Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase

Abstract

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg²⁺ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

Keywords: druggable cysteines; fast-cycling mutations in cancer; molecular dynamics simulations; point mutations; small-GTPases.

FIGURE 1

(a) Ribbon representations of GTP and GDP‐bound Rac1 with the main functional sites switch‐I, switch‐II, P‐loop, and insert‐region shown in cyan, green, yellow, and purple, respectively. The Mg²⁺ ion is shown as a pink sphere. (b) Schematic representation of Rac1 secondary structure, with the C‐terminal Hypervariable region (HVR) not shown in (a). On the bottom primary sequence with the color scheme according to (a). The mutation site is highlighted in cyan.

FIGURE 2

Root Mean Square Fluctuations (RMSF, Å) calculated on the backbone atoms, of wild type (gray) and P29L (magenta), P29Q (green) and P29S (cyan) mutants of the GTP‐bond (left) and GDP‐bound (right) Rac1 protein. The main functional sites p‐loop, switch‐I (swI), switch‐II (swII), and insert‐region (insert) are highlighted in pink, green, light‐blue, and orange, respectively.

FIGURE 3

The probability distribution function of the distance (Å) between (a) Cα:Phe37 and Cα:Thr58 accounting for switch‐I opening (b) Cα:Arg68 and Mg²⁺ atoms, accounting for switch‐II opening calculated along the 1 μs‐long classical MD trajectory of Rac1‐GDP wild type (gray) and P29L (magenta), P29Q (green), and P29S (cyan). The probability distribution function for replicas is reported in Figure S11.

FIGURE 4

Ribbon representations of GDP‐bound Rac1. The sphere sizes refer to per‐residue score and account for the differences in the Rac1/GDP interactions between Rac1 wt and the different mutants. Van deer Waals spheres, highlighting the residues experiencing the largest differences, are depicted in magenta, green, and cyan for P29, P29Q, and P29S, respectively. For clarity, only differences higher than 25% are reported. The GDP is shown in licorice surrounded by dotted spheres.

FIGURE 5

Essential dynamics of Rac1 in the GDP‐bound state. Eigenvectors of the first principal component extracted from PCA are shown as arrows whose direction and size represent the direction and amplitude of motion for (a) Rac1^wt, (b) Rac1^P29L, (c) Rac1^P29Q, and (d) Rac1^P29S. For clarity, we only report the arrows indicating a motion larger than 1 Å. (e) Density distribution of the positions visited by the Mg²⁺ ion plotted considering 4500 frames along the MD simulations trajectories. The gray square shows the population density of Mg²⁺ ion in Rac1^wt (silver transparent spheres). The magenta, green, and cyan squares show the position of the Mg²⁺ ion in Rac1^P29L, Rac1^P29Q, and Rac1^P29S, respectively. The image was generated by centering the MD trajectory with respect to the protein C‐alpha after verifying that the protein back‐bone fluctuations are less marked than those of the Mg²⁺ ion (Figure S20). The protein and GDP substrate are kept fixed in the visualization to enhance the clarity of the Mg²⁺ ion movement. However, the interaction between this ion and beta‐phosphate group is maintained throughout the MD simulation trajectory (Table S2).

FIGURE 6

Bar plots showing on the y‐axes the GDP residence time (ns) and on the x‐axes are reported the values for the three replicas of each system. The residence time was computed for (a) Rac1^wt and (c) Rac1^P29Q. The boxes represent the calculated residence time values, whiskers indicate the range of the data. The dots represent outliers values of τ, while median and mean are shown by orange and dashed red lines, respectively. The outliers are indicated, but they are not removed from the computation of the residence time. The mechanism of GDP dissociation along trajectory 1 for replica 1 is exemplified for (b) Rac1^wt and (d) Rac1^P29Q. The first snapshot represents reference state. The distance (Å) between Thr17 side‐chain oxygen and Mg²⁺ is reported to highlight Mg²⁺ ion dissociation along with that of the GDP.

FIGURE 7

Representation of the cropped model used for the density functional theory calculations, including GDP, Threonine 17, four water molecules and the magnesium ion.