Conformational Positioning of the LXCXE Motif of LTSV40 within an Ordered–Disordered Transition Drives pRb Binding Cleft Recognition

Abstract

The retinoblastoma protein (pRb) is a central negative regulator of the eukaryotic cell cycle. Oncoproteins bearing the LXCXE motif can inactivate pRb, thereby promoting cell-cycle progression. One such protein is the Large T antigen of the Simian Virus 40 (LTSV40), which contains extensive intrinsically disordered regions. Taking advantage of available structural information for the LTSV40–pRb complex, we generated computational models of pRb bound to different LTSV40 constructs and investigated their energetic and dynamical features using molecular dynamics simulations. Our results show that the isolated LXCXE motif has low affinity for pRb, that flanking residues enhance binding asymmetrically, and that the full-length protein binds more strongly than peptide fragments. Mechanistically, residues N-terminal to the LXCXE motif drive initial recognition by adopting an α-helical conformation, while C-terminal residues engage at later stages. Further analysis reveals that this behavior arises from an ordered–motif–disordered architecture within the LTSV40 region responsible for pRb interaction. Bioinformatic analysis shows that this feature is conserved across polyomavirus Large T antigens, suggesting a general strategy for efficient pRb targeting. Overall, this work provides a molecular and energetic framework for LXCXE-mediated recognition, highlighting the interplay between local structure, intrinsic disorder, and sequence context in protein–protein interactions.