MOLECULAR BASIS OF CALCIUM-INDUCED DIMERIZATION IN CLASSICAL CADHERINS
Neural (N-) and Epithelial (E-) cadherins are the preeminent members of the classical cadherin family, transmembrane proteins responsible for cell-cell adhesion through structures called adherens junctions, an adhesive structure that is responsible for dynamic communication between adherent cells in tissues. Adhesion between extracellular regions of cadherins from adherent cells must be communicated through tension in the molecule to the intracellular cytoskeleton1. Cytoplasmic factors, in turn control transcription in the cell and stable differentiation of tissue. Abnormal expression or mutation of cadherins can cause metastasis of cancer2-4. As such, understanding how cadherins “work” provides lessons in fundamental protein structure-function relationships and the molecular basis of disease.
Although these N-and E-cadherin are named for the tissues in which they were originally found, they occur in other tissues and in the case N-cadherin, its occurrence in tissues is developmentally regulated, as in angiogenesis in endothelial tissue5, 6 and in neural crest formation during development7. Of interest to our lab is the following. First, they are specifically located at neurological synapses in the central nervous system: N-cadherin at excitatory synapses and E-cadherin at inhibitory synapses. Second, these two proteins are remarkably similar in their calcium binding and dimerization interface, and yet they have different dimerization affinity and calcium dependence of dimerization. We argue that their biophysical properties suit them for the specific chemical milieu of the extracellular space of the synaptic cleft in which they are located. Our laboratory is currently focused on exploring the molecular determinants for their unique calcium dependent behavior with a long-term interest toward how the lessons from these studies translate into other members of the classical cadherin family.
My lab takes a biophysical approach to understanding the chemical driving forces that promote the calcium dependence of cadherin dimerization. This means that we form hypotheses about the role of specific amino acids in the folding, calcium binding or dimerization of cadherins. We then mutate those residues and study the effect on the protein’s structure and function.
Abundance of Prolines in the Dimerization Interface. We are systematically replacing prolines with alanines. We have found that these replacements can increase the dimerization affinity in N-cadherin by 25-fold, or decrease it to 1/3 its original value depending upon the position of the mutation. The position and abundance of prolines differ between N-and E-cadherins.
Trapped NCAD dimer in low calcium conditions. In the very low calcium conditions in the synaptic cleft of a potentiated synapse we suspect that the kinetically trapped dimer of NCAD (REF) dominates the dynamics of the synapse. Studies are underway to identify the exact molecular determinant of the molecular interaction that is responsible for the “trapping” of the dimeric form.
Anionic character of the dimer interface. We are interested in how pH affects the dimerization affinity of E-and N-cadherin. We have studied this effect in N-cadherin, but the data are incomplete for E-cadherin. Studies are underway.
The X-dimer Interface in N-cadherin. All we know it that it is not the same as in E-cadherin. Computational studies indicate a possible X-dimer interface for N-cadherin that has heretofore been unexplored. We would like to check it out.
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Prasad, A., Housley, N. A. and Pedigo, S. (2004) “Thermodynamic Stability of Domain 2 of Epithelial Cadherin” Biochemistry 43: 8055-8066.
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Vunnam, N. and Pedigo, S. (2011a) “Calcium-induced strain in the monomer promotes dimerization in neural cadherin” Biochemistry 50: 8437-8444.
Vunnam, N. and Pedigo, S. (2011b) “Prolines in betaA-Sheet of Neural Cadherin Act as a Switch To Control the Dynamics of the Equilibrium between Monomer and Dimer” Biochemistry 50: 6959-6965.
Vunnam, N. and Pedigo, S. (2011c) “Sequential binding of calcium leads to dimerization in neural cadherin” Biochemistry 50: 2973-82.
Vunnam, N., Flint, J., Balbo, A., Schuck, P. and Pedigo, S. (2011d) “Dimeric States of Neural-and Epithelial-Cadherins are Distinguished by the Rate of Disassembly” Biochemistry 50: 2951-61.
Vunnam, N., McCool, J.K., Williamson, M. and Pedigo, S. (2011e) “Stability Studies of Extracellular Domain Two of Neural-Cadherin” Biochim Biophys Acta 14:1841-1845.
Vunnam, N. and Pedigo, S. (2012) “X-interface is not the explanation for the slow disassembly of N-cadherin dimers in the apo state.” Protein Sci. 21:1006-14.
Jungles, J, Dukes, M.P., Vunnam, N. and Pedigo, S. (2014) “Impact of pH on Structure and Function of Neural Cadherin” Biochemistry 53:7436-7444.
Vunnam, N., Hammer, N. I. and Pedigo, S. (2015)“Basic residue at position 14 is not required for fast assembly and disassembly kinetics in Neural Cadherin” Biochemistry, 54:836–843.
Davila, S., Liu, P., Smith, A., Marshall, A.G., and Pedigo, S. “Spontaneous Calcium-Independent Dimerization of the Isolated First Domain of Neural Cadherin” (2018) Biochemistry 57:6404-6415.
Dukes, M.P., Rowe, R.K., Harvey, T., Rangel, W. and Pedigo, S. “Nickel reduces calcium dependent dimerization in neural cadherin” (2019) Metallomics11:475-482. https://dx.doi.org/10.1021/acs.biomac.0c00043.