(A) Residues identified in our genetic screen (see Figure 2) shown as colored spheres on a ribbon diagram of AP2 with outline of NECAPEx density (red, low isosurface threshold, see Figure 6—figure supplement 1). (B) Binding curves generated from pulldown depletion assays. Error bars represent mean ± SEM from three technical replicates. Inset: Calculated Kd values, variance is SEM. *p<0.05, **p<0.01, relative to control. (C) In the absence of NECAP (–), fcho-1 mutants take about 4 days to proliferate and consume a bacterial food source (fitness defect = 0). Expression of NECAP (+) increases the number of days to about 8 (fitness defect = 1). Data for interface mutants were normalized to this fitness defect; n = 10 biological replicates. (D) In fcho-1; apm-2 (E306K) mutant worms, NECAP is recruited to the nerve ring. Interface mutants disrupt nerve ring recruitment. Normalized RFP intensities plotted above representative confocal nerve ring images of ten biological replicates. (C–D) Error bars indicate mean ± SEM. Significance compared to NECAP (+); Student’s t-test performed on raw data (C) or normalized data (D). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) In vivo protease sensitivity assay to probe AP2 conformation in genetic backgrounds indicated. In the absence of NECAP (–), AP2 is protease sensitive (open). Expression of wild type NECAP (+) results in protease resistant AP2 (closed). All strains lack fcho-1. See also Figure 6—figure supplement 1.