Exploring the role of the phospholipid ligand in endothelial protein C receptor: A molecular dynamics study

Endothelial protein C receptor (EPCR) is a CD1‐like transmembrane glycoprotein with important regulatory roles in protein C (PC) pathway, enhancing PC's anticoagulant, anti‐inflammatory, and antiapoptotic activities. Similarly to homologous CD1d, EPCR binds a phospholipid [phosphatidylethanolamine (PTY)] in a groove corresponding to the antigen‐presenting site, although it is not clear if lipid exchange can occur in EPCR as in CD1d. The presence of PTY seems essential for PC γ‐carboxyglutamic acid (Gla) domain binding. However, the lipid‐free form of the EPCR has not been characterized. We have investigated the structural role of PTY on EPCR, by multiple molecular dynamics (MD) simulations of ligand bound and unbound forms of the protein. Structural changes, subsequent to ligand removal, led to identification of two stable and folded ligand‐free conformations. Compared with the bound form, unbound structures showed a narrowing of the A′ pocket and a high flexibility of the helices around it, in agreement with CD1d simulation. Thus, a lipid exchange with a mechanism similar to CD1d is proposed. In addition, unbound conformations presented a reduced interaction surface for Gla domain, confirming the role of PTY in establishing the proper EPCR conformation for the interaction with its partner protein. Single MD simulations were also obtained for 29 mutant models with predicted structural stability and impaired binding ability. Ligand affinity calculations, based on linear interaction energy method, showed that substitution‐induced conformational changes affecting helices around the A′ pocket were associated to a reduced binding affinity. Mutants responsible for this effect may represent useful reagents for experimental tests. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Molecular basis of the recognition of arachidonic acid by cytochrome P450 2E1 along major access tunnel

Cytochrome P450 2E1 is widely known for its ability to oxidize both low molecular weight xenobiotics and endogenous fatty acids (e.g., arachidonic acid (AA)). In this study, we investigated the structural features of the AA‐bound CYP2E1 complex utilizing molecular dynamics (MD) and found that the distinct binding modes for both AA and fatty acid analog are conserved. Moreover, multiple random acceleration MD simulations and steered MD simulations uncovered the most possible tunnel for fatty acids. The main attractions are derived from three key residues, His107, Ala108, and His109, whose side chains reorient to keep ligands bound via hydrogen bonds during the initial unbinding process. More importantly, based on the calculated binding free energy results, we hypothesize that the hydrogen bonds between the receptor and the ligand are the most important contributors involved in the binding affinity. Thus, it is inferred that the hydrogen bonds between these three residues and the ligand may help offer insights into the structural basis of the different ligand egress mechanisms for fatty acids and small weight compounds. Our investigation provides detailed atomistic insights into the structural features of human CYP2E1–fatty acid complex structures. Furthermore, the ligand‐binding characteristics obtained in the present study are helpful for both experimental and computational studies of CYPs and may allow future researchers to achieve desirable changes in enzymatic activities. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 53–66, 2015.     

Molecular dynamics simulation on the allosteric analysis of the c‐di‐GMP class I riboswitch induced by ligand binding

Riboswitches are RNA molecules that regulate gene expression using conformation change, affected by binding of small molecule ligands. Although a number of ligand‐bound aptamer complex structures have been solved, it is important to know ligand‐free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, we use dynamics simulations on a series of models to characterize the ligand‐free and ligand‐bound aptamer domain of the c‐di‐GMP class I (GEMM‐I) riboswitch. The results revealed that the ligand‐free aptamer has a stable state with a folded P2 and P3 helix, an unfolded P1 helix and open binding pocket. The first Mg ions binding to the aptamer is structurally favorable for the successive c‐di‐GMP binding. The P1 helix forms when c‐di‐GMP is successive bound. Three key junctions J1/2, J2/3 and J1/3 in the GEMM‐I riboswitch contributing to the formation of P1 helix have been found. The binding of the c‐di‐GMP ligand to the GEMM‐I riboswitch induces the riboswitch's regulation through the direct allosteric communication network in GEMM‐I riboswitch from the c‐di‐GMP binding sites in the J1/2 and J1/3 junctions to the P1 helix, the indirect ones from those in the J2/3 and P2 communicating to P1 helix via the J1/2 and J1/3 media.     

