : H-Ras and d) plexin-b1 : R-Ras. Shown is a superposition of 10 frames from each trajectory (spaced at 500 ps) on the initial structure using heavy atoms of the GTP nucleotide for the alignment. The nucleophilic water was identified as the one closest to the GTP γ-phosphate oxygens and closest to the Ras Gln sidechain. All simulations maintain the geometry of the GTP and the surrounding residues. A major difference is that in the case of plexin:h-ras complex the Arg finger moves away from the GTP, making only contacts with the terminal phosphate, but not with the O3B position. The solvent molecule closest to the sidechain of Q61 in H-Ras (Q87 in R-Ras) is maintained in a similar position but undergoes larger amplitude fluctuations in the case of the plexin:h-ras complex. These two effects are likely to result in a diminished catalytic efficiency in case of H-Ras (ref. S3). However, to understand whether the structures are configured in detail to promote GTP hydrolysis, would require a quantum mechanical approach, where the exact chemical mechanism of GTP hydrolysis and roles of sidechains continue to be debated (S4, S5). Overall, the close resemblance of the structure to that of p120:h-ras suggests that GAP function is likely also for plexin-b1:r-ras, but not for the plexin-b1:h-ras complex (consistent with the observed lack of binding affinity for the latter, see Fig. 4).

10 Figure S8. Comparison of several interatomic distances show differences in the trajectories of the complexes. Distances are plotted as a function of time for all 4 trajectories: a) distance between water and Gln sidechain oxygen for the water nearest to GTP and Gln, b) distance between Arg finger sidechain NH and GTP OB3 oxygen (P-O-P bond to terminal phosphate).. Additional computational details- Prior to the simulations MODELLER was used to build the missing loops in the intracellular region of plexin. The systems were then prepared as follows using CHARMM (ref. S6) : Large boxes filled with explicit TIP3P waters were used to solvate the structures leaving 16 Å to the boundary in each direction. Water molecules closer than 2.8 Å to any protein heavy atom were deleted. Random waters (> 5 Å away from the protein) were replaced by Cl - and Na + to yield a 100 mm salt solution and to neutralize the system. The crystallographic water that is located between GTP and Gln 61 in the p120gap : H-Ras complex (water 230 in the PDB structure 1WQ1) was also added to the system. NAMD 2.6 (S7) with the CHARMM22/CMAP force field (S8) was used for the simulations. For equilibration, the waters were first minimized, then heated to 300 K and equilibrated over 200 ps at constant temperature and volume, while the protein was held fixed. After another round of minimization the restraint on the GAP was removed and a 50 ps NVT simulation was run, after which the whole system was minimized and then equilibrated (at constant pressure) for 200 ps. (In the case of simulations with R-Ras, the last step of the preparation was split into two 100 ps simulations. During the first half, R-Ras and the crystallographic water were harmonically constrained to their equivalent positions in the p120gap:h-ras complex). The Berendsen thermostat and Langevin piston method were employed to keep the temperature (300 K) and pressure (1 atm) constant. Particle-Mesh Ewald (PME) was used for long-range electrostatic interactions, with a 12 Å cut-off for other non-bonded interactions. Time step was 2 fs, using SHAKE for X-H bonds.

11 Additional references: (other references see main text) S1. Krissinel, E., and Henrick, K (2007) J Mol Biol 372, S2. Adams, P.D., Grosse-Kunstleve, R.W., Hung, L-W., Ioerger, I.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.C. and Terwilliger, T.C. (2002) Acta Cryst. D58, S3. Resat, H., Straatsma, T. P., Dixon, D. A., and Miller, J. H. (2001) Proc. Natl. Acad. Sci. U. S. A 98, S4. Klahn, M., Braun-Sand, S., Rosta, E., and Warshel, A. (2005) J. Phys. Chem. B 109, S5. Grigorenko, B. L., Nemukhin, A. V., Shadrina, M. S., Topol, I. A., and Burt, S. K. (2007) Proteins 66, S6. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., and Karplus, M. (1983) J. Comput. Chem. 4, S7. Phillips, J. C., Braun, R., Wang, W., Gumbart, J., Tajkhorshid, E., Villa, E., Chipot, C., Skeel, R. D., Kale, L., and Schulten, K. (2005) J. Comput. Chem. 26, S8. Buck, M., Bouguet-Bonnet, S., Pastor, R. W., and MacKerell, A. D., Jr. (2006) Biophys. J. 90, L36-L38