Intrinsically Dominant Conformational Diversity in PDZ1 within the Tandem PDZ1-PDZ2 of Human Syntenin-1 Underlined by Crystal Structures

Abstract The intrinsic dynamic asymmetry between homologous PDZ domains in multidomain scaffold proteins offers critical insights into evolutionary mechanisms enabling multivalent partner recognition. Through systematic X-ray crystallographic analysis of human syntenin-1’s PDZ1-PDZ2 tandem, we resolve nine high-resolution structures that uncover fundamental differences in conformational plasticity between these sequentially similar domains. Pairwise root-mean-squared deviation (RMSD) analysis of 20 PDZ1 structures across multiple crystal forms reveals substantial structural variability concentrated in the Lys119-Ile125 and Ala181-Glu184 loops - key regions governing ligand specificity within PDZ1’s binding cleft. In stark contrast, PDZ2 maintains remarkable structural conservation across all crystallographic environments, indicating divergent evolutionary constraints on these tandem domains. Crucially, comparative analysis of isotropic B-factors demonstrates their inadequacy in capturing the full scope of conformational heterogeneity, emphasizing the necessity of multi-structure comparisons for mapping dynamic landscapes. Molecular dynamics (MD) simulations implemented through GROMACS corroborate these crystallographic observations, showing elevated residue-specific fluctuation (RMSF) values in PDZ1’s ligand-binding interface compared to analogous PDZ2 regions. This consistency across experimental and computational approaches confirms that PDZ1’s conformational diversity represents an inherent biophysical property rather than crystallographic artifact. The observed dynamic asymmetry suggests a functional division of labor: PDZ1’s structural plasticity enables broad ligand recognition via conformational selection mechanisms, while PDZ2’s rigid architecture likely stabilizes the tandem domain arrangement. These findings provide an atomic-level rationale for syntenin-1’s pleiotropic roles in cellular signaling and establish a structural blueprint for developing domain-selective therapeutics. Given syntenin-1’s clinical relevance in cancer metastasis, viral pathogenesis, and neurodevelopmental disorders, our work advances strategies for selectively modulating PDZ1-mediated interactions while preserving PDZ2’s scaffolding functions through structure-guided inhibitor design. Highlights ◊ Crystal structures of human Syntenin-1’s PDZ1-PDZ2 tandem reveal intrinsic conformational plasticity in PDZ1, particularly in ligand-binding loops, contrasting with PDZ2’s rigid architecture ◊ Pairwise RMSD analysis of 20 PDZ1 structures demonstrates substantial structural variability in the Lys119-Ile125 and Ala181-Glu184 loops, key regions governing ligand specificity ◊ Molecular dynamics simulations confirm that PDZ1’s conformational diversity is an inherent biophysical property, not a crystallographic artifact ◊ The asymmetric dynamics between PDZ1 and PDZ2 suggest a functional division: PDZ1’s plasticity enables broad ligand recognition while PDZ2 stabilizes the tandem arrangement ◊ These findings provide a structural basis for developing domain-selective Syntenin-1 inhibitors with potential applications in cancer metastasis, viral pathogenesis, and neurodevelopmental disorders Competing Interest Statement Among the authors, T.T. and H.H. are the founders of a Nagoya University-based spinoff startup company, called BeCellBar. LLC. The remaining authors declare no conflicts of interest. Footnotes Revised version includes PDB codes for all refined crystal structures (materials and methods section, 2, Crystallization and Data collection). Also, the severe mistakes on Figure 5A and 5B are now corrected. We re-analyzed the conserved hydrogen bonds between PDZ1 and PDZ2 in the ten crystal structures and also revised Figure 6A and 6B.