Identification of an adhesive interface for the non-clustered δ1 protocadherin-1 involved in respiratory diseases

Abstract

Cadherins form a large family of calcium-dependent adhesive proteins involved in morphogenesis, cell differentiation, and neuronal connectivity. Non-clustered δ1 protocadherins form a cadherin subgroup of proteins with seven extracellular cadherin (EC) repeats and cytoplasmic domains distinct from those of classical cadherins. Non-clustered δ1 protocadherins mediate homophilic adhesion and have been implicated in various diseases including asthma, autism, and cancer. Here we present X-ray crystal structures of human Protocadherin-1 (PCDH1), a δ1-protocadherin member essential for New World Hantavirus infection that is typically expressed in the brain, airway epithelium, skin keratinocytes, and lungs. The structures suggest a binding mode that involves antiparallel overlap of repeats EC1 to EC4. Mutagenesis combined with binding assays and biochemical experiments validated this mode of adhesion. Overall, these results reveal the molecular mechanism underlying adhesiveness of PCDH1 and δ1-protocadherins, also shedding light on PCDH1’s role in maintaining airway epithelial integrity, the loss of which causes respiratory diseases.

Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins

“…The mechanism of d2-protocadherin trans binding was revealed previously in a crystal structure of zebrafish pcdh19 EC1-EC4 (Cooper et al, 2016), which adopted an antiparallel trans dimer similar to clustered protocadherins (Goodman et al, 2016a(Goodman et al, , 2016b. Formation of a similar trans dimer by d1-protocadherins was also reported in a recent crystal structure of human pcdh1 EC1-EC4 (Modak and Sotomayor, 2019), representing the same crystal form as that described here. To compare binding determinants across the d-protocadherin family, we determined EC1-EC4 fragment crystal structures of human pcdh1, -10 (two crystal forms), -17, -18, and -19, with resolutions 2.3-3.7 Å (Table S1).…”

“…The clustered, d1-, and d2-protocadherin families bind in trans through topologically similar antiparallel EC1-EC4 dimers, each composed of two large EC1:EC4 and EC2:EC3 and one small EC3:EC3 interface region. This binding mode has been well characterized for the clustered protocadherins (Goodman et al, 2016a(Goodman et al, , 2016b and for d2-family member Pcdh19 from zebrafish (Cooper et al, 2016), while structures of pcdh1 reported here and in Modak and Sotomayor (2019) extend the mechanism to the d1-family. Despite topological similarity overall, we observed no cross-family interactions between d1-, d2-, and clustered protocadherins, likely reflecting differences in interface orientations and residue conservation.…”

“…Crystal structures of pcdh19 from zebrafish (Cooper et al, 2016) and a crystal structure of human pcdh1 published while this paper was under review (Modak and Sotomayor, 2019) revealed the trans binding mechanism for d1-and d2-protocadherins to be consistent with that observed for the related a-, b-, and g-clustered protocadherins (Goodman et al, 2016a(Goodman et al, , 2016b. In each trans-dimer structure, EC1-EC4 regions of partner molecules bind in an antiparallel orientation to form a dimer mediated by two EC1:EC4 and two EC2:EC3 interfaces related by 2-fold symmetry.…”

Structural determinants of protocadherin-15 mechanics and function in hearing and balance perception

“…The antiparallel interface observed in hs PCDH15 EC1-3 G16D/N369D/Q370N positions EC2 and EC3 from one subunit opposite to EC3 and EC2 from the other (interface area of 919 Å 2 with an aperture angle of 154.5°) ( SI Appendix , Fig. S4 A ), thus forming an antiparallel trans bond similar to that observed for clustered and δ protocadherins where EC1 and EC4 also play a role in the binding interface ( 48 – 51 ). The crystallographic contact observed in the hs PCDH15 EC2-3 WT structure reveals a potential “X” dimerization interface (∼1,052 Å 2 ) ( Fig.…”

Protocadherins at the Crossroad of Signaling Pathways

“…Structurally, the trans-dimers formed by cPcdhs and δ2-Pcdhs were proven to be quite similar. Indeed, X-ray crystallography analyses revealed that two zebrafish Pcdh19 molecules on adjacent cells interact via a "forearm handshake" involving EC1-EC4 domain binding (Cooper et al, 2016), and that human PCDH1 proteins also homophilically dimerize in an antiparallel manner through these domains (Modak and Sotomayor, 2019). Cell/bead aggregation studies using Pcdh7 EC1-4 or Pcdh9 single deletion mutants demonstrated that EC1-EC4 and EC2-EC3 interactions seem to confer, respectively, dimer binding affinity and binding specificity (Bisogni et al, 2018;Peng et al, 2018).…”