Published: June 28, 2011 r2011 American Chemical Society 10817 dx.doi.org/10.1021/ja1114198 | J. Am. Chem. Soc. 2011, 133, 10817–10825 ARTICLE pubs.acs.org/JACS Free Energy Calculations on the Two Drug Binding Sites in the M2 Proton Channel Ruo-Xu Gu, † Limin Angela Liu,* ,# Dong-Qing Wei,* ,† Jian-Guo Du, ‡ Lei Liu, ‡ and Hong Liu ‡ † State Key Laboratory of Microbial Metabolism, Luc Montagnier Biomedical Research Institute, and College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai Minhang District, China 200240 # Fred Hutchinson Cancer Research Center, Seattle WA, 98109, United States ‡ Institute of Earthquake Science, China Earthquake Administration, Beijing, China 100036 b S Supporting Information 1. INTRODUCTION Extensive experimental and computational studies have been carried out to elucidate the structure and function of the influenza A M2 proton channel 1À23 (briefly reviewed in the Supporting Information and summarized in Table 1) that is critical for the viral life cycle. Two adamantane-based antiviral drugs, amantadine and rimantadine (Figure S1C, Supporting Information), which inhibit the M2 channel, have been approved for treating influenza A viral infections. 1,2,24 However, the virus has quickly obtained drug resistance 8,9,25 in a number of naturally occurring M2 mutant strains due to the fast mutation rate of the M2 protein. Therefore, it is essential to understand how these drugs bind to the M2 channel and inhibit its proton conduction as well as how mutations affect drug binding so that better drugs may be found or designed. Two alternative binding sites of amantadine and rimantadine in the M2 channel have been reported recently, with one amanta- dine molecule bound in the channel pore (pore binding or P- binding) of a G34A M2 mutant at an environmental pH of 5.3 by crystallography 26 (PDB ID 3C9J) and with four mole- cules of rimantadine bound at the C-terminal surface of the transmembrane domain of the M2 channel (surface binding or S-binding) at pH 7.5 by solution NMR (sNMR) 27 (PDB ID 2RLF). Both models have received support from experimental and computational studies (reviewed in the Supporting Infor- mation), with the P-binding site being more widely accepted in the literature. These two 3D structures demonstrated two different drug binding conformations, suggested two possible inhibition mechanisms, and provided two alternative rationales for drug resistance in the S31N mutant. 28 In the former pore binding model, the drug molecule occludes the pore and prevents protons from conducting, thus inhibiting normal channel function. 26 Mutation S31N was believed to cause the pore size to decrease so that the drug molecule no longer binds in the pore. 13 In the latter surface binding model, the drug molecules stabilize the closed conformation of the channel and may inhibit proton transfer by an allosteric mecha- nism proposed by Schnell and Chou. 27 In the S31N mutant, the S-binding sites are allosterically perturbed so that drug molecules no longer bind effectively, thus leading to drug resistance. 27 Since the discovery of these two alternative drug binding sites, a series of experimental and computational studies have been carried out to investigate and compare these sites. 13,16À23,29À33 Received: December 19, 2010 ABSTRACT: Two alternative binding sites of adamantane-type drugs in the influenza A M2 channel have been suggested, one with the drug binding inside the channel pore and the other with four drug molecule S-binding to the C-terminal surface of the transmembrane domain. Recent computational and experimental studies have suggested that the pore binding site is more energetically favorable but the external surface binding site may also exist. Nonetheless, which drug binding site leads to channel inhibition in vivo and how drug-resistant mutations affect these sites are not completely understood. We applied molecular dynamics simulations and potential of mean force calculations to examine the structures and the free energies associated with these putative drug binding sites in an M2Àlipid bilayer system. We found that, at biological pH (∼7.4), the pore binding site is more thermodynamically favorable than the surface binding site by ∼7 kcal/mol and, hence, would lead to more stable drug binding and channel inhibition. This result is in excellent agreement with several recent studies. More importantly, a novel finding of ours is that binding to the channel pore requires overcoming a much higher energy barrier of ∼10 kcal/ mol than binding to the C-terminal channel surface, indicating that the latter site is more kinetically favorable. Our study is the first computational work that provides both kinetic and thermodynamic energy information on these drug binding sites. Our results provide a theoretical framework to interpret and reconcile existing and often conflicting results regarding these two binding sites, thus helping to expand our understanding of M2Àdrug binding, and may help guide the design and screening of novel drugs to combat the virus.