Abstract
Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition. Drosophila DIP and Dpr proteins mediate neuronal targeting in the fly through highly specific protein-protein interactions. We show here that DIPs/Dprs segregate into seven specificity subgroups defined by binding preferences between their DIP and Dpr members. We then describe a sequence-, structure- and energy-based computational approach, combined with experimental binding affinity measurements, to reveal how specificity is coded on the canonical DIP/Dpr interface. We show that binding specificity of DIP/Dpr subgroups is controlled by “negative constraints”, which interfere with binding. To achieve specificity, each subgroup utilizes a different combination of negative constraints, which are broadly distributed and cover the majority of the protein-protein interface. We discuss the structural origins of negative constraints, and potential general implications for the evolutionary origins of binding specificity in multi-protein families.
Highlights
Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition
Interfacial positions of DIP/Dpr complexes of unknown structure were inferred from multiple sequence alignment
We have described the structural and energetic origins of the partition of DIPs and Dprs into orthogonal specificity groups defined by Surface plasmon resonance (SPR)-derived binding affinity measurements
Summary
Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition. We carry out a comprehensive computational and experimental study of specificity determinants in two interacting families of Drosophila melanogaster neuronal recognition proteins, the 21-member Dpr (Defective proboscis extension response) and the 11-member DIP (Dpr Interacting Proteins). These proteins have been extensively characterized structurally[9,10,11], and their interactions were characterized quantitatively with biophysical measurements[11]. DIPs preferentially bind Dprs, and a network of specific heterophilic interactions is formed between members of the two families. Our DIP/Dpr grouping is somewhat different than that published by Cheng et al.[10] due in part to the fact that these authors did not include DIP-κ and DIP-λ, whose binding preferences had been previously mapped[11]
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