Evidence for dynamics in proteins as a mechanism for ligand dissociation

Abstract

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.

Figure 1

The series of reduced-affinity and previously characterized antifolates. (a) Chemical structures of the previously characterized antifolates – methotrexate (MTX), trimethoprim (TMP), and 1. (b) Chemical structures of the reduced-affinity antifolates 2–6. (c) The relationship between k_off and K_i for the series of reduced-affinity antifolates (R = 0.99).

Figure 2

High resolution crystal structures for the series. (a) Overlay of the crystal structures for E:NADPH:3 (blue), ENADPH:4 (teal), and E:NADPH:5 (maroon). NADPH is shown in cyan and bound antifolate in the colors designated per complex. (b) Expansion of the C-helix, now overlaying five inhibitor-bound complexes (E:NADPH:1 in dark grey and E:NADPH:MTX in light grey). PDB IDs are listed parenthetically. (c) Differential puckering of the saturated ring in the bound inhibitors, colored as in (a).

Figure 3

Slow timescale dynamics for the reduced-affinity inhibitor series. Sites along the backbone with detectable μs-ms motion are highlighted in colored spheres for each complex, ordered from left to right by increasing K_i value. The number of residues with significant R_ex is given parenthetically.

Figure 4

Internal motions vary with K_i and k_off. (a) The forward rate of motion (k_conf,forward) fit from relaxation dispersion data for each complex varies exponentially with the K_i value for the bound inhibitor (R = 0.97). The open circle represents the best fit for E:NADPH:4. (b) An exponential correlation is also seen between k_conf,forward and k_off (R = 0.97). Data points in red have predicted k_off values, as described in the text. k_off for TMP and 1 were calculated based on estimated values for k_on. For 1, the average k_on for the THQ series was used. For TMP, because of its greater similarity to MTX, k_on was taken to be intermediate between MTX and the average value for the THQ series. The data point for E:NADPH:4 (unfilled circle) does not fall along this exponential correlation, suggesting that this correlation may not always be predictive. The dashed curve represents what would be expected if the correlation were linear. Error bars represent standard deviations (originating from Monte Carlo simulations in the case of k_conf,forward).

Figure 5

Antifolate consensus sites sample a structurally similar excited state. (a) The twelve antifolate consensus sites are highlighted in yellow colored spheres. (b) Dynamic Δω values fitted from relaxation dispersion for these twelve sites cluster for each residue. The eight complexes are colored by the bound inhibitor, as indicated in the legend. Averages were calculated only from residues that have the dominant sign. No bar is shown if that residue did not exhibit significant slow motion while bound to a particular inhibitor. Error bars result from standard deviations derived from Monte Carlo simulations.