Insights into the Interaction Landscape of the EVH1 Domain of Mena

• Published in: Biochemistry (Easton) 2024-09, Vol.63 (17), p.2183-2195
• Main Authors: LaComb, Lanette, Ghosh, Agnidipta, Bonanno, Jeffrey B., Nilson, Daniel J., Poppel, Alex J., Dada, Lucas, Cahill, Sean M., Maianti, Juan Pablo, Kitamura, Seiya, Cowburn, David, Almo, Steven C.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; bacterial motility; Binding Sites; crystal structure; Crystallography, X-Ray; domain; fluorescence; Humans; hydrophobicity; Ligands; mammals; Microfilament Proteins - chemistry; Microfilament Proteins - metabolism; Models, Molecular; neoplasms; Nuclear Magnetic Resonance, Biomolecular; peptides; Peptides - chemistry; Peptides - metabolism; Proline - chemistry; Proline - metabolism; Protein Binding; Protein Domains
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.4c00331

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H–15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (K d = 3–8 μM). Inhibitor 6c displayed tighter binding (K d ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.