Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP protein ENAH

Area of Science:

• Molecular Biology
• Biochemistry
• Proteomics

Background:

• Short linear motifs (SLiMs) are crucial for protein-protein interactions but their surrounding sequence context is often overlooked.
• The EVH1 domain of human ENAH, an actin regulator implicated in cancer, interacts with SLiMs.

Purpose of the Study:

• To investigate how the sequence context of SLiMs influences protein-protein interactions.
• To discover new interaction partners of ENAH and elucidate context-dependent binding mechanisms.

Main Methods:

• A proteomic screen using 36-residue proteome-derived peptides was employed.
• Interactions with the ENAH EVH1 domain were analyzed, focusing on motif-flanking residues and extended binding regions.

Main Results:

• The ENAH EVH1 domain recognizes extended SLiMs and favors proline residues flanking motifs.
• High-affinity binders with multiple SLiMs utilize noncanonical binding sites, suggesting cooperative interactions.
• Photoreceptor cilium actin regulator (PCARE) demonstrated exceptionally high affinity through an extended motif.

Conclusions:

• Proteomic context plays a vital role in modulating SLiM-mediated interactions.
• SLiMs cannot be fully understood in isolation; their native sequence environment is critical.
• This study reveals novel mechanisms governing EVH1 domain interactions and highlights the importance of context in motif function.