Exploring the conformational and binding properties of unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 through docking and molecular dynamics simulations

B‐cell lymphoma (Bcl‐2) is commonly associated with the progression and preservation of cancer and certain lymphomas; therefore, it is considered as a biological target against cancer. Nevertheless, evidence of all its structural binding sites has been hidden because of the lack of a complete Bcl‐2 model, given the presence of a flexible loop domain (FLD), which is responsible for its complex behavior. FLD region has been implicated in phosphorylation, homotrimerization, and heterodimerization associated with Bcl‐2 antiapoptotic function. In this contribution, homology modeling, molecular dynamics (MD) simulations in the microsecond (µs) time‐scale and docking calculations were combined to explore the conformational complexity of unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 systems. Conformational ensembles generated through MD simulations allowed for identifying the most populated unphosphorylated/phosphorylated monomeric conformations, which were used as starting models to obtain trimeric complexes through protein–protein docking calculations, also submitted to µs MD simulations. Principal component analysis showed that FLD represents the main contributor to total Bcl‐2 mobility, and is affected by phosphorylation and oligomerization. Subsequently, based on the most representative unphosphorylated/phosphorylated monomeric and trimeric Bcl‐2 conformations, docking studies were initiated to identify the ligand binding site of several known Bcl‐2 inhibitors to explain their influence in homo‐complex formation and phosphorylation. Docking studies showed that the different conformational states experienced by FLD, such as phosphorylation and oligomerization, play an essential role in the ability to make homo and hetero‐complexes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 393–413, 2016.     

Stabilization of the integrase‐DNA complex by Mg2+ ions and prediction of key residues for binding HIV‐1 integrase inhibitors

The HIV‐1 integrase is an attractive target for the therapeutics development against AIDS, as no host homologue of this protein has been identified. The integrase strand transfer inhibitors (INSTIs), including raltegravir, specifically target the second catalytic step of the integration process by binding to the DDE motif of the catalytic site and coordinating Mg²⁺ ions. Recent X‐ray crystallographic structures of the integrase/DNA complex from prototype foamy virus allowed to investigate the role of the different partners (integrase, DNA, Mg²⁺ ions, raltegravir) in the complex stability using molecular dynamics (MD) simulations. The presence of Mg²⁺ ions is found to be essential for the stability, whereas the simultaneous presence of raltegravir and Mg²⁺ ions has a destabilizing influence. A homology model of HIV‐1 integrase was built on the basis of the X‐ray crystallographic information, and protein marker residues for the ligand binding were detected by clustering the docking poses of known HIV‐1 integrase inhibitors on the model. Interestingly, we had already identified some of these residues to be involved in HIV‐1 resistance mutations and in the stabilization of the catalytic site during the MD simulations. Classification of protein conformations along MD simulations, as well as of ligand docking poses, was performed by using an original learning method, based on self‐organizing maps. This allows us to perform a more in‐depth investigation of the free‐energy basins populated by the complex in MD simulations on the one hand, and a straightforward classification of ligands according to their binding residues on the other hand. Proteins 2014; 82:466–478. © 2013 Wiley Periodicals, Inc.     

