Fatty acid binding into the highest affinity site of human serum albumin observed in molecular dynamics simulation
Graphical abstract
Introduction
Fatty acids are structural components of cells and constitute a fundamental source for energy production when blood sugar is low. Only a tiny percentage (<0.01%) of fatty acids is free in the plasma, whereas a small fraction is bound to lipoproteins and the great majority to serum albumin [1]. This protein is the most abundant in the human blood and provides also the transport of other metabolites, drugs and ions [2], [3].
Long-chain non-esterified fatty acids attach non-covalently to albumin in seven binding sites [4]. Up to 6–7 fatty acids are bound to each albumin molecule under extreme conditions, such as fasting, intense stress or exercise, or in some disease conditions (see [5], [6], [7] and references therein). In contrast, an average of 0.1–2 molecules are present under normal physiological conditions [5]. The protein binding sites are not all equivalent: three have high affinity for fatty acids and four have lower affinity [6], [8]. High-affinity sites are named FA2, FA4 and FA5, and consist of long and narrow hydrophobic pockets in which the methylene chain of each fatty acid lies in an almost extended conformation [9], while the carboxylate head-group is hydrogen-bonded and additionally coordinated by a charged protein sidechain. In particular, the binding pocket with the highest affinity is site FA5 [8], [10], in which the fatty acid head-group forms a hydrogen bond (HB) with the Oη atom of Tyr401 and a salt bridge with the protonated amino group of Lys525.
Crystallographic structures [4], [9], [11] are available for human serum albumin complexed with saturated fatty acids ranging from capric (C10:0) to stearic acid (C18:0). Interaction with the latter is important not only for its nutritional relevance [12], but also from a molecular point of view. In fact, the relatively long chain of stearic acid is particularly suited to investigate the insertion of a fatty acid, because it can probe albumin binding sites in their full depth. Unfortunately, microscopic details concerning the dynamics of the lipid binding are currently not easily accessible to the experiment. Molecular dynamics (MD)1 represents an interesting tool to tackle this issue [13], [14] and has already been successfully employed in a few cases [15], [16], [17], [18], [19], [20].
Complete association events to fatty acid-binding proteins have been described in simulation in a small number of instances. Bello and García-Hernández performed a total of 20 unbiased MD runs to explore the binding of lauric and palmitic acid to β-lactoglobulin [18], starting with the ligand outside the protein binding site. The ligand was observed to have at least a partial penetration in approximately half of the simulations, and ended up forming a stable complex in three instances. In one case the penetration stage took approximately 1 ns, whereas in the other two cases the process was more gradual and slow. Tsfadia and coworkers reported an event of full insertion for palmitic acid [15] in a set of simulations on toad liver basic fatty acid binding protein (FABP). The estimated time for the completion of the process was on the order of 1 ns, and no uncertainty could be associated to this single occurrence.
In other studies, indirect information on the fatty acid association process was obtained ‘in reverse’ by simulating the dissociation process under non-equilibrium conditions. An extremely high temperature (1500 K) was used in an early report on intestinal FABP [21] to promote and speed up the dissociation process. More recently, a non-physical steering force was employed, either with constant direction [22] or randomly oriented [23], to favor dissociation of fatty acids from intestinal and liver FABP, respectively. Several details on structural intermediates and physical interactions involved along the association/dissociation pathway were obtained from these MD simulations, but no reliable kinetic features could be deduced.
Most of the previous results on protein-fatty acid association and dissociation involve proteins of the FABP family [24], which are relatively small (∼15 kDa) and with a simple structure (a single β-barrel), and generally bind only one fatty acid (two in the case of liver FABP). In contrast, in the present work MD simulations have been used to investigate the process of association of stearic acid into the highest-affinity binding site of albumin (66 kDa and multidomain), its major physiological carrier. By systematically varying the starting position of the ligand within the binding pocket, multiple association events were observed under equilibrium conditions. The results reveal key details of the kinetics and energetics of the association process, giving a comprehensive description of the binding pathway for the fatty acid in site FA5.
Section snippets
Molecular modeling and simulation
MD was performed by using the GROMACS simulation package [25] with the force field GROMOS 53a6 [26]. VMD was used for molecular modeling and visualization [27]. Human serum albumin was modeled on the basis of the fatty-acid loaded crystallographic structure [9], 1E7I entry of the Protein Data Bank. Ionizable amino acid residues were adapted to mimic protonation at neutral pH. The topology for stearic acid was built by combining the parameters used in GROMOS 53a6 for the carboxylate, methylene
Equilibrium dynamics of fully inserted stearic acid
An MD simulation was performed to characterize the coordination of a stearic acid molecule completely inserted into site FA5 in human serum albumin. The simulation indicates that albumin preserves its distinctive all-alpha secondary structure and heart-shaped tertiary structure, whereas the fatty acid molecule fluctuates around the starting configuration. Fig. 1 shows an ensemble of representative conformations of the fatty acid obtained during the simulation (Fig. 1a), compared with the
Discussion
The knowledge of the association process of fatty acids is fundamental to understand how transport proteins recognize and bind such ligands. In this work, the kinetic and energetic landscape of the binding reaction of stearic acid to site FA5 of human serum albumin was followed ‘backwards’. We first characterized the dynamics of the fatty acid in its unique binding position (i.e., fully inserted), and later investigated regions of the free energy funnel where the ligand is either only partly
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