MAGNet Driving Biological Project 1
Structural and energetic basis of cadherin binding specificity
Lawrence Shapiro and Barry Honig
This project investigated the structural and energetic basis of cell-cell adhesion, which helps shape tissues and “wire” the neural networks of the brain. Initially focused on adhesion mediated by the cadherin family of proteins, the project expanded to include the binding of neurexin and neuroligin proteins that play a role in synapse formation. For the cadherins, our research showed how "strand-swapping" between molecules affects their mutual affinity, and demonstrated for the first time a connection between molecular binding affinity and adhesive strength between cells. Further modeling showed how the affinities of two cadherins, toward each other and toward themselves, define the patterns formed by cells that carry these surface molecules. We also demonstrated how the precise sequence of two proteins changes their affinity, and used that information to convert a normally nonbinding cadherin to a binding form. Overall, the synergy between theory and experiment allowed us to make important connections between molecular and cellular properties to clarify how the differing binding energies of molecules presented on the surfaces of cells determine their adhesion and the tissues they form.
Adhesion between cadherin family members, primarily through preferential interaction between similar molecules, provides a key driving force in the development of tissue architecture. The crucial specificity that helps sculpt tissue shape arises from subtle differences among family members. In the past, neither sequence analysis nor observations on known crystal structures had revealed the structural and energetic origins of cadherin specificity. It became clear that computational studies were needed, both to generate testable hypotheses and to interpret experimental results. We pursued a joint program encompassing energetic analysis of protein-protein interfaces, structure prediction, x-ray crystallography, biophysical measurements of binding, and cell-sorting experiments. This program has produced several important results:
- Cadherin dimerization, which initiates cell-cell recognition, involves a protein-protein interface formed by the “swapping” of N-terminal beta-strands between partner molecules. We confirmed that the specificity of these interactions can be understood through sequence analysis, and identified the sequence determinants that enable strand swapping (Posy 2008). This work also demonstrated, for the first time, a relationship between cadherin dimerization affinities and cell-cell adhesive strength.
- Building on this work, we showed how strand swapping mediates dimerization affinities that are weak and yet highly specific (Katsamba 2009). The measurement of binding energies of E- and N-cadherins, both to themselves and each other, allowed theoretical modeling of the way these affinities give rise to patterns in cells expressing these molecules.
- Based on bioinformatics analysis, NMR studies, and molecular dynamics simulations, (Miloushev 2008) we arrived at a new model that explains how cadherins are designed to facilitate strand swapping. The model was validated with site-directed mutagenesis and through a remarkable experiment in which we were able to convert a naturally non-binding cadherin domain to a strand-swapping binding domain.
- A combination of biophysical, x-ray and computational analysis identified a new cadherin dimerization mode that is an intermediate on the path to the formation of the strand-swapped interface (Harrison, in press) . This work also reconciled previous x-ray and NMR results that appeared to be internally inconsistent.
- In order to explore the generality of our results, we also began investigations of the synaptic adhesion protein families, the neurexins and neuroligins (Koehnke 2008a, Koehnke 2008b). These two adhesive protein families appear on the pre- and post-synaptic sides of synapses, respectively. We are exploring how the wide diversity of variants available to these proteins through alternative splicing may contribute to their adhesive specificity.
Harrison OJ, Bahna F, Katsamba PS, Jin X, Brasch J, Vendome J, Ahlsen G, Carroll KJ, Price SR, Honig B, Shapiro L. Two step adhesive binding by classical cadherins. Nat Struct Mol Biol. 2010 Mar;17(3):348-57. Epub 2010 Feb 28.
Katsamba P, Carroll K, Ahlsen G, Bahna F, Vendome J, Posy S, Rajebhosale M, Price S, Jessell TM, Ben-Shaul A, Shapiro L, Honig BH. Linking molecular affinity and cellular specificity in cadherin-mediated adhesion. Proc Natl Acad Sci U S A. 2009;106(28):11594-9.
Koehnke J, Jin X, Budreck EC, Posy S, Scheiffele P, Honig B, Shapiro L. Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2. Proc Natl Acad Sci U S A. 2008;105(6):1873-8.
Koehnke J, Jin X, Trbovic N, Katsamba PS, Brasch J, Ahlsen G, Scheiffele P, Honig B, Palmer AG 3rd, Shapiro L. Crystal structures of beta-neurexin 1 and beta-neurexin 2 ectodomains and dynamics of splice insertion sequence 4. Structure. 2008;16(3):410-21.
Miloushev VZ, Bahna F, Ciatto C, Ahlsen G, Honig B, Shapiro L, Palmer AG 3rd. Dynamic properties of a type II cadherin adhesive domain: implications for the mechanism of strand-swapping of classical cadherins. Structure. 2008;16(8):1195-205.
Patel SD, Ciatto C, Chen CP, Bahna F, Rajebhosale M, Arkus N, Schieren I, Jessell TM, Honig B, Price SR, Shapiro L Type II cadherin ectodomain structures: implications for classical cadherin specificity. Cell. 2006;124(6):1255-68
Posy S, Shapiro L, Honig B. Sequence and structural determinants of strand swapping in cadherin domains: do all cadherins bind through the same adhesive interface? J Mol Biol. 2008;378(4):954-68.