A positive cooperativity binding model between Ly49 natural killer cell receptors and the viral immunoevasin m157: kinetic and thermodynamic studies

Abstract

Natural killer (NK) cells discriminate between healthy and virally infected or transformed cells using diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 NK receptors, which can adopt two distinct conformations (backfolded and extended), are of particular importance for detecting cells infected with mouse cytomegalovirus (CMV) via recognition of the viral immunoevasin m157. The interaction of m157 with activating (Ly49H) and inhibitory (Ly49I) receptors governs the spread of mouse CMV. We carried out kinetic and thermodynamic experiments to elucidate the Ly49/m157 binding mechanism. Combining surface plasmon resonance, fluorescence anisotropy, and circular dichroism (CD), we determined that the best model to describe both the Ly49H/m157 and Ly49I/m157 interactions is a conformational selection mechanism where only the extended conformation of Ly49 (Ly49*) is able to bind the first m157 ligand followed by binding of the Ly49*/m157 complex to the second m157. The interaction is characterized by strong positive cooperativity such that the second m157 binds the Ly49 homodimer with a 1000-fold higher sequential constant than the first m157 (∼10(8) versus ∼10(5) M(-1)). Using far-UV CD, we obtained evidence for a conformational change in Ly49 upon binding m157 that could explain the positive cooperativity. The rate-limiting step of the overall mechanism is a conformational transition in Ly49 from its backfolded to extended form. The global thermodynamic parameters from the initial state (backfolded Ly49 and m157) to the final state (Ly49*/(m157)2) are characterized by an unfavorable enthalpy that is compensated by a favorable entropy, making the interaction spontaneous.

Keywords: Fluorescence Anisotropy; Innate Immunity; Ly49; Murine Cytomegalovirus; Natural Killer (NK) Cell; Surface Plasmon Resonance (SPR); Thermodynamics; Viral Immunology; m157.

FIGURE 1.

Conformations adopted by Ly49 receptors and interaction with MHC-I and the cytomegalovirus immunoevasin m157. A, the backfolded conformation of Ly49 on the NK cell membrane (bottom). The NKDs of the Ly49 homodimer (blue ovals) are backfolded onto the α-helical stalk region (blue rectangles) with interchain disulfides drawn as red bars (21). B, the extended conformation of Ly49 in which the NKDs extend away from the stalk. C, Ly49 in the backfolded conformation binding in trans to two MHC-I molecules (green) exposed on the target cell membrane (top) (21). The interaction with MHC-I is mediated by the NKDs. D, Ly49 in the extended conformation binding in cis to one MHC-I molecule on the same NK cell. E, Ly49 in the extended conformation binding in trans to two m157 molecules (red) on the target cell. The interaction with m157 is mediated by the Ly49 stalk region, not the NKDs (22).

FIGURE 2.

SPR equilibrium analysis. Plots of the maximum response (R) in RU obtained at each m157 total concentration assayed by injection over immobilized ∼100 RU Ly49H (A) and Ly49I (B) at 25 °C. The curves describe the fitting to the Hill equation. The n_H coefficient obtained for the Ly49H/m157 pair is 1.7 ± 0.1 and for the Ly49I/m157 pair is 1.9 ± 0.2. Residual errors are indicated below each panel.

FIGURE 3.

Equilibrium fluorescence anisotropy (r) as a function of m157 concentration. 1 μm Ly49H-FITC (A), 1 μm Ly49I-FITC (B), and 2.6 μm Ly49I-FITC (C) were titrated at 25 °C with m157, and fluorescence anisotropy was measured after the interaction had reached equilibrium. The curves describe the fitting to an equation that links the anisotropy (r) to the total m157 molar concentration. The constants (K_G1, K_G2, K_s1, K_s2, k, and k′) are shown in Table 1. Error bars represent S.D.

FIGURE 4.

