NOH…O, OOH…O, and COH…O Hydrogen Bonds in Protein–Ligand Complexes: Strong and Weak Interactions in Molecular Recognition Sanjay Sarkhel and Gautam R. Desiraju * School of Chemistry, University of Hyderabad, Hyderabad, India ABSTRACT The characteristics of NOH…O, OOH…O, and COH…O hydrogen bonds are exam- ined in a group of 28 high-resolution crystal struc- tures of protein–ligand complexes from the Protein Data Bank and compared with interactions found in small-molecule crystal structures from the Cam- bridge Structural Database. It is found that both strong and weak hydrogen bonds are involved in ligand binding. Because of the prevalence of multi- furcation, the restrictive geometrical criteria set up for hydrogen bonds in small-molecule crystal struc- tures may need to be relaxed in macromolecular structures. For example, there are definite devia- tions from linearity for the hydrogen bonds in pro- tein–ligand complexes. The formation of COH…O hydrogen bonds is influenced by the activation of the C  OH atoms and by the flexibility of the side- chain atoms. In contrast to small-molecule struc- tures, anticooperative geometries are common in the macromolecular structures studied here, and there is a gradual lengthening as the extent of furcation increases. COH…O bonds formed by Gly, Phe, and Tyr residues are noteworthy. The numbers of hydrogen bond donors and acceptors agree with Lipinski’s “rule of five” that predicts drug-like prop- erties. Hydrogen bonds formed by water are also seen to be relevant in ligand binding. Ligand COH…O w interactions are abundant when com- pared to NOH…O w and OOH…O w . This suggests that ligands prefer to use their stronger hydrogen bond capabilities for use with the protein residues, leaving the weaker interactions to bind with water. In summary, the interplay between strong and weak interactions in ligand binding possibly leads to a satisfactory enthalpy– entropy balance. The implica- tions of these results to crystallographic refinement and molecular dynamics software are discussed. Proteins 2004;54:247–259. © 2003 Wiley-Liss, Inc. Key words: intermolecular interaction; enthalpy– entropy; Protein Data Bank; Cambridge Structural Database; multifurcation; water INTRODUCTION The binding properties of proteins are the essence of functional genomics. It is necessary to know where a protein is localized and when it is expressed, but to find out what it does, one needs to find out to what it binds—and more importantly, how. The specificity of biological pro- cesses suggests that the intermolecular interactions in- volved in the underlying recognition events are also spe- cific, with conserved orientation. 1–3 Hydrogen bonding is directional, and this is what makes it so important in the whole domain of biomolecular recognition. 4,5 Hydrogen bonds are instrumental not only in mediating drug- receptor binding, but they also affect physicochemical properties of a molecule, such as solubility, partitioning, distribution, and permeability, that are crucial to drug development. 6 Another compelling aspect of the hydrogen bond is its composite character. The hydrogen bond, or rather the “hydrogen bridge,” is viewed as an interaction that has covalent, electrostatic, and van der Waals charac- ter and spans an energy range from 40 kcal/mol to 0.25 kcal/mol. 7 The composite nature of the hydrogen bond means that the relative proportions of covalency, electro- statics, and van der Waals character in the XOH…A interaction vary smoothly depending on the nature of X and A. This in turn renders the interactions chemically “tunable” with the corresponding implications for func- tion. No less important in biological processes than specificity is reversibility. Weaker interactions can be made and broken more easily than stronger interactions. In this context, it is of interest to assess the relative significance of strong and weak interactions in the macromolecular recog- nition process. Is protein–ligand binding governed by conventional, that is, electrostatic, NOH…O and OOH…O hydrogen bonds, or do weaker interactions with a greater dispersive component such as COH…O also play a role? If so, to what extent are they significant? While several recent studies have concentrated on identifying or validat- ing the presence of COH…O and other weak hydrogen bonds in macromolecular structures, 8 –16 the purpose of this article is twofold: (1) to describe strong and weak hydrogen bonds in a particular category of biological structures that is of importance in drug design, namely, Grant sponsor: Department of Science and Technology, Government of India; Project number: VI-D&P/2/99/TT. *Correspondence to: Gautam R. Desiraju, School of Chemistry, University of Hyderabad, Hyderabad 500 046, India. E-mail: desiraju@uohyd.ernet.in Received 13 May 2003; Accepted 16 June 2003 PROTEINS: Structure, Function, and Bioinformatics 54:247–259 (2004) © 2003 WILEY-LISS, INC.