Absolute Binding Free Energy Calculations of Sparsomycin Analogs to the Bacterial Ribosome Xiaoxia Ge † and Benoı ˆt Roux* ,‡,§ Department of Physiology and Biophysics, Weill Medical College of Cornell UniVersity, New York, New York 10065, Department of Biochemistry and Molecular Biology, The UniVersity of Chicago, 929 East 57th Street, Chicago, Illinois 60637, and Biosciences DiVision, Argonne National Laboratory, 9700 South Cass AVenue, Argonne, Illinois 60439 ReceiVed: January 20, 2010; ReVised Manuscript ReceiVed: June 1, 2010 The interactions of the 50S subunit of bacterial ribosome with antibiotic sparsomycin (SPS) and five analogs (AN) are investigated through the calculation of the standard (absolute) binding free energy and the characterization of conformational dynamics. The standard binding free energies of the complexes are computed using free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent. Restraining potentials are applied and then released during the simulation to efficiently sample the changes in translational, orientational, and conformational freedom of the ligand and receptor upon binding. The biasing effects of the restraining potentials are rigorously removed. The loss of conformational freedom of the ligand upon binding is determined by introducing a potential of mean force (PMF) as a function of the root-mean-square deviation (rmsd) of the ligand relative to its conformation in the bound state. To reduce the size of the simulated system, the binding pocket of the ribosome is simulated in the framework of the generalized solvent boundary potential (GSBP). The number of solvent molecules in the buried binding site is treated via grand canonical Monte Carlo (GCMC) during the FEP/MD simulations. The correlation coefficient between the calculated and measured binding free energies is 0.96, and the experimentally observed ranking order for the binding affinities of the six ligands is reproduced. However, while the calculated affinities of the strong binders agree well with the experimental values, those for the weak binders are underestimated. Introduction The ribosome is the largest and most complex enzyme in nature. Ribosomes synthesize proteins in every cell of all living organisms. These complicated molecular machines comprise two nonequivalent subunits composed of RNA strands and more than 50 different proteins with a total mass of over 2.5 MDa. The 30S small subunit functions mainly to decode the genetic information in mRNA and control the translation fidelity. 1,2 The 50S large subunit performs the main ribosomal catalytic function in the peptidyl-transferase center (PTC) and provides the protein exit tunnel. The tRNA substrates join the two subunits at each of their three binding sites, A (aminoacyl), P (peptidyl), and E (exit), and functionally decode the genetic information and carry amino acids to be incorporated in the nascent protein. 3,4 Nearly half of known antibiotic therapeutics target the ribosome. Antibiotics inhibit protein synthesis by interfering with tRNA binding in either of the ribosomal subunits. 5,6 Among many ribosome-targeting drugs, sparsomycin (SPS) has been used as a cancer drug due to its inhibition activity at peptide bond formation by binding to the PTC of large ribosomal subunit in both prokaryotic and eukaryotic cells. 7,8 High-resolution crystal structures of ribosome-antibiotic complexes published in recent years have revolutionized our understanding of the mechanism of ribosomal function and inhibition. Significant efforts have been made in trying to derivatize existing drugs to improve the interactions at their binding site, which is illustrated by the macrolide drugs and ketolide derivatives. 9-11 The crystal structures 12-15 of ribosome- SPS complex capture the binding mode of SPS in the presence of P-site tRNA and support to some extent the hypothesis suggested from the earlier biochemical and kinetic studies. 16-20 Despite the extensive experimental investigations, the energetic driving forces for the ribosome-antibiotic interaction are still not clear. Computations based on atomic models can help shed some light on these issues. Alchemical free energy perturbation molecular dynamics (FEP/MD) simulations, in particular, is a powerful approach to study the ligand-macromolecule associa- tion processes at the atomic level 21-26 (for recent reviews, see refs 27 and 28). However, calculation of the absolute binding free energy of antibiotics to the ribosome encounters extreme challenges due to the large size of the ribosomal target, hydration of the buried binding pocket, conformational flexibility of the ligands, and the interaction with counterions. These factors also increase the difficulties to accurately predict the binding affinity of antibiotics to the ribosome with scoring functions in traditional docking approaches. 29-32 For these reasons, few computational studies have been reported to date on investiga- tion of small-molecule binding to the ribosome, 33-35 especially for the large subunit as a target. 36 The present study is aimed at clarifying the microscopic interactions responsible for the binding of SPS analogs to the ribosome. A set of SPS derivatives (Figure 1), created by chemical modifications, is used as probes to study the mecha- nism of ribosome-antibiotic interaction based on the atomic resolution structure of ribosome-SPS complex. The standard binding free energies are calculated using FEP/MD simulations * Corresponding author. E-mail: roux@uchicago.edu. † Weill Medical College of Cornell University. ‡ The University of Chicago. § Argonne National Laboratory. J. Phys. Chem. B 2010, 114, 9525–9539 9525 10.1021/jp100579y  2010 American Chemical Society Published on Web 07/07/2010