Rimantadine Binds to and Inhibits the Influenza A M2 Proton Channel without Enantiomeric Specificity.

The influenza A M2 wild-type (WT) proton channel is the target of the anti-influenza drug rimantadine. Rimantadine has two enantiomers, though most investigations into drug binding and inhibition have used a racemic mixture. Solid-state NMR experiments using the full length-M2 WT have shown significant spectral differences that were interpreted to indicate tighter binding for (R)- vs (S)-rimantadine. However, it was unclear if this correlates with a functional difference in drug binding and inhibition. Using X-ray crystallography, we have determined that both (R)- and (S)-rimantadine bind to the M2 WT pore with slight differences in the hydration of each enantiomer. However, this does not result in a difference in potency or binding kinetics, as shown by similar values for kon, koff, and Kd in electrophysiological assays and for EC50 values in cellular assays. We concluded that the slight differences in hydration for the (R)- and (S)-rimantadine enantiomers are not relevant to drug binding or channel inhibition. To further explore the effect of the hydration of the M2 pore on binding affinity, the water structure was evaluated by grand canonical ensemble molecular dynamics simulations as a function of the chemical potential of the water. Initially, the two layers of ordered water molecules between the bound drug and the channel's gating His37 residues mask the drug's chirality. As the chemical potential becomes more unfavorable, the drug translocates down to the lower water layer, and the interaction becomes more sensitive to chirality. These studies suggest the feasibility of displacing the upper water layer and specifically recognizing the lower water layers in novel drugs.

X-ray Crystal Structures of the Influenza M2 Proton Channel Drug-Resistant V27A Mutant Bound to a Spiro-Adamantyl Amine Inhibitor Reveal the Mechanism of Adamantane Resistance.

The V27A mutation confers adamantane resistance on the influenza A matrix 2 (M2) proton channel and is becoming more prevalent in circulating populations of influenza A virus. We have used X-ray crystallography to determine structures of a spiro-adamantyl amine inhibitor bound to M2(22-46) V27A and also to M2(21-61) V27A in the Inwardclosed conformation. The spiro-adamantyl amine binding site is nearly identical for the two crystal structures. Compared to the M2 "wild type" (WT) with valine at position 27, we observe that the channel pore is wider at its N-terminus as a result of the V27A mutation and that this removes V27 side chain hydrophobic interactions that are important for binding of amantadine and rimantadine. The spiro-adamantyl amine inhibitor blocks proton conductance in the WT and V27A mutant channels by shifting its binding site in the pore depending on which residue is present at position 27. Additionally, in the structure of the M2(21-61) V27A construct, the C-terminus of the channel is tightly packed relative to that of the M2(22-46) construct. We observe that residues Asp44, Arg45, and Phe48 face the center of the channel pore and would be well-positioned to interact with protons exiting the M2 channel after passing through the His37 gate. A 300 ns molecular dynamics simulation of the M2(22-46) V27A-spiro-adamantyl amine complex predicts with accuracy the position of the ligands and waters inside the pore in the X-ray crystal structure of the M2(22-46) V27A complex.

XFEL structures of the influenza M2 proton channel: Room temperature water networks and insights into proton conduction.

The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.

X-ray Crystal Structure of the Influenza A M2 Proton Channel S31N Mutant in Two Conformational States: An Open and Shut Case.

The amantadine-resistant S31N mutant of the influenza A M2 proton channel has become prevalent in currently circulating viruses. Here, we have solved an X-ray crystal structure of M2(22-46) S31N that contains two distinct conformational states within its asymmetric unit. This structure reveals the mechanism of adamantane resistance in both conformational states of the M2 channel. In the Inwardopen conformation, the mutant Asn31 side chain faces the channel pore and sterically blocks the adamantane binding site. In the Inwardclosed conformation, Asn31 forms hydrogen bonds with carbonyls at the monomer-monomer interface, which twists the monomer helices and constricts the channel pore at the drug binding site. We also examine M2(19-49) WT and S31N using solution NMR spectroscopy and show that distribution of the two conformational states is dependent on both detergent choice and experimental pH.

Inhibitors of the M2 Proton Channel Engage and Disrupt Transmembrane Networks of Hydrogen-Bonded Waters.

