Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2.
The adaptor proteins AP-2 and AP-1/GGAs are essential components of clathrin coats at the plasma membrane and trans-Golgi network, respectively. The adaptors recruit accessory proteins to clathrin-coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF-based motif that functions to mediate the binding of an array of accessory proteins to the alpha-adaptin ear domain of AP-2. Using nuclear magnetic resonance and mutational studies, we identified WXXF-based motifs as major ligands for a site on the alpha-ear previously shown to bind the DPW-bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the alpha-ear motif to AP-2 as compared to those that allow a highly related WXXF-based motif to bind to the ear domains of AP-1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin-coated vesicle formation.
Ritter
Brigitte
B
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, Canada.
Denisov
Alexei Yu
AY
Philie
Jacynthe
J
Deprez
Christophe
C
Tung
Elaine C
EC
Gehring
Kalle
K
McPherson
Peter S
PS
D033965:Adaptor Protein Complex alpha Subunits / Q000378:metabolism*; D033982:Adaptor Protein Complex gamma Subunits / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism
D033965:Adaptor Protein Complex alpha Subunits; D033982:Adaptor Protein Complex gamma Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D004268:DNA-Binding Proteins; D008024:Ligands; D010446:Peptide Fragments; D018808:Transcription Factor AP-1; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; C114154:epsin