DUSP15 exhibits structural and dynamical signatures inconsistent with catalytic phosphatase activity.

Dual-specificity phosphatases (DUSPs) require two conserved motifs, the HCX₅R nucleophilic loop and a WPD/FPD-type general-acid loop, to support cysteine-dependent dephosphorylation. Although annotated as a DUSP, the catalytic competence of DUSP15 has remained ambiguous, with only weak activity reported against artificial substrates and paradoxical roles in sustaining ERK and Jak1-STAT3 signalling. Here, sequence analysis, crystallographic inspection, structural modelling, evolutionary profiling, interaction-network inference, and molecular dynamics (MD) simulations are integrated to reassess the functional properties of DUSP15. Motif analysis identifies two defining deviations: a phenylalanine immediately following the catalytic cysteine within a divergent HCFAGISR loop, and complete absence of a WPD/FPD-type general-acid loop. Structural examination of a DUSP15 crystal fragment, together with AlphaFold predictions, shows that the inserted phenylalanine projects into and sterically occludes the active-site cleft, in contrast to the open catalytic pocket of the active phosphatase DUSP7. Comparative analysis of 11 mammalian orthologs reveals absolute conservation of both anomalies, indicating long-standing selective maintenance of a catalytically divergent architecture. 100-ns all-atom MD simulations reveal a globally stable and compact fold with a conformationally rigid, tightly packed, and selectively dehydrated catalytic motif, lacking the flexibility and solvent accessibility typically required for productive cysteine-based catalysis. Comparative MD simulations performed under identical conditions further distinguish DUSP15 from the catalytically competent phosphatase DUSP7. Interaction-network analysis places DUSP15 within phosphatase-, transcriptional-, and metabolism-associated modules, consistent with scaffold-like regulatory roles. Together, these convergent structural, evolutionary, and dynamical features support a model in which DUSP15 functions predominantly as a non-catalytic adaptor, providing a mechanistic framework for its non-canonical regulation of ERK and Jak1-STAT3 signalling and its tumour-selective expression in chromophobe renal cell carcinoma.