Fig 2: CD spectral analysis of Syx. Experimental spectra is shown as black dots in the plot below. Fits of CDSSTR (SMP180), BeStSel, and K2D3 CD analysis algorithms are shown as purple dashes, green line and red + respectively. The predicted spectra of Syx based on the homology model is shown in blue dashes.

Fig 3: (A) The architecture of the docked Syx-RhoA complex as was simulated, complete with PI(4,5)P2 (light green spheres at the top of the GEF, interacting with membrane lipids), GDP, and Mg2+ cofactors and post translationally geranylgeranylated Cys-190 (light yellow spheres attached to the N-terminus of RhoA), simulated on a POPC and PI(4,5)P2 bilayer (transparent sticks at the top of the protein complex). (B) Dynamic network analysis of the DH domain interacting with RhoA Switch I and Switch II residues (shown as cyan sticks at the interface between the two proteins) illustrate how these interactions (yellow tubes) influence the residues responsible for complexing Mg2+ (purple sphere indicated by figure label) and GDP (teal sticks indicated by figure label). (C) The figure shows the geranylgeranyl group (green) conjugated to RhoA (red) residue C190. The geranylgerany group formed transient interactions with the PH domain of Syx (blue) at the interface of the membrane and the cytoplasm during the 1 microsecond all atom molecular dynamics simulation.

Fig 4: (A) SEC absorbance trace at 280 nm of mixtures of MBP-Syx393–792 and RhoA after using varying strategies for removing GDP from RhoA to induce the formation of a high affinity MBP-Syx393–792–RhoA complex. The composition of each SEC trace is as follows: Shown in purple, “Mix 1” contains MBP-Syx393–792–RhoA with 10 mM of EDTA to chelate magnesium out of the GDP binding site of RhoA; shown in red, “Mix 2” used cleaved Syx393–792 mixed with RhoA, also with 10 mM EDTA; shown in black, “Mix 3” contained MBP-Syx393–792 mixed with RhoA which was buffer exchanged six times in a 10k MWCO spin concentrator to remove GDP; shown in light blue, “mix 4” an excess of ammonium sulfate was used to precipitate a protein mixture containing both MBP-Syx393–792 and RhoA to remove GDP; Shown as a grey trace “mix 5” the process was the same as “mix 4” except that the ammonium sulfate precipitation was performed on RhoA alone before being mixed with MBP-Syx393–792. All complex formation mixes were made as 1:1 mixtures and each combination was allowed to equilibrate for 1 h. The green trace labeled “RhoA” shows RhoA control and orange trace labeled “Syx” shows MBP-Syx393–792 control. MW of N-term MBP-Syx is 90.8 kDa. RhoA is 22 kDa. The protein standard shown in dark blue and labled as “standard” consists of A. Thyroglobulin (Mr 669 000), B. Ferritin (Mr 440 000), C. Aldolase (Mr 158 000), D. Conalbumin (Mr 75 000), E. Ovalbumin (Mr 44 000), F. Carbonic anhydrase (Mr 29 000), G. Ribonuclease A (Mr 13 700). (B) Cartoons depict expressed protein fusion constructs of both MBP-Syx393–792 and RhoA. RhoA is truncated at residue 184 to remove its highly charged linker and geranylgeranyl transferase recognition site. (C) MBP-Syx393–792 (lane A) cleavage with TEV protease produces an approximately 48 kDa band (lane B) corresponding to the molecular weight of Syx393–792 (48 kDa and MBP 42 kDa), indicative of successful cleavage. (D) Coomassie Gel of fractions from SEC run “mix 4” shows the presence of bands corresponding to the correct molecular weight for RhoA and MBP-Syx393–792. Bands in fractions at 8 and 9 ml indicate the presence of MBP-Syx393–792 and RhoA where bands in fraction 15, 16, and 17 ml indicate the presence of RhoA and cleaved MBP-Syx393–792 due to background cleavage activity at the TEV site.