Absolute binding free energy calculations using molecular dynamics simulations with restraining potentials

The absolute (standard) binding free energy of eight FK506-related ligands to FKBP12 is calculated using free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent. A number of features are implemented to improve the accuracy and enhance the convergence of the calculations. First, the absolute binding free energy is decomposed into sequential steps during which the ligand-surrounding interactions as well as various biasing potentials restraining the translation, orientation, and conformation of the ligand are turned "on" and "off." Second, sampling of the ligand conformation is enforced by a restraining potential based on the root mean-square deviation relative to the bound state conformation. The effect of all the restraining potentials is rigorously unbiased, and it is shown explicitly that the final results are independent of all artificial restraints. Third, the repulsive and dispersive free energy contribution arising from the Lennard-Jones interactions of the ligand with its surrounding (protein and solvent) is calculated using the Weeks-Chandler-Andersen separation. This separation also improves convergence of the FEP/MD calculations. Fourth, to decrease the computational cost, only a small number of atoms in the vicinity of the binding site are simulated explicitly, while all the influence of the remaining atoms is incorporated implicitly using the generalized solvent boundary potential (GSBP) method. With GSBP, the size of the simulated FKBP12/ligand systems is significantly reduced, from approximately 25,000 to 2500. The computations are very efficient and the statistical error is small ( approximately 1 kcal/mol). The calculated binding free energies are generally in good agreement with available experimental data and previous calculations (within approximately 2 kcal/mol). The present results indicate that a strategy based on FEP/MD simulations of a reduced GSBP atomic model sampled with conformational, translational, and orientational restraining potentials can be computationally inexpensive and accurate.     

Distal mutation modulates the heme sliding in mouse neuroglobin investigated by molecular dynamics simulation

Neuroglobin (Ngb), a hexa‐coordinated hemoprotein primarily expressed in the brain and retina, is thought to be involved in neuroprotection and signal transduction. Ngb can reversibly bind small ligands such as O₂ and CO to the heme iron by replacing the distal histidine which is bound to the iron as the endogenous ligand. In this work, molecular dynamics (MD) simulations were performed to investigate the functionally related structural properties and dynamical characteristics in carboxy mouse neuroglobin and three distal mutants including single mutants H64V, K67T and double mutant H64V/K67T. MD simulations suggest that the heme sliding motion induced by the binding of exogenous ligand is affected by the distal mutation obviously. Accompanying changes in loop flexibility and internal cavities imply the structural rearrangement of Ngb. Moreover, the solvent accessibility of heme and some crucial residues are influenced revealing an interactive network on the distal side. The work elucidates that the key residues K67 at E10 and H64 at E7 are significant in modulating the heme sliding and hence the structural and physiological function of Ngb. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium‐bound and calcium‐free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can “clamp” the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure‐based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Peptide binding to the PDZ3 domain by conformational selection

The PDZ domains, a large family of peptide recognition proteins, bind to the C‐terminal segment of membrane ion channels and receptors thereby mediating their localization. The peptide binding process is not known in detail and seems to differ among different PDZ domains. For the third PDZ domain of the synaptic protein PSD‐95 (PDZ3), a lock‐and‐key mechanism was postulated on the basis of the almost perfect overlap of the crystal structures in the presence and absence of its peptide ligand. Here, peptide binding to PDZ3 is investigated by explicit solvent molecular dynamics (MD) simulations (for a total of 1.3 μs) and the cut‐based free energy profile method for determining free energy barriers and basins. The free energy landscape of apo PDZ3 indicates that there are multiple basins within the native state. These basins differ by the relative orientation of the α2 helix and β2 strand, the two secondary structure elements that make up the peptide binding site. Only the structure with the smallest aperture of the binding site is populated in the MD simulations of the complex whose analysis reveals that the peptide ligand binds to PDZ3 by selecting one of three conformations. Thus, the dynamical information obtained by the atomistic simulations increment the static, that is, partial, picture of the PDZ3 binding mechanism based on the X‐ray crystallography data. Importantly, the simulation results show for the first time that conformational selection is a possible mechanism of peptide binding by PDZ domains in general. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Molecular dynamics simulations of phenylimidazole inhibitor complexes of cytochrome P450 cam

