Learning from cadherin structures and sequences: affinity determinants and protein architecture

Cadherins are a family of cell-surface proteins mediating adhesion that are important in development and maintenance of tissues. The family is defined by the repeating cadherin domain (EC) in their extracellular region, but they are diverse in terms of protein size, architecture and cellular function. The best-understood subfamily is the type I classical cadherins, which are found in vertebrates and have five EC domains. Among the five different type I classical cadherins, the binding interactions are highly specific in their homo- and heterophilic binding affinities, though their sequences are very similar. As previously shown, E- and N-cadherins, two prototypic members of the subfamily, differ in their homophilic K_D by about an order of magnitude, while their heterophilic affinity is intermediate. To examine the source of the binding affinity differences among type I cadherins, we used crystal structures, analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and electron paramagnetic resonance (EPR) studies. Phylogenetic analysis and binding affinity behavior show that the type I cadherins can be further divided into two subgroups, with E- and N-cadherin representing each. In addition to the affinity differences in their wild-type binding through the strand-swapped interface, a second interface also shows an affinity difference between E- and N-cadherin. This X-dimer interface, which is a weakly binding kinetic intermediate in E-cadherin, has a much stronger affinity in N-cadherin: nearly as strong as N-cadherin wild-type binding. In the swapped and X-dimer interactions of E- and N-cadherin, differences in hydrophobic surface area can mostly account for the affinity difference. However, several mutants of N-cadherin have a K_D an order of magnitude stronger even than the wild-type N-cadherin. In these mutants, the source of the strong affinity seems to be entropic stabilization through an equilibrium between multiple conformations with similar energies. We thus have a molecular-level understanding of vertebrate classical cadherins, with a detailed understanding of their adhesive mechanism and their binding affinity determinants. However, the adhesive mechanisms of cadherins from invertebrates, which are structurally divergent yet function in similar roles, remain unknown. We present crystal structures of the predicted N-terminal region of Drosophila N-cadherin (DN-cadherin). Of the 16 total predicted EC domains, we have crystallized the EC1-3 and EC1-4 segments. While the linker regions for the EC1-EC2 and EC3-EC4 pairs display binding of three Ca^2+ ions similar to that in vertebrate cadherins, domains EC2 and EC3 are joined in a bent orientation by a novel, previously uncharacterized Ca^2+-free linker. Based on sequence analysis of the further ECs of DN-cadherin, we predict another such Ca^2+-free linker between EC7 and EC8. Biophysical analysis demonstrates that a construct containing the first nine predicted EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins. Intriguingly, this segment contains both the crystallized and predicted Ca^2+-free linkers, suggesting a complex binding interface. Sequence analysis of the cadherin family reveals that similar Ca^2+-free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins. In cases of long cadherins, there are frequently multiple Ca^2+-free linkers in a single protein chain. It thus appears that a combination of calcium-binding and calcium-free linkers can allow cadherins to form three-dimensional arrangements that are more complex than the extended, calcium-rigidified structures in classical cadherins. Discovery of the Ca^2+-free linker, together with the differing numbers and arrangements of ECs and other domain types, implies that the cadherin superfamily is more structurally diverse than previously thought. Because little is known about the function and even less about the structure of the majority of the superfamily, studying the linear architecture (i.e. the precise sequence of ECs and the characteristics of the interdomain linkers) at the scale of the superfamily would give significant new insights on the structure and function of less-understood cadherins. With this motivation, we have constructed a cadherin database with relevant information on two different scales: the protein and the domain. On the whole protein level, we represent the architecture of each cadherin by recording the arrangement of ECs, different linker types, and other (non-EC) domain types in the protein. On the individual EC level, based on the sequence, we record the domain characteristics that give rise to the different structural features at the protein level. We have annotated over 9,600 proteins from 560 organisms, containing over 69,000 ECs; and built an online interface to search and access this information. Our aim is to provide a tool for understanding the protein architecture, function, and relationships among cadherins, a structurally diverse protein family. Together, these studies examine the relationships between sequence, structure and function of cadherins at different scales. In the classical cadherin study, small changes of one or two residues can dramatically alter the dimer conformations and thus lead to large differences in binding affinity between highly related cadherins, or between wild-type and mutant proteins. These seemingly small mutations can result in even higher binding affinity with the effect of entropic stabilization by multiple conformations. In DN-cadherin, the absence of certain calcium-binding motifs in adjacent ECs leads to a new linker type and a new interdomain orientation. This, in turn, has great implications in the global shape, and possibly the binding mechanism of the protein. The cadherin database aims to provide information at different structural levels in order to allow users to draw connections between primary sequence, domain structure and protein architecture, to ultimately learn about protein function

