proteins STRUCTURE O FUNCTION O BIOINFORMATICS The role of Li 1 , Na 1 , and K 1 in the ligand binding inside the human acetylcholinesterase gorge Gabriele Petraglio, 1 Manuela Bartolini, 2 Davide Branduardi, 1 Vincenza Andrisano, 2 Maurizio Recanatini, 2 Francesco Luigi Gervasio, 1 * Andrea Cavalli, 2 * and Michele Parrinello 1 1 Computational Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, USI Campus, CH-6900 Lugano, Switzerland 2 Department of Pharmaceutical Sciences, University of Bologna, I-40126 Bologna, Italy INTRODUCTION Acetylcholinesterase (AChE, EC 3.1.1.7) is a fast serine hydrolase enzyme that plays a key role in terminating the nervous signal, by hydrolyzing the neurotransmitter acetylcholine (ACh), 1 and, for this reason, its inhibition is one of the most exploited pharmacological strategies for the reinforcement of cholinergic transmission. In the last 2 decades, AChE has been the focus of intense pharmaceutical research, culminating in the development of a number of drugs currently in clinical use for the symptomatic treatment of Alzhei- mer’s disease. 2 ACh bears a positive charge that interacts with W86 by means of a cation-p interaction leading to the correct positioning of the substrate into the catalytic site of the enzyme. It has been shown that aromatic and negatively charged resi- dues on the external surface of the enzyme play an important role in capturing and funneling the substrate toward the catalytic site. 3–7 Given these electrostatic features one might expect the cations in solution to be subject to a strong attraction, possibly influencing the ligand binding at the enzyme gorge and the catalytic activity. Indeed, early experiments have shown that, at low ionic strength, monovalent and divalent metal ions enhanced the AChE enzymatic activity. 8 More recently, in a molecular dynamics (MD) investigation of the interaction of tetramethylamonium (TMA) with the AChE gorge, 6 we noticed that Na 1 could enter the active site. This prompted us to perform a combined computa- tional and experimental study to investigate the effects of alkali cations (Li 1 , Na 1 , and K 1 ) in the ligand binding at the enzyme gorge. Notably, Li 1 , Na 1 , and K 1 represent the counterions of the most commonly used phosphate buf- fers. We carried out metadynamics simulations on the human isoform (hAChE) of the enzyme 9 to estimate the free energy surface (FES) of the TMA penetra- tion in the presence of the three selected cations. It has been shown that TMA is an appropriate probe for studying such dynamical processes, as it carries a formal positive charge and resembles the bulky part of the natural substrate. 6,10 In parallel, experimental measures of the catalytic efficiency of recombinant Grant sponsor: FIRB. *Correspondence to: Francesco Luigi Gervasio, Computational Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, USI Campus, Via Giuseppe Buffi 13, CH-6900 Lugano, Switzerland. E-mail: fgervasi@phys.chem.ethz.ch or Andrea Cavalli, Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy. E-mail: andrea.cavalli@unibo.it Received 7 December 2006; Accepted 29 March 2007 Published online 29 August 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21560 ABSTRACT Alkali cations can affect the catalytic efficiency of enzymes. This is particu- larly true when dealing with enzymes whose substrate bears a formal posi- tive charge. Computational and bio- chemical approaches have been com- bined to shed light on the atomic aspects of the role of Li 1 , Na 1 , and K 1 on human acetylcholinesterase (hAChE) ligand binding. In this respect, molecular dynamics simula- tions and our recently developed metadynamics method were applied to study the entrance of the three cat- ions in the gorge of hAChE, and their effect on the dynamical motion of a ligand (tetramethylammonium) from the bulk of the solvent into the deep narrow enzyme gorge. Furthermore, in order to support the theoretical results, K M and k cat for the acetylcho- line hydrolysis in the presence of the three cations were evaluated by using an approach based on the Ellman’s method. The combination of compu- tational and biochemical experiments clearly showed that Li 1 , Na 1 , and K 1 may influence the ligand binding at the hAChE gorge. Proteins 2008; 70:779–785. V V C 2007 Wiley-Liss, Inc. Key words: molecular dynamics; meta- dynamics; inhibition assays; Alzheimer’s disease, cation-p interactions; ligand penetration; alkali cations; catalytic ef- ficiency; enzymatic catalysis. V V C 2007 WILEY-LISS, INC. PROTEINS 779