Molecular dynamics simulations have been performed on three phenylimidazole inhibitor complexes ofP450 _cam, utilizing the X-ray structures and the AMBER suite of programs. Compared to their corresponding optimized X-ray structures, very similar features were observed for the 1-phenylimidazole (1-PI) and 2-phenylimidazole (2-PI) complexes during a 100 ps MD simulation. The 1-PI inhibitor binds as a Type II complex with the imidazole nitrogen as a ligand of the heme iron. Analysis of the inhibitor-enzyme interctions during the MD simulations reveals that electrostatic interactions of the imidazole with the heme and van der Waals interactions of the phenyl ring with nearby hydrophobic residues are dominant. By contrast, 2-PI binds as a Type I inhibitor in the substrate binding pocket, but not as a ligand of the iron. The interactions of this inhibitor are qualitatively different from that of the Type II 1-PI, being mainly electrostatic/H-bonding interactions with a bound water and polar residues. Although the third compound, 4-PI, in common with 1-PI, also binds as a Type II inhibitor, with one nitrogen of the imidazole as a ligand to the iron, the MD average binding orientation deviates significantly from the X-ray structure. The most important changes observed include: (1) the rotation of the imidazole ring of this inhibitor by about 90° to enhance electrostatic interactions of the imidazole NH group with the carbonyl group of LEU244, and (2) the rotation of the carbonyl group of ASP251 to form a H-bond with VAL254. An analysis of the H-bonding network surrounding this substrate in the optimized crystal structure revealed that there is no H-bonding partner either for the free polar NH group in the imidazole ring of 4-phenylimidazole or for the polar carbonyl group of the nearby ASP251 residue. The deviation of the dynamically averaged inhibitor-enzyme structure of the 4-PI complex from the optimized crystal structure can therefore be rationalized as a consequence of the optimization of the electrostatic interactions among the polar groups.     

The xenograft antigen bound to Griffonia simplicifolia lectin 1-B(4). X-ray crystal structure of the complex and molecular dynamics characterization of the binding site.

The shortage of organs for transplantation into human patients continues to be a driving force behind research into the use of tissues from non-human donors, particularly pig. The primary barrier to such xenotransplantation is the reaction between natural antibodies present in humans and Old World monkeys and the Gal alpha(1-3)Gal epitope (xenograft antigen, xenoantigen) found on the cell surfaces of the donor organ. This hyperacute immune response leads ultimately to graft rejection. Because of its high specificity for the xenograft antigen, isolectin 1-B(4) from Griffonia simplicifolia (GS-1-B(4)) has been used as an immunodiagnostic reagent. Furthermore, haptens that inhibit natural antibodies also inhibit GS-1-B(4) from binding to the xenoantigen. Here we report the first x-ray crystal structure of the xenograft antigen bound to a protein (GS-1-B(4)). The three-dimensional structure was determined from orthorhombic crystals at a resolution of 2.3 A. To probe the influence of binding on ligand properties, we report also the results of molecular dynamics (MD) simulations on this complex as well as on the free ligand. The MD simulations were performed with the AMBER force-field for proteins augmented with the GLYCAM parameters for glycosides and glycoproteins. The simulations were performed for up to 10 ns in the presence of explicit solvent. Through comparison with MD simulations performed for the free ligand, it has been determined that GS-1-B(4) recognizes the lowest energy conformation of the disaccharide. In addition, the x-ray and modeling data provide clear explanations for the reported specificities of the GS-1-B(4) lectin. It is anticipated that a further understanding of the interactions involving the xenograft antigen will help in the development of therapeutic agents for application in the prevention of hyperacute xenograft rejection.     

