Mena EVH1 Interacts with PTP1B Via a Distributed Interface Without Regulating Enzymatic Activity.

Abstract

The interaction between mammalian enabled protein (Mena), an actin regulatory protein, and protein tyrosine phosphatase 1B (PTP1B) is critical for maintaining epidermal growth factor receptor (EGFR) signaling homeostasis and regulating cellular motility. Mena recruits PTP1B to activated EGFR in vivo, facilitating receptor dephosphorylation and limiting invasive signaling. However, overexpression of wild-type Mena or the invasive isoform, MenaINV, perturbs this regulatory mechanism by altering PTP1B localization and EGFR signaling dynamics. Here, we report the solution NMR structure of the unliganded Mena EVH1 domain, which contains a conserved aromatic triad (Y16, W23, F77) that forms the canonical polyproline-binding cleft. Building on prior evidence that the PTP1B polyproline region serves as an EVH1 ligand, we investigated the structural basis of the interaction between EVH1 and wild-type PTP1B. NMR chemical shift perturbation analyses using multiple PTP1B variants, including full-length and truncated constructs, revealed that binding affects not only the canonical EVH1 hydrophobic cleft but also residues on the opposite surface, suggesting an expanded interaction interface. Diffusion-ordered spectroscopy (DOSY) and size-exclusion chromatography further indicate that PTP1B variants containing the disordered C-terminal tail form more compact complexes with EVH1, consistent with a disorder-to-order transition. Complementary perturbation studies using [2H,15N] PTP1B demonstrate that EVH1 binding induces changes not only in the polyproline region but also across the phosphatase core, α7 helix, and C-terminal tail. Despite these structural perturbations, enzymatic assays show that EVH1 binding does not affect the catalytic activity of PTP1B, supporting a model in which Mena functions as a scaffold to spatially organize PTP1B within EGFR signaling complexes.