Plot of the normalized fluorescence anisotropy signal Q as a function of the bound ligand density (v̄). An analysis of the fluorescence anisotropy data using two Ly49I-FITC concentrations (1 and 2.6 μm) allowed us to calculate Q and v̄. The dotted line indicates that the stoichiometry of the Ly49I/m157 interaction is 1:2, the point where the response Q reaches its maximum value.

FIGURE 5.

Far-UV CD spectra of Ly49H and Ly49I in combination with m157. Ly49H (A) and Ly49I (B) preincubated with m157 at molar ratios of 1:1 (solid green lines) and 1:2 (solid red lines) and the algebraic sum of spectra of Ly49 and one m157 molecule (dashed green lines) or of spectra of Ly49 and two m157 molecules (dashed red lines) are shown. The difference between the preincubated spectra and the algebraic sum spectra indicates a conformational change upon m157 binding. deg, degrees.

FIGURE 6.

Analysis of SPR kinetic data at 25 °C. A and B show the association and dissociation SPR data for the Ly49H/m157 and Ly49I/m157 pairs, respectively, fitted with two-exponential functions. The residual errors appear randomly distributed and are shown below each panel. C, first derivative of the response (R) as a function of the response for the association data for the Ly49H/m157 (left panel) and Ly49I/m157 pairs (right panel). D, semilog plots of the response versus time for the dissociation data for the two couples analyzed. These plots (C and D) describe biphasic curves and indicate at least a two-step mechanism. This is in agreement with the fitting of two-exponential functions (A and B).

FIGURE 7.

Analysis of SPR association kinetic data at 25 °C. k_obs was obtained after the fitting of a two-exponential function to the association data from the sensorgrams versus m157 concentration. A, k_obs1 versus m157 concentration corresponding to the slow exponential function component for the Ly49H/m157 and Ly49I/m157 couples. These distributions correspond to a conformational selection mechanism. B, k_obs2 versus m157 concentration corresponding to the fast exponential function component for the two interactions studied. The linear dependence of the points indicates a simple 1:1 interaction of the Ly49*/m157 complex with a second m157 ligand.

FIGURE 8.

Proposed models for the binding mechanism between Ly49 and m157. Models of the Ly49H/m157 and Ly49I/m157 interactions should include at least two steps according to the assumptions described in the text. A, backfolded Ly49; A*, extended Ly49; B, m157.

FIGURE 9.

Sensorgrams for Ly49/m157 interactions at different temperatures. Sensorgrams for the Ly49H/m157 and for Ly49I/m157 interactions at 5, 10, 15, 25, and 30 °C are shown. On the side of each sensorgram, the total m157 molar concentration assayed is indicated. Black lines indicate the fitted curves using numerical integration with BIAevaluation 4.1 software from the differential equations of model D. The kinetic rate constants and macroscopic sequential equilibrium constants are shown in Tables 2 and 3, respectively, for the Ly49H/m157 and Ly49I/m157 pairs. R, response.

FIGURE 10.

Eyring analysis using kinetic rate constants at different temperatures. Eyring analysis using the kinetic rate constants obtained after applying model D to the SPR data for the Ly49H/m157 (A) and Ly49I/m157 interactions (B) is shown. The Eyring equation was fitted to experimental data points at 25 °C to obtain the thermodynamic activation parameters ΔH_o^‡, ΔS_o^‡, and ΔCp_o^‡ (supplemental Table S3). Filled circles indicate association data, and empty circles indicate dissociation data. Error bars represent S.D.

FIGURE 11.

Reaction progress landscapes at 25 °C. Reaction progress landscapes at 25 °C for the free energy (A) together with the deconvolution into enthalpic (B) and entropic (C) components for the Ly49H/m157 (red) and Ly49I/m157 pairs (green) are shown. This figure shows that the global rate-limiting step is a conformational change from the backfolded to the extended state of Ly49. A, backfoldedLy49; A*, extended Ly49; B, m157; A^T, Ly49 in transition state for the conformational change step; A*–B, transition state for the binding to the first m157; A*B, extended Ly49 bound to one m157 molecule; A*B–B, transition state for the binding to the second m157; A*B₂, extended Ly49 bound to two m157 molecules.