Forced unfolding modulated by disulfide bonds in the Ig domains of a cell adhesion molecule

Abstract

Cell adhesion molecules (CAMs) mediate cell attachment and stress transfer through extracellular domains. Here we forcibly unfold the Ig domains of a prototypical Ig superfamily CAM that contains intradomain disulfide bonds. The Ig domains of all such CAMs have conformations homologous to cadherin extracellular domains, titin Ig-type domains, and fibronectin type-III (FNIII) domains. Atomic force microscopy has been used to extend the five Ig domains of Mel-CAM (melanoma CAM)--a protein that is overexpressed in metastatic melanomas--under conditions where the disulfide bonds were either left intact or disrupted through reduction. Under physiological conditions where intradomain disulfide bonds are intact, partial unfolding was observed at forces far smaller than those reported previously for either titin's Ig-type domains or tenascin's FNIII domains. This partial unfolding under low force may be an important mechanism for imparting elasticity to cell-cell contacts, as well as a regulatory mechanism for adhesive interactions. Under reducing conditions, Mel-CAM's Ig domains were found to fully unfold through a partially folded state and at slightly higher forces. The results suggest that, in divergent evolution of all such domains, stabilization imparted by disulfide bonds relaxes requirements for strong, noncovalent, folded-state interactions.

Figure 1

Experimental AFM set-up and proposed reduction-coupled mechanism for forced unfolding of a multidomain Ig-CAM. (a) A single surface-to-tip adsorbed protein is shown with intact intradomain disulfide bonds. In separate experiments, the disulfide bonds are chemically reduced by DTT present in the surrounding medium. A prototypical ribbon structure of an Ig-CAM domain shows, in red, the disulfide-delimited cores. Protein is adsorbed to substrates of freshly cleaved mica or amino-silanized glass and nonadsorbed excess is washed away before AFM experiments. The use of mica or glass substrates here and elsewhere (13), rather than gold substrates, avoids artifactual formation of thio-gold bonds. Inset (i) shows fluorescently labeled Mel-CAM homogeneously adsorbed to mica at an estimated surface density of less than ≈10 protein molecules per 100 nm². Inset (ii) shows a tapping-mode AFM image subsequent to contact-mode scraping over half the domain (arrow indicates direction); the image is consistent with no more than a monolayer of Mel-CAM molecules (1–2 nm thick) adsorbed to the surface. DTT does not affect the extent of fluorescent-Mel-CAM adsorption, and 1 mM DTT treatments of tenascin's FNIII domains (6) also indicate that DTT does not significantly alter the unfolding forces of Ig-like domains lacking intradomain disulfides. (b) Proposed unfolding pathway for a single domain under force. Under physiological conditions, only the first step can occur. In the presence of DTT, however, the partially unfolded domain exposes the disulfide bond to DTT so that, after reduction, the S–S-secluded region unfolds.

Figure 2

Representative force–extension curves for Mel-CAM, under either physiological (a) or reducing conditions (+DTT; b). Various numbers of unfolding peaks (N_pk) are shown; fewer N_pk are observed when Mel-CAM is oxidized. The peak-to-peak spacing also appears shorter with intact S–S. The exponential increase in force, for peaks beyond the first that may or may not be initial tip or chain desorption from the substrate, are fitted with the worm-like chain (WLC) equation (15): f(x) = (k_BT/p)[(x/L_c) + 0.25/(1 − x/L_c)² − 0.25]. A differential contour length between peaks averaged ΔL_c = 16.5 nm for intact disulfides and ΔL_c = 26.0 nm for reduced samples. For both oxidized and reduced samples, the persistence length that characterizes the minimal flexible length of an unfolded domain was determined to average P = 0.3–0.5 nm, consistent with reports of others (7). Dynamic MC simulations of unfolding, shown as Inset for oxidized Mel-CAM, were performed as prescribed by Rief et al. (14) by using a force-free, unfolding rate constant of k_u^o = 3 × 10⁻³ s⁻¹ and a transition state length of x_u = 1.6 nm (see Figure 6). Various contour lengths were taken from Table 1. For reference, titin Ig domain unfolding at much higher forces has previously been fit with k_u^o = 2.8 − 3.3 × 10⁻⁵ s⁻¹ and x_u = 0.25 − 0.3 nm (7, 14, 17)

