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InDrosophila, two interacting adhesion protein families, Defective proboscis responses (Dprs) and Dpr interacting proteins (DIPs), coordinate the assembly of neural networks. While intercellular DIP::Dpr interactions have been well characterized, DIPs and Dprs are often co-expressed within the same cells, raising the question as to whether they also interact incis. We show, in cultured cells andin vivo, that DIP-α and DIP-δ can interact inciswith their ligands, Dpr6/10 and Dpr12, respectively. When co-expressed inciswith their cognate partners, these Dprs regulate the extent oftransbinding, presumably through competitivecisinteractions. We demonstrate the neurodevelopmental effects ofcisinhibition in fly motor neurons and in the mushroom body. We further show that a long disordered region of DIP-α at the C-terminus is required forcisbut nottransinteractions, likely because it alleviates geometric constraints oncisbinding. Thus, the balance betweencisandtransinteractions plays a role in controlling neural development. 

Abstract Differential binding affinities among closely related protein family members underlie many biological phenomena, including cell-cell recognition.DrosophilaDIP 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.