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. 2011 Apr 12;50(14):2951-61.
doi: 10.1021/bi2001246. Epub 2011 Mar 21.

Dimeric states of neural- and epithelial-cadherins are distinguished by the rate of disassembly

Nagamani Vunnam  1 , Jon Flint, Andrea Balbo, Peter Schuck, Susan Pedigo
Affiliations

Dimeric states of neural- and epithelial-cadherins are distinguished by the rate of disassembly

Nagamani Vunnam et al. Biochemistry. .

Abstract

Epithelial- and neural-cadherins are specifically localized at synapses in neurons which can change the shape and contact surface on a time scale of seconds to months. We have focused our studies on the role of the extracellular domains of cadherins in the dynamics of synapses. The kinetics of dimer disassembly of the first two extracellular domains of E- and N-cadherin, ECAD12 and NCAD12, were studied with analytical size exclusion chromatography and sedimentation velocity. NCAD12 forms three different dimers that are distinguished by assembly conditions and kinetics of dissociation. ECAD12 dimer disassembles rapidly regardless of the calcium concentration, whereas the disassembly of NCAD12 dimers was strongly dependent on calcium concentration. In addition to the apo- and saturated-dimeric forms of NCAD12, there is a third dimeric form that is a slow exchange dimer. This third dimeric form for NCAD12, formed by decalcification of the calcium-saturated dimer, was kinetically trapped in apo-conditions and did not disassemble over a period of months. Sedimentation velocity experiments showed that this dimer, upon addition of calcium, had similar weighted averages as a calcium-saturated dimer. These studies provide evidence that the kinetics of dimer disassembly of the extracellular domains may be a major contributor to the morphological dynamics of synapses in vivo.

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Figures

FIGURE 1
FIGURE 1
Linked equilibria and calcium titrations of ECAD12 and NCAD12. (A) Model showing the four-linked equilibria for the ECAD12 and NCAD12 (Mapo- calcium-free monomer, Dapo- calcium-free dimer, Msatd – calcium-bound monomer and Dsatd – calcium-bound dimer). Literature values for the dimer dissociation equilibrium constant, Kd, for saturated dimers (Dsatd to Msatd) is 25 µM for NCAD12 and 100 µM for ECAD12 (52). The Kd values for the dissociation of apo dimers (Dapo to Mapo) are not known. (B) Calcium titrations of NCAD12 (black) and ECAD12 (red) monitored by circular dichroism at 229 nm. Protein concentration was 2.5 µM. Solid curves were simulated based on best fit parameters to Eq 1. The average resolved free energy change for 3 titrations of both proteins was −26 ± 1 kJ/mol.
FIGURE 2
FIGURE 2
Analytical SEC to determine the monomer-dimer equilibria as a function of protein and calcium concentrations. Dilute NCAD12 and ECAD12 stocks (17 and 30 µM, respectively; dashed) were concentrated (180 and 100 µM, respectively; solid) in apo (1 µM calcium; top) and calcium-bound states (1 mM calcium; bottom) to show the affect of protein and calcium concentration on the monomer and dimer equilibria. These samples were analyzed under apo-buffer conditions. Insets: Calcium-saturated samples analyzed by SEC in a chromatography buffer containing 1 mM calcium. Scale is identical to the chromatograms under apo-buffer conditions.
FIGURE 3
FIGURE 3
Characterization of kinetically-trapped dimeric species of NCAD12. Sedimentation coefficient distributions c(s) of NCAD12 stocks from preparative SEC enriched in (A) kinetically-trapped dimer and (B) in monomer under 4 different experimental conditions: Apo-dilute (0.2 mg/ml; red dashed), Apo-concentrated (1 mg/mL; red solid), 1 mM calcium-dilute (0.2 mg/ml; black dashed) and 1 mM calcium-concentrated (1 mg/ml; black solid). (C) SEC analysis of kinetically-trapped dimer enriched NCAD12 stocks from preparative SEC. Top panel shows the concentrated (black, 68% dimer) and 1 to 5 diluted (red, 68% dimer) apo-NCAD12 dimer-enriched stock. Bottom panel shows the concentrated (black, 54% dimer) and 1 to 5 diluted (red, 47% dimer) calcium bound NCAD12 dimer-enriched stock.
FIGURE 4
FIGURE 4
Determination of the equilibrium dimer dissociation constant. The analytical SEC technique was adapted to measure the equilibrium dimer dissociation constant for NCAD12. EDTA was added to form the kinetically-trapped dimeric species in a set of protein solutions of differing concentrations. (A) Representative chromatograms showing D*apo (12.1 mL) and monomer (13.2 mL) over a range of protein concentrations (5 µM (black), 25 µM (blue), 110 µM (green) and 220 µM (red)). (B) Dimer fraction is plotted versus the total protein concentration. The line is simulated based on the best fit values resolved from fitting the data to Eq 2.
FIGURE 5
FIGURE 5
Thermal-denaturation of ECAD12 and NCAD12. (A) Thermal-denaturation of ECAD12 (triangles) and NCAD12 (circles) at 230 nm. Normalized CD signal versus temperature (°C) at 1 µM (filled) and 1 mM (open) calcium at 5 µM protein concentrations. (B) CD spectra of ECAD12 (triangles) and NCAD12 (circles) in the Apo-state (140 mM NaCl, 10 mM HEPES, pH 7.4).
FIGURE 6
FIGURE 6
Exchange dynamics of monomer-dimer equilibria for ECAD12 and NCAD12. Exchange dynamics of monomer-dimer equilibrium for ECAD12 (top panel) and NCAD12 (bottom panel). Solid arrows are reactions that were observed experimentally. Calcium titrations of monomeric NCAD12 and ECAD12 are represented by solid red arrows (from Figure 1B). Dsatd is formed through calcium addition to Mapo-Dapo stocks (blue; from Figure 2). The formation of Dapo depends upon the construct. For NCAD12 the green arrows represent the decalcification of Dsatd to form D*apo. This was observed through decalcification of calcium saturated stocks on the chromatographic column or by addition of EDTA (from Figure 4). The activation energy barrier between Dapo and D*apo is too high to overcome without addition of heat or calcium (dashed arrows).
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