Fatty acid binding into the highest affinity site of human serum albumin observed in molecular dynamics simulation

Multiple molecular dynamics simulations were performed to investigate the association of stearic acidinto the highest affinity binding site of human serum albumin.All binding events ended with a rapid (<10 ps) lock-in of the fatty acid due to formation of a hydrogenbond with Tyr401. The kinetics and energetics of the penetration process both depended linearly on thepositional shift of the fatty acid, with an average insertion time and free energy reduction of, respectively,32 ± 20 ps and 0.70 ± 0.15 kcal/mol per methylene group absorbed. Binding events of longer duration(tbind > 1 ns) were characterized by a slow exploration of the pocket entry and, frequently, of a nearby proteincrevice corresponding to a metastable state along the route to the binding site.Taken all together, these findings reconstruct the following pathway for the binding process of stearicacid: (i) contact with the protein surface, possibly facilitated by the presence of an intermediate location,(ii) probing of the site entry, (iii) insertion into the protein, and (iv) lock-in at the final position. This generaldescription may also apply to other long-chain fatty acids binding into any of the high-affinity sites ofalbumin, or to specific sites of other lipid-binding proteins.