Figure 5. Analysis of GTP binding site in N-GTPase.

a) Ribbon diagram of N-GTPase structure (green) with consensus GTPase G motifs differently colored and labeled. P-loop: magenta, Switch I: pink Switch II: yellow, G4: teal, G5: purple. b) Ribbon diagram of H-Ras G12V (Grey) bound to GMP-PNP (PDB ID: 5P21). G motifs are colored as in part a. c) Zoom-in showing the superposition of H-Ras with N-GTPase and the catalytic Q61 of H-Ras (grey) sterically clashing with N-GTPase residues S22, T24, and E98. d) Map of N-GTPase interaction with GTP and Mg²⁺ adapted from Ligplot (Wallace et al., 1995). e) Detailed view of GTP gamma phosphate and Mg²⁺ binding site in N-GTPase (left) and H-Ras (right). Region shown is indicated by a dashed box in parts a and b. f) Solubility and total expression of N-GTPase wild-type (WT) and mutant proteins. Clarified (top) or total (bottom) lysates are resolved by SDS-PAGE and proteins visualized by Coomassie Blue staining. g) Final purified N-GTPase wild-type and mutant proteins, resolved by SDS-PAGE and stained with Coomassie Blue. h) Thermal denaturation curves from a representative experiment of N-GTPase proteins. Fluorescence signal has been normalized in each sample. i) Scatter dot plot of melting temperatures calculated as the mean of four measurements for each sample (error bars indicate standard deviation) determined by fitting the melting curve to a sigmoidal model. P-values from One-way ANOVA comparisons of the melting temperatures to wild-type is indicated above each bar (****: p <0.0001. n.s.: no significant difference).