Quantitative Analysis of T Cell Receptor Complex Interaction Sites Using Genetically Encoded Photo-Cross-Linkers

The T cell receptor (TCR)-cluster of differentiation 3 (CD3) signaling complex plays an important role in initiation of adaptive immune responses, but weak interactions have obstructed delineation of the individual TCR-CD3 subunit interactions during T cell signaling. Here, we demonstrate that unnatural amino acids (UAA) can be used to photo-cross-link subunits of TCR-CD3 on the cell surface. Incorporating UAA in mammalian cells is usually a low efficiency process. In addition, TCR-CD3 is composed of eight subunits and both TCR and CD3 chains are required for expression on the cell surface. Photo-cross-linking of UAAs for studying protein complexes such as TCR-CD3 is challenging due to the difficulty of transfecting and expressing multisubunit protein complexes in cells combined with the low efficiency of UAA incorporation. Here, we demonstrate that by systematic optimization, we can incorporate UAA in TCR-CD3 with high efficiency. Accordingly, the incorporated UAA can be used for site-specific photo-cross-linking experiments to pinpoint protein interaction sites, as well as to confirm interaction sites identified by X-ray crystallography. We systemically compared two different photo-cross-linkersp-azido-phenylalanine (pAzpa) and H-p-Bz-Phe-OH (pBpa)for their ability to map protein subunit interactions in the 2B4 TCR. pAzpa was found to have higher cross-linking efficiency, indicating that optimization of the selection of the most optimal cross-linker is important for correct identification of protein–protein interactions. This method is therefore suitable for studying interaction sites of large, dynamic heteromeric protein complexes associated with various cellular membrane systems

Structural Model of the Extracellular Assembly of the TCR-CD3 Complex

SummaryAntigen recognition of peptide-major histocompatibility complexes (pMHCs) by T cells, a key step in initiating adaptive immune responses, is performed by the T cell receptor (TCR) bound to CD3 heterodimers. However, the biophysical basis of the transmission of TCR-CD3 extracellular interaction into a productive intracellular signaling sequence remains incomplete. Here we used nuclear magnetic resonance (NMR) spectroscopy combined with mutational analysis and computational docking to derive a structural model of the extracellular TCR-CD3 assembly. In the inactivated state, CD3γε interacts with the helix 3 and helix 4-F strand regions of the TCR Cβ subunit, whereas CD3δε interacts with the F and C strand regions of the TCR Cα subunit in this model, placing the CD3 subunits on opposing sides of the TCR. This work identifies the molecular contacts between the TCR and CD3 subunits, identifying a physical basis for transmitting an activating signal through the complex

Single-Cell Identity Generated by Combinatorial Homophilic Interactions between α, β, and γ Protocadherins

Individual mammalian neurons express distinct repertoires of protocadherin (Pcdh) -α, -β and -γ proteins that function in neural circuit assembly. Here we show that all three types of Pcdhs can engage in specific homophilic interactions, that cell surface delivery of alternate Pcdhα isoforms requires cis interactions with other Pcdh isoforms, and that the extracellular cadherin domain EC6 plays a critical role in this process. Analysis of specific combinations of up to five Pcdh isoforms showed that Pcdh homophilic recognition specificities strictly depend on the identity of all of the expressed isoforms, such that mismatched isoforms interfere with cell-cell interactions. We present a theoretical analysis showing that the assembly of Pcdh-α, −β and −γ isoforms into multimeric recognition units, and the observed tolerance for mismatched isoforms can generate the cell surface diversity necessary for single-cell identity. However, competing demands of non-self discrimination and self-recognition place limitations on the mechanisms by which recognition units can function

Structural and energetic determinants of adhesive binding specificity in type I cadherins

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell-cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles

Structural and energetic determinants of adhesive binding specificity in type I cadherins

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell–cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles