Hydration of a Hydrophobic Cavity and Its Functional Role: A Simulation Study of Human Interleukin-1b Sandeep Somani, Choon-Peng Chng, and Chandra S. Verma * Biomolecular Modeling & Design Group, Bioinformatics Institute, Singapore 138671 ABSTRACT Molecular dynamics simulations reveal that the hydrophobic cavity in human cyto- kine Interleukin-1b is hydrated and can dynami- cally accommodate between one and four water molecules. These waters have residence times > 500 ps and can give rise to detectable NOEs, in agreement with NMR observations of Ernst et al. (Science 1995; 267:1813–1817). The waters also dis- play high positional disorder within the cavity, which explains why they have not been resolved crystallographically. The average distribution of water molecules over time within the cavity matches well the low resolution electron density extracted by Yu et al. (Proc Natl Acad Sci 1999; 96:103–108). The water molecules hydrate the hydrophobic cavity preferentially as complex clus- ters. These clusters result from a combination of hydrogen bonds between the waters and stabiliz- ing interactions between the waters and aromatic rings forming the cavity. Free energy estimates suggest that it takes 4-waters to hydrate the cavity in a thermodynamically stable manner leading to a gain in free energy of transfer from bulk of 3.6 kcal/mol. This arises from the existence of the water clusters in multiple hydrogen bonded states. In addition, the waters are found to migrate either individually or as clusters out of the cavity through several pathways. The upper limit for one-dimensional diffusion of the waters within the protein matrix is 4 A ˚ /ps (relative to 6 A ˚ /ps for bulk). Simulations reveal pathways in addition to those identified crystallographically, with motions controlled by the rotations of sidechains. We find that only when the hydrophobic cavity is hy- drated, do correlated motions couple distant sites with the sites that make contact with the receptor and this data partly offers an explanation of experimental mutagenesis data. Simulations, to- gether with recent observations based on muta- genesis by Heidary et al. (J Mol Biol 2005; 353:1187–1198) that hydrogen bond networks cou- ple motions across long distances in interleukin-1 b, lead us to hypothesize that the hydration of the cavity (conserved across mammals) can thermody- namically enhance hydrogen bond networks to enable coupling across long distances by acting as a plug and this in turn enables a kinetic control of the rate of transmission of signals. Proteins 2007;67:868–885. V V C 2007 Wiley-Liss, Inc. Key words: interleukin-1b; molecular dynamics; hydrophobic cavity; water clusters; exchange pathway; correlated motions INTRODUCTION Water molecules buried in internal cavities of proteins have been known both for their functional and structural roles. 1–7 However, importance has only been ascribed to waters that have been observed structurally, that is, by crystallography and/or NMR; these have largely been water molecules found in polar cavities. Such waters are relatively immobile because they are usually localized by forming direct hydrogen bonds to cavity atoms 8,9 and/or indirectly with other buried water molecules. Crystallo- graphically, they are largely characterized by thermal parameters or B-values smaller than 80 A ˚ 2 (which trans- lates into mean squared fluctuations (MSF) smaller than 1A ˚ 2 ), the upper limit for resolving electron densities. However, proteins are also often shown to contain seem- ingly empty cavities that are lined with hydrophobic res- idues and yet are large enough to contain water mole- cules. But because such water molecules are not local- ized by hydrogen bonds to protein atoms, their detection by crystallography has been difficult if not impossible. Indeed, until recently, hydrophobic cavities were gener- ally believed to be empty primarily because of the belief that the absence of crystallographic electron density was evidence for an empty cavity 10 ; in fact if such hydration exists, it has invariably been associated with events The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/0887-3585/suppmat Sandeep Somani and Choon-Peng Chng contributed equally to this work. Sandeep Somani’s current address is Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850. Choon-Peng Chng’s present address is Laboratory of Molecular Design, Institute of Molecular and Cellular Biosciences, the Univer- sity of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. *Correspondence to: Chandra S. Verma, Bioinformatics Institute, 30 Biopolis Street, no. 07-01 Matrix, Singapore 138671. E-mail: chandra@bii.a-star.edu.sg Received 29 December 2005; Revised 10 October 2006; Accepted 23 October 2006 Published online 22 March 2007 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/prot.21320 V V C 2007 WILEY-LISS, INC. PROTEINS: Structure, Function, and Bioinformatics 67:868–885 (2007)