Figure 3

Frequency distributions for the number of peaks observed under oxidizing or reducing conditions where the imposed extension rate was either 1 nm/msec (a) or 5 nm/msec (b). Exponential decays at the higher values of N_pk were fit with P(N_pk) ∝ m^N_pk, where m may be interpreted as the number of ways that the AFM tip, after randomly contacting one domain in a chain, can extend and unfold a subset of all of the multiple domains between tip and substrate (before desorption). Extension of single chains is assumed to predominate under the adsorption conditions used; furthermore, chains are known from separate ultracentrifugation experiments to exist only as monomers in solution. For the intact disulfide bonds, m_S-S | _1Hz = 4.6 and m_S-S | _5Hz = 4.5, signifying, perhaps, that each of four or five domains has an equal chance of partially unfolding or not in a two-state process before domain detachment. Desorption, however, also seems likely to be accelerated at higher N_pk because protein–tip or protein–substrate interactions are at a minimum in this limit. For the reduced disulfide bonds, sequential partial unfolding (see Figure 1b), in addition to full unfolding, appears implicated by higher values of m_SH | _1Hz = 5.6 and m_SH | _5Hz = 5.5.

Figure 4

Histograms of full unfolding lengths for Mel-CAM under oxidizing (a) and reducing (b) conditions. Unfolding lengths summed between all identified unfolding peaks were cumulated for extension curves containing four and five peaks for the oxidized conditions and curves containing four, five, six, seven, and eight peaks for the reduced conditions. The right-most hatched regions correspond to the theoretical full unfolding lengths of the protein in either oxidized or reduced states, as given by the respective contour lengths minus a maximum of 4 nm per domain for the folded state. Consistent with extension of monomeric chains, < 0.25% of the data collected exceeds the theoretical limits for a single chain.

Figure 5

Histograms of unfolding lengths and unfolding forces for Mel-CAM under oxidizing (a and b) and reducing (c and d) conditions. Extension curves with three or more, four or more, etc., peaks are cumulated separately, but only for those intervals beyond the first interval (l₁₂) because the initial peak may well reflect chain desorption. By including the interval between the next-to-last and the last peak, a significant percentage (< 20%) of the l_pk-pk are in excess of 25 nm, as apparent in the N_pk = 5 extension curve in Fig. 2a. However, the distributions of unfolding force do not include the first or last peaks. The Upper Insets show corresponding histograms from MC simulations: Both partial unfolding and full domain unfolding simulations were separately carried out by using data in Table 1 and the parameters listed in the legends of Figs. 2 and 6. In c and d, simulated distributions for full and partial unfolding were scaled to reflect experimental proportions; and the measured distributions were fitted with Gaussians to account for measurement noise as described in the text. The Lower Inset in b shows the log-rate dependence for the unfolding force under oxidizing and reducing conditions. The experimental data points (circles) fall well within one standard deviation of the average disruption force calculated by simulation at various substrate velocities and indicated by the two gray bands.

Figure 6

Model and simulation results for unfolding of Mel-CAM. (a) Free energy profiles (in the absence of force) for unfolding of a domain. The kinetic barriers in these one-dimensional models are overcome in forced unfolding over a distance x_u. DTT is assumed to effectively modulate the barrier height. In theory, x_u^SH ≤ x_u^SS. MC simulations in b and c follow the algorithm in ref. and show isoforce curves for oxidized or reduced Mel-CAM extended at either 1 nm/ms (black lines) or 5 nm/ms (gray lines) for a broad range of the fitting parameters x_u and k_u^o. Overlap between the simulations and rate-dependant measurements of peak forces (± 1 pN) are shown as black regions. Under either condition, the overlap region starts at x_u ≈ 1.5 − 1.7 nm, indicating that x_u^SH ≈ x_u^SS. Parameters fit to titin unfolding (★) agree with previous fits to measured forces in the literature (7, 16) and suggest that suitable x_u^SS and x_u^SH for Mel-CAM are likely to be in the range 1.5–1.7 nm.

Figure 7

Generic model of Ig-CAM domain unfolding in cell–cell contact sites. Under the stresses encountered during metastatic detachment, organogenesis, cell migration, etc. Additional extensibility and elasticity is provided by partial unfolding of Ig domains that bridge two cell membranes. (a) Model depicting heterotypic association between the N-terminal domain of a five-Ig domain CAM and a receptor on an opposing cell. (b) Under moderate stress, up to four domains could partially unfold without breaking cell–cell contact. (c) When tensile forces are sufficient to unfold domains directly involved in receptor associations, cell adhesion would be disrupted or at least moderated. The core regions delimited by the disulfides are purple. These regions may act as nuclei for domain refolding when tensile stresses are removed or reduced. Applying the model to Mel-CAM, which interacts with a different type of receptor but efficiency of unknown identity and at unknown site(s) on Mel-CAM, the clear suggestion is that molecular extensibility contributes to adhesive efficiency in metastases.