Metal Cation-Binding Mechanisms of Q-Proline Peptoid Macrocycles in Solution

Abstract

The rational design of foldable and functionalizable peptidomimetic scaffolds requires the concerted application of both computational and experimental methods. Recently, a new class of designed peptoid macrocycle incorporating spiroligomer proline mimics (Q-prolines) has been found to preorganize when bound by monovalent metal cations. To determine the solution-state structure of these cation-bound macrocycles, we employ a Bayesian inference method (BICePs) to reconcile enhanced-sampling molecular simulations with sparse ROESY correlations from experimental NMR studies to predict and design conformational and binding properties of macrocycles as functional scaffolds for peptidomimetics. Conformations predicted to be most populated in solution were then simulated in the presence of explicit cations to yield trajectories with observed binding events, revealing a highly preorganized all-trans amide conformation, whose formation is likely limited by the slow rate of cis/trans isomerization. Interestingly, this conformation differs from a racemic crystal structure solved in the absence of cation. Free energies of cation binding computed from distance-dependent potentials of mean force suggest Na⁺ has a higher affinity to the macrocycle than K⁺, with both cations binding much more strongly in acetonitrile than water. The simulated affinities are able to correctly rank the extent to which different macrocycle sequences exhibit preorganization in the presence of different metal cations and solvents, suggesting our approach is suitable for solution-state computational design.

Figure 1:

The Q-proline macrocycle (QPM) scaffold as described by Northrup et al. Shown are the three macrocycle sequences simulated in this study. QPM-1 and QPM-3 bind metal cations, while QPM-9 (the control) does not. All are three-fold symmetric.

Figure 2:

A flowchart of methods used to model macrocycle cation-binding, starting from the generation of initial structures.

Figure 3:

The autocorrelation function g(τ), shown for QPM-1, shows dihedral angle decorrelation times around 20 ns, indicating well-converged sampling of amide dihedrals over the much longer (>600 ns) tREMD trajectory lengths.

Figure 4:

ROESY ¹H correlations, shown here for QPM-1. Strong and weak ROEs are denoted by blue and red arrows, respectively.

Figure 5:

Results of the BICePs algorithm. (a) A comparison of estimated conformational state populations from BICePs when only experimental restraints are used (‘exp’), versus a combination of tREMD simulation and experiment (‘sim+exp’). States 11, 33, and 92 (labeled in green) have the highest predicted populations. (b) The marginal posterior distributions of P(σ_d) for ‘exp’ and ‘sim+exp’ scenarios. (c) The marginal posterior distributions for P(γ′).

Figure 6:

Distributions of backbone amide cis/trans isomers for QPM-1, shown for (a) all tREMD temperature replicas, (b) the three lowest-temperature replicas, and (c) after reweighting populations by the BICePs algorithm. The inclusion of experimental ROE restraints results in much higher population estimates for backbone trans-amides, with the mostly trans tctttt as the most populated state.

Figure 7:

Potentials of mean force (PMF) as a function of cation distance to the centers of macrocycles QPM-1 and QPM-3, computed from explicit cation-binding trajectories simulated in acetonitrile and water.

Figure 8:

Projection of explicit-water QPM-1 trajectories to the first two tICA components reveals three slowly-interconverting conformational states: “open” and “closed” states with weak affinity to cations, and a “bound” conformation with stronger affinity to cation. Representative structures are taken from simulations of QPM-1 with Na⁺. The slowest conformational motions (along tIC₁) correspond to the formation of an unobstructed cation binding site, while the next slowest motion (along tIC₂) corresponds to reversible cation binding to the “bound” conformation. Binding is defined as the cation being < 0.35 nm from the center of macrocycle.

Figure 9:

Projection of explicit-acetonitrile QPM-1 trajectories to the first two tICA components similarly reveals three metastable conformational states: “open” and “closed” states, which contain at least one cis amide, and an all-trans “bound” conformation with stronger affinity to cation. Representative structures are taken from simulations of QPM-1 with K⁺. The slowest conformational motions (along tIC₁) correspond to the formation of favorable cation binding site, while the next slowest motion (along tIC₂) corresponds to transitions to a cation-bound all-trans amide “bound” conformation.

Figure 10:

Simulations of QPM-1 in acetonitrile reveals a highly stable, two-cation structure that forms in the presence of both (A) sodium and (B) potassium cations. The bound conformation features all-trans backbone amides, with one cation above the macrocycle plane in a “basket” of Q-proline residues coordinated by triflate sulfonate groups, and the second cation below the plane, stabilized by coordinating peptoid N-methoxyethyl substituents.