Water-mediated interactions play key roles in drug binding. In protein sites with sparse polar functionality, a small-molecule approach is often viewed as insufficient to achieve high affinity and specificity. Here we show that small molecules can enable potent inhibition by targeting key waters. The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine. Structural studies of drug binding to the channel using X-ray crystallography have been limited because of the challenging nature of the target, with the one previously solved crystal structure limited to 3.5 Å resolution. Here we describe crystal structures of amantadine bound to M2 in the Inwardclosed conformation (2.00 Å), rimantadine bound to M2 in both the Inwardclosed (2.00 Å) and Inwardopen (2.25 Å) conformations, and a spiro-adamantyl amine inhibitor bound to M2 in the Inwardclosed conformation (2.63 Å). These X-ray crystal structures of the M2 proton channel with bound inhibitors reveal that ammonium groups bind to water-lined sites that are hypothesized to stabilize transient hydronium ions formed in the proton-conduction mechanism. Furthermore, the ammonium and adamantyl groups of the adamantyl-amine class of drugs are free to rotate in the channel, minimizing the entropic cost of binding. These drug-bound complexes provide the first high-resolution structures of drugs that interact with and disrupt networks of hydrogen-bonded waters that are widely utilized throughout nature to facilitate proton diffusion within proteins.

High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction.

The matrix 2 (M2) protein from influenza A virus is a proton channel that uses His37 as a selectivity filter. Here we report high-resolution (1.10 Å) cryogenic crystallographic structures of the transmembrane domain of M2 at low and high pH. These structures reveal that waters within the pore form hydrogen-bonded networks or "water wires" spanning 17 Å from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium via second-shell interactions involving bridging water molecules. In addition, room temperature crystallographic structures indicate that water becomes increasingly fluid with increasing temperature and decreasing pH, despite the higher electrostatic field. Complementary molecular dynamics simulations reveal a collective switch of hydrogen bond orientations that can contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle.

Transient water wires mediate selective proton transport in designed channel proteins.

Selective proton transport through proteins is essential for forming and using proton gradients in cells. Protons are conducted along hydrogen-bonded 'wires' of water molecules and polar side chains, which, somewhat surprisingly, are often interrupted by dry apolar stretches in the conduction pathways, inferred from static protein structures. Here we hypothesize that protons are conducted through such dry spots by forming transient water wires, often highly correlated with the presence of the excess protons in the water wire. To test this hypothesis, we performed molecular dynamics simulations to design transmembrane channels with stable water pockets interspersed by apolar segments capable of forming flickering water wires. The minimalist designed channels conduct protons at rates similar to viral proton channels, and they are at least 106-fold more selective for H+ over Na+. These studies inform the mechanisms of biological proton conduction and the principles for engineering proton-conductive materials.

Packing of apolar side chains enables accurate design of highly stable membrane proteins.

The features that stabilize the structures of membrane proteins remain poorly understood. Polar interactions contribute modestly, and the hydrophobic effect contributes little to the energetics of apolar side-chain packing in membranes. Disruption of steric packing can destabilize the native folds of membrane proteins, but is packing alone sufficient to drive folding in lipids? If so, then membrane proteins stabilized by this feature should be readily designed and structurally characterized-yet this has not been achieved. Through simulation of the natural protein phospholamban and redesign of variants, we define a steric packing code underlying its assembly. Synthetic membrane proteins designed using this code and stabilized entirely by apolar side chains conform to the intended fold. Although highly stable, the steric complementarity required for their folding is surprisingly stringent. Structural informatics shows that the designed packing motif recurs across the proteome, emphasizing a prominent role for precise apolar packing in membrane protein folding, stabilization, and evolution.

Crystal structure of the drug-resistant S31N influenza M2 proton channel.

The M2 protein is a small proton channel found in the influenza A virus that is necessary for viral replication. The M2 channel is the target of a class of drugs called the adamantanes, which block the channel pore and prevent the virus from replicating. In recent decades mutations have arisen in M2 that prevent the adamantanes from binding to the channel pore, with the most prevalent of these mutations being S31N. Here we report the first crystal structure of the S31N mutant crystallized using lipidic cubic phase crystallization techniques and solved to 1.59 Å resolution. The Asn31 residues point directly into the center of the channel pore and form a hydrogen-bonded network that disrupts the drug-binding site. Ordered waters in the channel pore form a continuous hydrogen bonding network from Gly34 to His37.

Detection of drug-induced conformational change of a transmembrane protein in lipid bilayers using site-directed spin labeling.

As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high-resolution structural data available for the M2 protein to design and interpret site-directed spin-labeling electron paramagnetic resonance spectroscopy experiments on drug-induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22-46 and M2TMC 23-60). M2TM peptides were spin labeled at the N-terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C-terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C-terminal amphipathic helices.