The RNA-binding protein KhpB binds dsRNA and retains binding activity at elevated temperatures

RNA-binding proteins are central regulators of post-transcriptional gene expression, mediating RNA recognition, processing, and regulation. While canonical domains such as dsRBDs are well characterized, alternative motifs including the heterogeneous nuclear ribonucleoprotein K-homology (KH) and arginine spaced histidine (R3H) remain less understood, particularly in their ability to interact with double-stranded RNA (dsRNA). Here, we engineered recombinant variants of the thermostable bacterial RNA-binding protein KhpB from Marinithermus hydrothermalis (mhKhpB), containing either KH, R3H, or both domains, and assessed their dsRNA binding capacity in vitro using electrophoretic mobility shift assays. The interaction between recombinant mhKhpB dsRNA remained stable across temperature variations up to 95 °C. Constructs displayed distinct dsRNA-binding affinities and protein-dsRNA complex formation behaviors, supported by structural predictions from in silico modeling and physicochemical analyses. Among the variants tested, the R3H-only protein (KhpB2) showed the strongest interaction with dsRNA, suggesting that this domain alone could serve as a minimal functional module. Molecular dynamics demonstrated that the interaction was maintained primarily by basic residues, which, when mutated, led to the decoupling of the dsRNA:KhpB complex. Our findings evidentiate a thermostable KhpB as a novel dsRNA-binding protein and provide new insights into how KH and R3H containing proteins engage dsRNA, expanding the understanding of modular RNA-binding architectures and highlighting their potential in nucleic acid based biotechnological applications.