Human serum albumin possesses multiple binding sites and transports a wide range of ligands that include the anti-inflammatory drug ibuprofen. A complete map of the binding sites of ibuprofen in albumin is difficult to obtain in traditional experiments, because of the structural adaptability of this protein in accommodating small ligands. In this work, we provide a set of predictions covering the geometry, affinity of binding and protonation state for the pharmaceutically most active form (S- isomer) of ibuprofen to albumin, by using absolute binding free energy calculations in combination with classical molecular dynamics (MD) simulations and molecular docking. The most favorable binding modes correctly reproduce several experimentally identified binding locations, which include the two Sudlows drug sites (DS2 and DS1) and the fatty acid binding sites 6 and 2 (FA6 and FA2). Previously unknown details of the binding conformations were revealed for some of them, and formerly undetected binding modes were found in other protein sites. The calculated binding affinities exhibit trends which seem to agree with the available experimental data, and drastically degrade when the ligand is modeled in a protonated (neutral) state, indicating that ibuprofen associates with albumin preferentially in its charged form. These findings provide a detailed description of the binding of ibuprofen, help to explain a wide range of results reported in the literature in the last decades, and demonstrate the possibility of using simulation methods to predict ligand binding to albumin.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC=\"FIGDIR/small/068502_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (31K):\norg.highwire.dtl.DTLVardef@1b04be9org.highwire.dtl.DTLVardef@147e748org.highwire.dtl.DTLVardef@1a13803org.highwire.dtl.DTLVardef@164624f_HPS_FORMAT_FIGEXP M_FIG C_FIG FocusAlchemical free energy methods can identify favored binding modes of a ligand within a large protein with multiple binding sites\n\nHighlightsO_LIHuman serum albumin binds the anti-inflammatory drug ibuprofen in multiple sites\nC_LIO_LIAlchemical free energy calculations predicted favored binding modes of ibuprofen\nC_LIO_LIBound geometry, affinity and protonation state of the ligand were determined\nC_LIO_LISimulations identified a number of previously undetected binding sites for ibuprofen\nC_LIO_LIFree energy methods can be used to study large proteins with multiple binding sites\nC_LI
