ABSTRACT
The early phases of clathrin mediated endocytosis are organized through a highly complex interaction network mediated by clathrin associated sorting proteins (CLASPs) that comprise long intrinsically disordered regions (IDRs). AP180 is a CLASP exclusively expressed in neurons and comprises a long IDR of around 600 residues, whose function remains partially elusive. Using NMR spectroscopy, we discovered an extended and strong interaction site within AP180 with the major adaptor protein AP2, and describe its binding dynamics at atomic resolution. We find that the 70 residue-long site determines the overall interaction between AP180 and AP2 in a dynamic equilibrium between its bound and unbound states, while weaker binding sites contribute to the overall affinity at much higher concentrations of AP2. Our data suggest that this particular interaction site might play a central role in recruitment of adaptors to the clathrin coated pit, whereas more transient and promiscuous interactions allow reshaping of the interaction network until cargo uptake inside a coated vesicle.
Subject(s)
Adaptor Protein Complex 2 , Clathrin , Endocytosis , Monomeric Clathrin Assembly Proteins , Protein Binding , Adaptor Protein Complex 2/metabolism , Clathrin/metabolism , Binding Sites , Monomeric Clathrin Assembly Proteins/metabolism , Monomeric Clathrin Assembly Proteins/genetics , Humans , Animals , Magnetic Resonance Spectroscopy , Clathrin-Coated Vesicles/metabolism , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/geneticsABSTRACT
Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.
Subject(s)
Breast Neoplasms/drug therapy , Cell Adhesion Molecules/metabolism , DNA-Binding Proteins/metabolism , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Protein Interaction Domains and Motifs/drug effects , Small Molecule Libraries/pharmacology , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Female , Humans , Jurkat Cells , Proline/metabolism , Protein Binding/drug effects , ZebrafishABSTRACT
GYF domains are conserved eukaryotic adaptor domains that recognize proline-rich sequences. Although the structure and function of the prototypic GYF domain from the human CD2BP2 protein have been characterized in detail, very little is known about GYF domains from other proteins and species. Here we describe the binding properties of four GYF domains of various origins. Phage display in combination with SPOT analysis revealed the PPG(F/I/L/M/V) motif as a general recognition signature. Based on these results, the proteomes of human, yeast, and Arabidopsis thaliana were searched for potential interaction sites. Binding of several candidate proteins was confirmed by pull-down experiments or yeast two-hybrid analysis. The binding epitope of the GYF domain from the yeast SMY2 protein was mapped by NMR spectroscopy and led to a structural model that accounts for the different binding properties of SMY2-type GYF domains and the CD2BP2-GYF domain.
Subject(s)
Arabidopsis/metabolism , Proteins/chemistry , Proteins/metabolism , Proteomics , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Motifs , Amino Acid Sequence , Arabidopsis/genetics , Binding Sites , Carrier Proteins/chemistry , Epitope Mapping , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptide Library , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RNA Splicing Factors , RNA-Binding Proteins/analysis , RNA-Binding Proteins/chemistry , Repressor Proteins/analysis , Repressor Proteins/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/chemistry , Sequence Alignment , Substrate Specificity , Two-Hybrid System Techniques , Vesicular Transport ProteinsABSTRACT
The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B' were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis.
