Interaction specificity of clustered protocadherins inferred from sequence covariation and structural analysis

Abstract

Clustered protocadherins are a large family of paralogous proteins that play important roles in neuronal development. The more than 50 clustered protocadherin isoforms have remarkable homophilic specificity for interactions between cellular surfaces that is controlled by a large antiparallel dimer interface formed by the first four extracellular cadherin (EC) domains. To understand how specificity is achieved between the numerous paralogs, we used a combination of structural and computational approaches. Molecular dynamics simulations revealed that individual EC interactions are weak and go through binding and unbinding events, but together they form a stable complex through polyvalency. Using sequence coevolution, we generated a statistical model of interaction energy for the clustered protocadherin family that measures the contributions of all amino acid pairs in the interface. Our interaction energy model assesses specificity for all possible pairs of isoforms, recapitulating known pairings and predicting the effects of experimental changes in isoform specificity that are consistent with literature results. Our results show that sequence coevolution can be used to understand specificity determinants in a protein family and prioritize interface amino acid substitutions to reprogram specific protein-protein interactions. Clustered protocadherins (Pcdhs) are a large protein family (53 isoforms in humans) that play roles in vertebrate nervous system development, including neuronal survival, axon targeting, neuronal arborization, and dendritic self-avoidance (1-9). Dendritic self-avoidance is mediated by formation of a clustered Pcdh assembly between two dendrites (10). This assembly relies on individual recognition units formed in trans across two cellular membranes that consist of homodimers of the first four extracellular cadherin-repeat (EC) domains in an antiparallel arrangement ( Figure 1A). These homodimers are highly specific such that no cross interactions are observed in even the most similar isoforms (11-13). The trans EC1-4 interaction is also found in other non-clustered Pcdhs (14-16), which have roles in nervous system development and maintenance (17), indicating the importance of this recognition unit in cognitive function. Given that there are many isoforms per vertebrate genome, we sought to understand how specificity is achieved in this large interface.Structures of these recognition units (14,(18)(19)(20) have revealed idiosyncratic characteristics of individual dimer structures, such as the lack of the EC1/EC4 interaction in the structure of PcdhγA1 and PcdhγA8 (20) and the small interface of the EC2/EC3 interaction in PcdhγB3 (14). More subtle structural differences can be observed broadly between the three clustered Pcdh subfamilies, α, β, and γ (14,19,20). Based on the variety of interfaces found in the existing crystal structures (14,(18)(19)(20), it is possible that every isoform achieves specificity by adopting a different static interface conformation, or that isoforms sample a distributi...

Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition

“…cPcdh recognition is mediated by a mechanism coupling nonspecific cis and specific trans interactions (29,30,41,42,45). cPcdh isoforms engage homophilically in trans via four membrane distal domains (EC1-EC4) and in cis via a nonoverlapping and independent interface involving the membrane proximal EC5-EC6 domains (30,41,42,(45)(46)(47)(48)(49). Our data indicate that although Chelicerata sDscams and cPcdhs are evolutionarily unrelated, they form similar interactions.…”

The conserved serine transporter SdaC moonlights to enable self recognition

“…From studies on eukaryotes, we know that protein-protein interactions affect how selfrecognition proteins change over time. For clustered protocadherins, residues co-evolve across the homophilic interaction interface (Nicoludis et al, 2015(Nicoludis et al, , 2016(Nicoludis et al, , 2019. Allelic diversity of self-recognition genes results from balancing selection (Gruenheit et al, 2017;Noonan et al, 2003;Wu, 2005;Zhao et al, 2015).…”

How clustered protocadherin binding specificity is tuned for neuronal self/non-self-recognition

“…Even the non-clustered delta protocadherins, which are preferentially homophilic and utilize an antiparallel EC1-4 interface like the cPcdhs, show heterophilic intra-family trans interactions, though they show no crossreactivity with cPcdhs (Harrison et al, 2020). 3 4 High fidelity homophilic interaction is a strict requirement of the chain termination model for the 5 barcoding of vertebrate neurons and has been accomplished through the exploitation of a 6 multidomain interface of almost 4000 Å 2 (Nicoludis et al, 2019) that enables the positioning of enough "negative constraints" (Sergeeva et al, 2020) to preclude the dimerization of about 1600 heterophilic pairs of 58 mouse cPcdh isoforms (Rubinstein et al, 2017). Dscams accomplish the same task for thousands of isoforms but this is accomplished by exploiting the combinatorics made possible by a three-domain interface where each domain interacts largely independently with an identical domain on its interacting partner (see discussion in (Zipursky and Grueber, 2013)).…”