Use of a Molecular Switch Probe to Activate or Inhibit GIRK1 Heteromers In Silico Reveals a Novel Gating Mechanism.

G protein-gated inwardly rectifying K+ (GIRK) channels form highly active heterotetramers in the body, such as in neurons (GIRK1/GIRK2 or GIRK1/2) and heart (GIRK1/GIRK4 or GIRK1/4). Based on three-dimensional atomic resolution structures for GIRK2 homotetramers, we built heterotetrameric GIRK1/2 and GIRK1/4 models in a lipid bilayer environment. By employing a urea-based activator ML297 and its molecular switch, the inhibitor GAT1587, we captured channel gating transitions and K+ ion permeation in sub-microsecond molecular dynamics (MD) simulations. This allowed us to monitor the dynamics of the two channel gates (one transmembrane and one cytosolic) as well as their control by the required phosphatidylinositol bis 4-5-phosphate (PIP2). By comparing differences in the two trajectories, we identify three hydrophobic residues in the transmembrane domain 1 (TM1) of GIRK1, namely, F87, Y91, and W95, which form a hydrophobic wire induced by ML297 and de-induced by GAT1587 to orchestrate channel gating. This includes bending of the TM2 and alignment of a dipole of two acidic GIRK1 residues (E141 and D173) in the permeation pathway to facilitate K+ ion conduction. Moreover, the TM movements drive the movement of the Slide Helix relative to TM1 to adjust interactions of the CD-loop that controls the gating of the cytosolic gate. The simulations reveal that a key basic residue that coordinates PIP2 to stabilize the pre-open and open states of the transmembrane gate flips in the inhibited state to form a direct salt-bridge interaction with the cytosolic gate and destabilize its open state.

De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule-binding proteins with tuned interaction energies is feasible entirely from computation.

De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small-molecule-binding proteins has seen exciting recent progress; however, the ability to achieve exquisite affinity for binding small molecules while tuning specificity has not yet been demonstrated directly from computation. Here, we develop a computational procedure that results in the highest affinity binders to date with predetermined relative affinities, targeting a series of PARP1 inhibitors. Two of four designed proteins bound with affinities ranging from < 5 nM to low µM, in a predictable manner. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free-energy calculations performed directly on the designed models are in excellent agreement with the experimentally measured affinities, suggesting that the de novo design of small-molecule-binding proteins with tuned interaction energies is now feasible entirely from computation. We expect these methods to open many opportunities in biomedicine, including rapid sensor development, antidote design, and drug delivery vehicles.