Binding Site Flexibility Research Articles

Dynamics of Neurotoxin II Provides Adaptivity of its Functional Sites

The details of protein internal motions could be of crucial importance to understanding its function. Such a details can't be directly obtained by conventional NMR methods: the set of NMR structures and relaxation order parameters do not provide motional models. One of the viable approaches is the analysis of molecular dynamics trajectories verified by NMR structural and relaxation data sets. The technique was implemented to the peripheral protein Neurotoxin II (NTII). High-quality experimental structures of 13С/15N labeled NTII were used as starting points for a set of MD trajectories covering a total of 7 microseconds. 3JHNHA, 3JHAHB and 3JHBHG coupling constants together with backbone and sidechain 15N-/13C-relaxation rates, heteronuclear NOEs and chemical shifts were back-calculated from MD trajectories and found to be in a good agreement with experimental data. The analysis of MD trajectories revealed a number of highly flexible protein regions with locally correlated motions in the nanosecond time scale. Two of the regions, as was shown previously, provide NTII binding to the environment of the Nicotinic Acetylcholine Receptor (nAChR) and to the receptor itself. Flexibility of protein binding sites could lead to their adaptivity favoring binding to target molecules. In the case of NTII two flexible binding sites of different targeting (membrane binding site and nAChR inhibition site) act successively. High flexibility of the binding site of NTII provides its adaptivity to the specific interaction with the lipid head groups of phosphatidylserine in the environment of nAChR enabling the membrane catalysis mechanism for NTII/nAChR interaction. Then high flexibility and, accordingly, adaptivity of the nAChR inhibition site of NTII plays its role in the final step of the membrane catalysis - high affinity binding of NTII to nAChR.

Computational Simulations Reveal How Calmodulin Methionine Oxidation Triggers Large-Scale Changes in Structural Dynamics

We have examined the structural consequences of methionine (Met) oxidation in the calcium-sensing muscle regulatory protein calmodulin (CaM) using molecular dynamics simulations. Protein oxidation by reactive oxygen species (ROS), and subsequent reduction by the antioxidant enzyme methionine sulfoxide reductase, has emerged as a crucial cell regulatory mechanism. In the context of oxidative stress, protein oxidation is implicated in disease progression and biological aging. Our goal is to bridge our understanding of muscle dysfunction and protein oxidation with atomic-level insights into site-specific methionine oxidation and calmodulin structural dynamics.We have carried out multiple 50 ns molecular dynamics simulations of explicitly solvated calmodulin, starting from the calcium-bound (1cll) and apo (1cfd) structures. Simulations were performed using NAMD (University of Illinois) and the CHARMM27 force-field. Simulations suggest that Met oxidation alters the flexibility of calcium binding sites, the conformation of the linker helix connecting the lobes, and the relative orientation of the lobes. This work is a component of a larger study in which spectroscopic distance measurements and NMR experiments have been used to resolve the structural impact of site-specific CaM Met oxidation. We expect that our in silico results will bring atomic-level insight to spectroscopic measurements, and will be integral to creating a more complete model for oxidation-induced changes in CaM structural dynamics.This work is supported by a University of Wisconsin-La Crosse Faculty Research Grant to JC Klein, NIH grants to DD Thomas (R37AG026160), and the Minnesota Supercomputing Institute.

Evaluation and Structural Basis for the Inhibition of Tankyrases by PARP Inhibitors.

Tankyrases, an enzyme subfamily of human poly(ADP-ribosyl)polymerases, are potential drug targets especially against cancer. We have evaluated inhibition of tankyrases by known PARP inhibitors and report five cocrystal structures of the most potent compounds in complex with human tankyrase 2. The inhibitors include the small general PARP inhibitors Phenanthridinone, PJ-34, and TIQ-A as well as the more advanced inhibitors EB-47 and rucaparib. The compounds anchor to the nicotinamide subsite of tankyrase 2. Crystal structures reveal flexibility of the ligand binding site with implications for drug development against tankyrases and other ADP-ribosyltransferases. EB-47 mimics the substrate NAD(+) and extends from the nicotinamide to the adenosine subsite. The clinical ARTD1 inhibitor candidate rucaparib was the most potent tankyrase inhibitor identified (24 and 14 nM for tankyrases), which indicates that inhibition of tankyrases would affect the cellular responses of this compound.

Does bromodomain flexibility influence histone recognition?

Bromodomains are protein modules that selectively recognize histones by binding to acetylated lysines. Here, we have carried out multiple molecular dynamics simulations of 20 human bromodomains to investigate the flexibility of their binding site. Some bromodomains show alternative side chain orientations of three evolutionarily conserved residues: the Asn involved in acetyl-lysine binding and two conserved aromatic residues. Furthermore, for the BAZ2B and CREBBP bromodomains we observe occlusion of the binding site which is coupled to the displacement of the two aromatic residues. In contrast to available structures, the simulations reveal large variability of the binding site accessibility. The simulations suggest that the flexibility of the bromodomain binding site and presence of self-occluded metastable states influence the recognition of acetyl-lysine on histone tails.

