No abstract

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.cadherin dimerization | protein family design | entropy contribution I n metazoans, the elaboration and maintenance of multicellular architectures relies upon the ability of cells to specifically adhere to one another. Cadherins constitute a superfamily of singlepass transmembrane proteins that can confer such specific adhesive properties to cells (1). In particular, the classical type I and type II cadherins, which are only found in vertebrates and are characterized by an extracellular region comprised of five extracellular cadherin (EC) domains, have been shown to help drive cell-patterning behavior in numerous settings: for example, in morphogenesis (2-4) and in neural patterning (5, 6). Cells expressing the same classical cadherin on their surface generally aggregate through homophilic interactions, whereas cells expressing different cadherins segregate into distinct layers that, in at least some instances, remain in contact with each other through heterophilic binding (7-9).Cell adhesion by classic cadherins is mediated by the dimerization of cadherin extracellular domains emanating from apposed cell surfaces through an interface confined to the N-terminal EC1 domain (Fig. 1A). Numerous crystal structures have revealed the atomic details of the trans (i.e., between cells) dimerization interface for three type I cadherins: C-, E-, and N-cadherins (10-13). In all three cases, the dimer partner molecules swap their N-terminal β-strand (the A*-strand), whose conserved Trp2 residues provide an "anchor" for the adhesive interface by docking into a complementary hydrophobic pocket in the partner protomer (Fig. 1A). A second dimerization interface that can form in the trans orientation has been observed in crystal structures of mutants of both type I and type II classical cadherins. Specifically, numerous mu...

SUMMARY Type II cadherins are cell-cell adhesion proteins critical for tissue patterning and neuronal targeting but whose molecular binding code remains poorly understood. Here, we delineate binding preferences for type II cadherin cell-adhesive regions, revealing extensive heterophilic interactions between specific pairs, in addition to homophilic interactions. Three distinct specificity groups emerge from our analysis with members that share highly similar heterophilic binding patterns and favor binding to one another. Structures of adhesive fragments from each specificity group confirm near-identical dimer topology conserved throughout the family, allowing interface residues whose conservation corresponds to specificity preferences to be identified. We show that targeted mutation of these residues converts binding preferences between specificity groups in biophysical and co-culture assays. Our results provide a detailed understanding of the type II cadherin interaction map and a basis for defining their role in tissue patterning and for the emerging importance of their heterophilic interactions in neural connectivity.

Individuals who receive a third mRNA vaccine dose show enhanced protection against severe COVID-19, but little is known about the impact of breakthrough infections on memory responses. Here, we examine the memory antibodies that develop after a third or fourth antigenic exposure by Delta or Omicron BA.1 infection, respectively. A third exposure to antigen by Delta breakthrough increases the number of memory B cells that produce antibodies with comparable potency and breadth to a third mRNA vaccine dose. A fourth antigenic exposure with Omicron BA.1 infection increased variant-specific plasma antibody and memory B cell responses. However, the fourth exposure did not increase the overall frequency of memory B cells or their general potency or breadth compared to a third mRNA vaccine dose. In conclusion, a third antigenic exposure by Delta infection elicits strain-specific memory responses and increases in the overall potency and breadth of the memory B cells. In contrast, the effects of a fourth antigenic exposure with Omicron BA.1 are limited to increased strain-specific memory with little effect on the potency or breadth of memory B cell antibodies. The results suggest that the effect of strain-specific boosting on memory B cell compartment may be limited.

SUMMARY Transcription factors bind to their binding sites over a wide range of affinities, yet how differences in affinity are encoded in DNA sequences is not well understood. Here, we report X-ray crystal structures of four heterodimers of the Hox protein AbdominalB bound with its cofactor Extradenticle to four target DNA molecules that differ in affinity by up to ~20-fold. Remarkably, despite large differences in affinity, the overall structures are very similar in all four complexes. In contrast, the predicted shapes of the DNA binding sites (i.e., the intrinsic DNA shape) in the absence of bound protein are strikingly different from each other and correlate with affinity: binding sites that must change conformations upon protein binding have lower affinities than binding sites that have more optimal conformations prior to binding. Together, these observations suggest that intrinsic differences in DNA shape provide a robust mechanism for modulating affinity without affecting other protein-DNA interactions.