With Proton Channel Structure, Flu Fighters Record Hit Against Infection

After more than a decade of seemingly futile efforts, researchers now have two scenarios to explain how the adamantane family of antiviral agents blocks the action of a tiny surface pore that plays a big part in the infection and spread of the common flu virus.

Detailed snapshots of the virus pore, an ion channel known as M2, show the drug stuck to the side, jamming the gating mechanism shut. Another set of molecular pictures reveals the drug directly plugging the channel, blocking the ion flow.

These opposite mechanisms of drug inhibition come from two groups in side-by-side papers that otherwise generally agree on the overall structure of M2. The studies are published in the Jan. 31 Nature by independent groups from HMS and the University of Pennsylvania.

The structures are the first such detailed views of a proton channel. The contrasting hypotheses of drug action arise from the complexity of the biological system and should not overshadow the main advance of the papers, other experts said.

“Both of these papers are major accomplishments,” said Christopher Miller, a Howard Hughes investigator at Brandeis University who wrote an accompanying perspective on the papers. “These are tough proteins to work with.”

The importance of knowing more about M2 in particular arises from its crucial role in influenza A infections. In people, the virus sneaks in and out of cells in the endosome bubbles that normally carry other cargo, such as iron. The acidic endosome sends protons through the M2 channel, causing the virus to release its genetic material into the host cell. After it replicates, the virus employs M2 in the Golgi membrane in the reverse direction to shunt out protons long enough for its defining hemagglutinin protein to fold properly.

Interest in M2 grew in the 1980s when researchers discovered it was the target of the flu-fighting staple amantadine. It took another decade to learn that M2 was a proton channel. Changes to amino acids in the M2 protein appear responsible for most of the widespread global drug resistance in common influenza A strains.

Three years ago, the U.S. Centers for Disease Control and Prevention (CDC) declared amantadine and closely related rimantadine virtually useless for now because of the current viral resistance to the drugs. As of early February, 99 percent of influenza A (H3N2) viruses and 8.3 percent of influenza A (H1N1) viruses were resistant to the adamantanes, according to the CDC. A single class of antiviral agents remains, including oseltamivir (Tamiflu). (Vaccination remains the first and strongest line of defense against the flu.)

“If we can understand how the drug blocks the channel and how mutations evade the effect of the drug, we can come up with better approaches to block it,” said James Chou, HMS assistant professor of biological chemistry and molecular pharmacology and co-author of one of the papers.

The new structural details of M2, how it opens and closes, and the possible mechanisms of inhibition come from different techniques for analyzing different truncations of the full-length protein, which is nearly 100 amino acids long.

In Chou’s lab, postdoctoral fellow Jason Schnell used nuclear magnetic resonance (NMR), which measures distances and angles between atoms in free-floating molecules, allowing researchers to calculate each statistical ensemble by repeated computer calculations that converged on a common structure. Schnell and Chou’s structure shows the closed channel within the membrane and a tail section anchoring it inside the virus. It is one of the few membrane-embedded proteins solved by NMR.

“It is a big deal in the NMR field,” Chou said. The structure demonstrates the value of NMR for small to medium-sized ion channels, especially those that resist crystallization.

Researchers in the lab of William DeGrado at the UPenn School of Medicine approached the problem with X-ray crystallography, a technique that images molecules frozen into a repeating lattice and computationally reorders the duplicated X-ray diffraction patterns into a cohesive whole. This paper reports the structure of the shorter transmembrane section.

The basic results from both groups are consistent with predictions and data from experiments by other researchers using the full-length protein. Four molecular columns make a tunnel for protons. The crystal structure shows a “tepee” configuration, with a wider interior base. In the NMR structure, the longer tail section secures the M2 transmembrane pore into a parallel bundle. At the interior base, a four-door tryptophan gate blocks the channel, the NMR structure shows, locked by four aspartate molecules. In the channel, the protons charge the histidine pH sensors with so much positive energy, the histidines repel one another, releasing the gate. The crystal structure captures a channel in the act of opening or closing like a camera shutter, with a bent helix directing the action, said co-first author Amanda Stouffer.

The different ideas about how the drug works arise from interpretations of where the drugs stuck to each structure. DeGrado’s group reports the drug inside the channel, plugging the pore. Schnell and Chou were surprised to see the drug lodged outside the channel in a greasy pocket on top of the gate-locking residues. Chou and DeGrado, who have spent long hours on the phone puzzling over the data and their interpretations, can each make persuasive and biologically plausible cases.

Most important to Schnell and Chou, the NMR structure makes a definitive identification of the drug. The crystal structure, on the other hand, sees a molecule matching the size and shape of the drug plugging the channel, which their experiments and calculations show is likely to be amantadine.

“Neither [mechanism] is unprecedented,” said Miller, who pointed out that pain-numbing local anesthetics plug a sodium channel in nerves, and painful scorpion venom corks a potassium channel. On the other hand, some calcium channel inhibitors lower blood pressure by stabilizing the closed gate.

There are also plenty of cases of perfectly good drug binding sites that have no pharmacological significance, said Robert Lamb, a Howard Hughes investigator at Northwestern University, who first discovered the M2 protein and later proved it was a proton channel. Referring to the question of which apparent blocking mechanism seems more likely, Lamb said, at this stage, there are “no definitive experiments to sort this out.”

All the researchers cited the need for functional studies to measure the ion channel function under many different conditions. In the meantime, Miller said, “One of the fun parts of doing science in this field is that you are always navigating ambiguities that arise from complexity.”