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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. 

The stochastic expression of fewer than 60 clustered protocadherin (cPcdh) isoforms provides diverse identities to individual vertebrate neurons and a molecular basis for self-/nonself-discrimination. cPcdhs form chains mediated by alternating cis and trans interactions between apposed membranes, which has been suggested to signal self-recognition. Such a mechanism requires that cPcdh cis dimers form promiscuously to generate diverse recognition units, and that trans interactions have precise specificity so that isoform mismatches terminate chain growth. However, the extent to which cPcdh interactions fulfill these requirements has not been definitively demonstrated. Here, we report biophysical experiments showing that cPcdh cis interactions are promiscuous, but with preferences favoring formation of heterologous cis dimers. Trans homophilic interactions are remarkably precise, with no evidence for heterophilic interactions between different isoforms. A new C-type cPcdh crystal structure and mutagenesis data help to explain these observations. Overall, the interaction characteristics we report for cPcdhs help explain their function in neuronal self-/nonself-discrimination. 

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. 

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions.