Journal of Molecular Graphics and Modelling 22 (2004) 335–348 Computational analysis of ligand binding dynamics at the intermolecular hot spots with the aid of simulated tempering and binding free energy calculations Gennady M. Verkhivker ∗ Pfizer Global Research and Development, La Jolla Laboratories, 10777 Science Center Drive, San Diego, CA 92121-1111, USA Abstract Equilibrium binding dynamics is studied for a panel of benzimidazole-containing compounds at the remodeled interface between human growth hormone (hGH) and the extracellular domain of its receptor (hGHbp), engineered by mutating to glycine hot spot residues T175 from the hormone and W104 from the receptor. Binding energetics is predicted in a good agreement with the experimental data for a panel of these small molecules that complement the engineered defect and restore the binding affinity of the wild-type hGH–hGHbp complex. The results of simulated tempering ligand dynamics at the protein–protein interface reveals a diversity of ligand binding modes that is consistent with the structural orientation of the benzimidazole ring which closely mimics the position of the mutated W104 hot spot residue in the wild-type hGH–hGHbp complex. This structural positioning of the benzimidazole core motif is shown to be a critical feature of the low-energy ligand conformations binding in the engineered cavity. The binding free energy analysis provides a plausible rationale behind the experimental dissociation constants and suggests a key role of ligand–protein van der Waals interactions in restoring binding affinity. © 2004 Elsevier Inc. All rights reserved. Keywords: Computational analysis; Human growth hormone; Ligand binding dynamics 1. Introduction Understanding mechanisms and fundamental biophysical principles of molecular ecognition continues to present a fundamental experimental and theoretical challenge [1–6]. Alanine scanning mutagenesis of protein–protein interfacial residues, combined with structural and thermodynamic stud- ies, have enabled discovery of energetically important hot spot regions at the intermolecular interfaces that are criti- cal in determining binding affinity, i.e. alanine mutation of a hot spot residue in the binding site results in a pronounced drop in binding affinity of the complex [7]. A comprehen- sive analysis of protein–protein interfaces [8,9] and a sur- vey of hot spots compiled from various protein binding sites [10] have demonstrated a diversity of interaction patterns and a lack of general rules for hydrophobicity, polarity, or shape, that can be used to unambiguously predict hot spots at the intermolecular interfaces. A recent analysis of con- served residues in 11 clustered interface families comprising a total of 97 crystal structures has shown that the compo- sition of hot spots is typically enriched by certain residues, such as Trp, Tyr, Arg, His, Gln, Asn, and Pro and can be sur- ∗ Tel.: +1-858-622-3008; fax: +1-858-678-8244. E-mail address: gennady.verkhivker@pfizer.com (G.M. Verkhivker). rounded by a shell of less important residues [10–12]. The discovery of hot spots appears to be broadly relevant in a variety of protein–protein recognition events where, despite a typically large size of intermolecular interface, binding affinity and specificity may be determined by a functional epitope consisting of only a small fraction of the interfa- cial residues [7,10]. An overlap between flexible consensus binding sites and energetically critical hot spots, discovered through a combination of structural and mutagenesis stud- ies, have triggered experimental and computational studies aiming to understand the nature of structural flexibility and functional diversity in molecular recognition [7–13]. Molecular recognition between proteins and flexible tar- get molecules, including other proteins and small molecules is often accompanied by a considerable flexibility of the protein binding sites and structural rearrangements upon binding between the associated partners [14–18]. Protein dynamics at the intermolecular interfaces can have a pro- found effect in determining binding thermodynamics, ki- netics and consequently in modulating binding affinity and specificity of molecular recognition [19–22]. Protein bind- ing interfaces can be not only structurally flexible, but also functionally adaptive, with a diverse repertoire of protein systems capable of binding with high affinity to ligands, different from their natural binding partners in composition, 1093-3263/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jmgm.2003.12.001