Probing binding and allosteric mechanisms of the KRAS interactions with monobodies and affimer proteins: ensemble-based mutational profiling and thermodynamic analysis of binding energetics and allostery reveal diversity of functional hotspots and cr

KRAS, a historically "undruggable" oncogenic driver, has eluded targeted therapies due to its lack of accessible binding pockets in its active state. This study investigates the conformational dynamics, binding mechanisms, and allosteric communication networks of KRAS in complexes with monobodies (12D1, 12D5) and affimer proteins (K6, K3, K69) to characterize the binding and allosteric mechanisms and hotspots of KRAS binding. Through molecular dynamics simulations, mutational scanning, binding free energy analysis and network-based analyses, we identified conserved allosteric hotspots that serve as critical nodes for long-range communication in KRAS. Key residues in β-strand 4 (F78, L80, F82), α-helix 3 (I93, H95, Y96), β-strand 5 (V114, N116), and α-helix 5 (Y157, L159, R164) consistently emerged as hotspots across diverse binding partners, forming contiguous networks linking functional regions of KRAS. Notably, β-strand 4 acts as a central hub for propagating conformational changes, while the cryptic allosteric pocket centered around H95/Y96 positions targeted by clinically approved inhibitors was identified as a universal hotspot for both binding and allostery. The study also reveals the interplay between structural rigidity and functional flexibility, where stabilization of one region induces compensatory flexibility in others, reflecting KRAS's adaptability to perturbations. We found that monobodies stabilize the switch II region of KRAS, disrupting coupling between switch I and II regions and leading to enhanced mobility in switch I of KRAS. Similarly, affimer K3 leverages the α3-helix as a hinge point to amplify its effects on KRAS dynamics. Mutational scanning and binding free energy analysis highlighted the energetic drivers of KRAS interactions. Revealing key hotspot residues, including H95 and Y96 in the α3 helix, as major contributors to binding affinity and selectivity. Network analysis identified β-strand 4 as a central hub for propagating conformational changes, linking distant functional sites. The predicted allosteric hotspots strongly aligned with experimental data, validating the robustness of the computational approach. Despite distinct binding interfaces, shared hotspots highlight a conserved allosteric infrastructure, reinforcing their universal importance in KRAS signaling. The results of this study can inform rational design of small-molecule inhibitors that mimic the effects of monobodies and affimer proteins, challenging the "undruggable" reputation of KRAS.