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Human MAP1LC3B (LC3B) binds proteins involved in autophagy and other cellular processes using a degenerate four-residue short linear motif known as the LC3-interacting region (LIR). Biochemical and structural studies have identified LIRs in many LC3B interaction partners, but the sequence features that contribute to binding have not been systematically explored. To discover peptides that interact with LC3B and deeply profile the key binding determinants, we screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data, coupled with structural studies and site-directed mutagenesis, revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR, which we visualized in a newly determined structure of LC3B bound to a peptide isolated in our screen. Guided by our screening data, we designed synthetic LIR-containing peptides that bind LC3B with affinities comparable to the tightest measured natural binder. Finally, we determined that mutations in LC3B commonly thought to abrogate binding of LIR-containing peptides instead alter LC3B binding specificity, leading to enhanced binding of some LIR-containing sequences. Taken together, our results refine the LIR motif definition, expand the network of candidate LC3B interaction partners, and highlight how mutations at the LC3B- LIR interface can modulate affinity and specificity.