Molecular Modeling-Based Evaluation of hTLR10 and Identification of Potential Ligands in Toll-Like Receptor Signaling

Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogens based on distinct molecular signatures. The human (h)TLR1, 2, 6 and 10 belong to the hTLR1 subfamilies, which are localized in the extracellular regions and activated in response to diverse ligand molecules. Due to the unavailability of the hTLR10 crystal structure, the understanding of its homo and heterodimerization with hTLR2 and hTLR1 and the ligand responsible for its activation is limited. To improve our understanding of the TLR10 receptor-ligand interaction, we used homology modeling to construct a three dimensional (3D) structure of hTLR10 and refined the model through molecular dynamics (MD) simulations. We utilized the optimized structures for the molecular docking in order to identify the potential site of interactions between the homo and heterodimer (hTLR10/2 and hTLR10/1). The docked complexes were then used for interaction with ligands (Pam₃CSK₄ and PamCysPamSK₄) using MOE-Dock and ASEDock. Our docking studies have shown the binding orientations of hTLR10 heterodimer to be similar with other TLR2 family members. However, the binding orientation of hTLR10 homodimer is different from the heterodimer due to the presence of negative charged surfaces at the LRR11-14, thereby providing a specific cavity for ligand binding. Moreover, the multiple protein-ligand docking approach revealed that Pam₃CSK₄ might be the ligand for the hTLR10/2 complex and PamCysPamSK_4, a di-acylated peptide, might activate hTLR10/1 hetero and hTLR10 homodimer. Therefore, the current modeled complexes can be a useful tool for further experimental studies on TLR biology.     

Unraveling the molecular basis for ligand binding in truncated hemoglobins: The trHbO Bacillus subtilis case

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2‐over‐2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O₂ bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

How maltose influences structural changes to bind to maltose‐binding protein: Results from umbrella sampling simulation

A well‐studied periplasmic‐binding protein involved in the abstraction of maltose is maltose‐binding protein (MBP), which undergoes a ligand‐induced conformational transition from an open (ligand‐free) to a closed (ligand‐bound) state. Umbrella sampling simulations have been us to estimate the free energy of binding of maltose to MBP and to trace the potential of mean force of the unbinding event using the center‐of‐mass distance between the protein and ligand as the reaction coordinate. The free energy thus obtained compares nicely with the experimentally measured value justifying our theoretical basis. Measurement of the domain angle (N‐terminal‐domain – hinge – C‐terminal‐domain) along the unbinding pathway established the existence of three different states. Starting from a closed state, the protein shifts to an open conformation during the initial unbinding event of the ligand then resides in a semi‐open conformation and later resides predominantly in an open‐state. These transitions along the ligand unbinding pathway have been captured in greater depth using principal component analysis. It is proposed that in mixed‐model, both conformational selection and an induced‐fit mechanism combine to the ligand recognition process in MBP. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Effects of substrate binding on the dynamics of RNase A: Molecular dynamics simulations of UpA bound and native RNase A

RNase A has been extensively used as a model protein in several biophysical and biochemical studies. Using the available structural and biochemical results, RNase A-UpA interaction has been computationally modeled at an atomic level. In this study, the molecular dynamics (MD) simulations of native and UpA bound RNase A have been carried out. The gross dynamical behavior and atomic fluctuations of the free and UpA bound RNase A have been characterized. Principal component analysis is carried out to identify the important modes of collective motion and to analyze the changes brought out in these modes of RNase A upon UpA binding. The hydrogen bonds are monitored to study the atomic details of RNase A-UpA interactions and RNase A-water interactions. Based on these analysis, the stability of the free and UpA bound RNase A are discussed. © 1997 John Wiley & Sons, Inc. Biopoly 42: 505–520, 1997     

Curcumin specifically binds to the human calcium–calmodulin-dependent protein kinase IV: fluorescence and molecular dynamics simulation studies

