Mapping Allosteric Rewiring in Viral RNA: Sequence-Encoded Control of Protein Binding Mechanisms

Abstract

RNA recognition by proteins is governed not only by static structure but also by allostery encoded within non-local dynamic motifs. In this study, we systematically identify allosteric communication hubs in RNA and map multiple residue-connected pathways, revealing how these networks are rewired upon mutation and protein binding. To capture these effects under physiological salt conditions, we performed tens of microseconds of atomistic and steered molecular dynamics simulations and computed binding free energies for Tat-TAR complexes across three immunodeficiency virus variants, BIV, HIV-1, and HIV-2. Allosterically coupled sites were identified using contact-based principal component analysis, and communication pathways were traced through an extended graph-network algorithm--the first such application to RNA systems. Two distant motifs--the bulge and the apical loop--emerge as allosteric switches and information hubs: the bulge engages Tat, while the loop interacts with another protein partner, CycT1, both essential for transcriptional activation and antiviral targeting. We find that HIV-2 TAR, with strong loop-bulge coupling and high self-integrity, favour conformational selection and exhibits lower Tat-binding affinity. In contrast, a single C24 insertion in HIV-1 TAR reconfigures communication pathways, enabling an induced-fit mechanism with enhanced affinity. The study not only elucidates an allosteric rewiring between the loop and bulge but also highlights how this communication is dynamically reconfigured upon protein binding. Tat association at the bulge reorganizes and reorients loop residues, thereby promoting the subsequent recruitment of CycT1. This work overall underscores how sequence (even a single mutation) encoded RNA allostery can modulate not only a proteins binding mechanism and affinity but also influence downstream molecular events within transcriptional signalling cascades. STATEMENT OF SIGNIFICANCEIn vitro selection strategies such as SELEX have long underscored the therapeutic relevance of the TAR RNA-Tat protein interface, yet they remain experimentally demanding and often overlook how subtle sequence variations reshape molecular recognition and RNA-protein binding affinity. By integrating atomistic and free-energy simulations with a graph-network-based framework, the study reveals that even a single-nucleotide change can rewire RNAs allosteric communication pathways, switching between conformational selection and induced-fit mechanisms and alter binding affinity. These findings bridge molecular dynamics with experimental selection, offering a predictive, thermodynamics-based framework to rationalize sequence-structure-function relationships and guide the rational design of high-affinity RNA-protein complexes and next-generation RNA therapeutics.