Calcium–calmodulin-dependent protein kinase IV (CAMK4) plays significant role in the regulation of calcium-dependent gene expression, and thus, it is involved in varieties of cellular functions such as cell signaling and neuronal survival. On the other hand, curcumin, a naturally occurring yellow bioactive component of turmeric possesses wide spectrum of biological actions, and it is widely used to treat atherosclerosis, diabetes, cancer, and inflammation. It also acts as an antioxidant. Here, we studied the interaction of curcumin with human CAMK4 at pH 7.4 using molecular docking, molecular dynamics (MD) simulations, fluorescence binding, and surface plasmon resonance (SPR) methods. We performed MD simulations for both neutral and anionic forms of CAMK4-curcumin complexes for a reasonably long time (150 ns) to see the overall stability of the protein–ligand complex. Molecular docking studies revealed that the curcumin binds in the large hydrophobic cavity of kinase domain of CAMK4 through several hydrophobic and hydrogen-bonded interactions. Additionally, MD simulations studies contributed in understanding the stability of protein–ligand complex system in aqueous solution and conformational changes in the CAMK4 upon binding of curcumin. A significant increase in the fluorescence intensity at 495 nm was observed (λ_exc = 425 nm), suggesting a strong interaction of curcumin to the CAMK4. A high binding affinity (K_D = 3.7 × 10^?8 ± .03 M) of curcumin for the CAMK4 was measured by SPR further indicating curcumin as a potential ligand for the CAMK4. This study will provide insights into designing a new inspired curcumin derivatives as therapeutic agents against many life-threatening diseases.     

Probing the structural and energetic basis of kinesin-microtubule binding using computational alanine-scanning mutagenesis

Kinesin-microtubule (MT) binding plays a critical role in facilitating and regulating the motor function of kinesins. To obtain a detailed structural and energetic picture of kinesin-MT binding, we performed large-scale computational alanine-scanning mutagenesis based on long-time molecular dynamics (MD) simulations of the kinesin-MT complex in both ADP and ATP states. First, we built three all-atom kinesin-MT models: human conventional kinesin bound to ADP and mouse KIF1A bound to ADP and ATP. Then, we performed 30 ns MD simulations followed by kinesin-MT binding free energy calculations for both the wild type and mutants obtained after substitution of each charged residue of kinesin with alanine. We found that the kinesin-MT binding free energy is dominated by van der Waals interactions and further enhanced by electrostatic interactions. The calculated mutational changes in kinesin-MT binding free energy are in excellent agreement with results of an experimental alanine-scanning study with a root-mean-square error of ~0.32 kcal/mol [Woehlke, G., et al. (1997) Cell 90, 207-216]. We identified a set of important charged residues involved in the tuning of kinesin-MT binding, which are clustered on several secondary structural elements of kinesin (including well-studied loops L7, L8, L11, and L12, helices α4, α5, and α6, and less-explored loop L2). In particular, we found several key residues that make different contributions to kinesin-MT binding in ADP and ATP states. The mutations of these residues are predicted to fine-tune the motility of kinesin by modulating the conformational transition between the ADP state and the ATP state of kinesin.     

Exploration of human serum albumin binding sites by docking and molecular dynamics flexible ligand–protein interactions

Five‐nanosecond molecular dynamics (MD) simulations were performed on human serum albumin (HSA) to study the conformational features of its primary ligand binding sites (I and II). Additionally, 11 HSA snapshots were extracted every 0.5 ns to explore the binding affinity (K_d) of 94 known HSA binding drugs using a blind docking procedure. MD simulations indicate that there is considerable flexibility for the protein, including the known sites I and II. Movements at HSA sites I and II were evidenced by structural analyses and docking simulations. The latter enabled the study and analysis of the HSA–ligand interactions of warfarin and ketoprofen (ligands binding to sites I and II, respectively) in greater detail. Our results indicate that the free energy values by docking (K_d observed) depend upon the conformations of both HSA and the ligand. The 94 HSA–ligand binding K_d values, obtained by the docking procedure, were subjected to a quantitative structure‐activity relationship (QSAR) study by multiple regression analysis. The best correlation between the observed and QSAR theoretical (K_d predicted) data was displayed at 2.5 ns. This study provides evidence that HSA binding sites I and II interact specifically with a variety of compounds through conformational adjustments of the protein structure in conjunction with ligand conformational adaptation to these sites. These results serve to explain the high ligand‐promiscuity of HSA. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 161–170, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com