Ligand Binding Site Similarity Identification Based on Chemical and Geometric Similarity

The similarity comparison of binding sites based on amino acid between different proteins can facilitate protein function identification. However, Binding site usually consists of several crucial amino acids which are frequently dispersed among different regions of a protein and consequently make the comparison of binding sites difficult. In this study, we introduce a new method, named as chemical and geometric similarity of binding site (CGS-BSite), to compute the ligand binding site similarity based on discrete amino acids with maximum-weight bipartite matching algorithm. The principle of computing the similarity is to find a Euclidean Transformation which makes the similar amino acids approximate to each other in a geometry space, and vice versa. CGS-BSite permits site and ligand flexibilities, provides a stable prediction performance on the flexible ligand binding sites. Binding site prediction on three test datasets with CGS-BSite method has similar performance to Patch-Surfer method but outperforms other five tested methods, reaching to 0.80, 0.71 and 0.85 based on the area under the receiver operating characteristic curve, respectively. It performs a marginally better than Patch-Surfer on the binding sites with small volume and higher hydrophobicity, and presents good robustness to the variance of the volume and hydrophobicity of ligand binding sites. Overall, our method provides an alternative approach to compute the ligand binding site similarity and predict potential special ligand binding sites from the existing ligand targets based on the target similarity.

FTFlex: accounting for binding site flexibility to improve fragment-based identification of druggable hot spots

Computational solvent mapping finds binding hot spots, determines their druggability and provides information for drug design. While mapping of a ligand-bound structure yields more accurate results, usually the apo structure serves as the starting point in design. The FTFlex algorithm, implemented as a server, can modify an apo structure to yield mapping results that are similar to those of the respective bound structure. Thus, FTFlex is an extension of our FTMap server, which only considers rigid structures. FTFlex identifies flexible residues within the binding site and determines alternative conformations using a rotamer library. In cases where the mapping results of the apo structure were in poor agreement with those of the bound structure, FTFlex was able to yield a modified apo structure, which lead to improved FTMap results. In cases where the mapping results of the apo and bound structures were in good agreement, no new structure was predicted. FTFlex is freely available as a web-based server at http://ftflex.bu.edu/.

Drug resistance mechanism of PncA in Mycobacterium tuberculosis

Tuberculosis continues to be a global health threat. Pyrazinamide (PZA) is an important first-line drug in multidrug-resistant tuberculosis treatment. The emergence of strains resistant to PZA represents an important public health problem, as both first- and second-line treatment regimens include PZA. It becomes toxic to Mycobacterium tuberculosis when converted to pyrazinoic acid by the bacterial pyrazinamidase (PncA) enzyme. Resistance to PZA is caused mainly by the loss of enzyme activity by mutation, the mechanism of resistance is not completely understood. In our studies, we analysed three mutations (D8G, S104R and C138Y) of PncA which are involved in resistance towards PZA. Binding pocket analysis solvent accessibility analysis, molecular docking and interaction analysis were performed to understand the interaction behaviour of mutant enzymes with PZA. Molecular dynamics simulations were conducted to understand the three-dimensional (3D) conformational behaviour of native and mutants PncA. Our analysis clearly indicates that the mutation (D8G, S104R and C138Y) in PncA is responsible for rigid binding cavity which in turn abolishes conversion of PZA to its active form and is the sole reason for PZA resistance. Excessive hydrogen bonding between PZA binding cavity residues and their neighbouring residues are the reason of rigid binding cavity during simulation. We present an exhaustive analysis of the binding site flexibility and its 3D conformations that may serve as new starting points for structure-based drug design and helps the researchers to design new inhibitors with consideration of rigid criterion of binding residues due to mutation of this essential target.An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:11

Spectroscopy of hydroxyphenyl benzazoles in solution and human serum albumin: detecting flexibility, specificity and high affinity of the warfarin drug binding site

The complex photophysics of 2-(2′-hydroxyphenyl)benzoxazole (HBO), 2-(2′-hydroxyphenyl)benzimidazole (HBI), and 2-(2′-hydroxyphenyl)benzothiazole (HBT) in different media makes them suitable as fluorescent probes to study the nature of binding sites in macromolecular systems. In this work, we investigate the spectroscopy of the three benzazole derivatives (HBXs) in different solvents and in human serum albumin (HSA) in order to understand the binding mechanism in subdomain IIA of HSA which has the ability to host a large variety of natural and pharmaceutical compounds. The three probes are found to specifically bind close to W214, the sole tryptophan residue in HSA, in a mode similar to that of the binding of the anticoagulant drug warfarin. The current results show that the structural differences between the three HBX molecules did not produce any measurable effects when binding with HSA. In particular, the change in planarity of the molecular backbone, from a perfectly planar and more rigid structure (HBO) to a twisted structure (HBI) and a flexible structure (HBT) has no effect on the mode of binding. Also, the strength of the intramolecular hydrogen bonds in HBXs (HBO > HBT > HBI) is shown not to intervene with the ability of HSA to ionize the ligands via through-space interaction with polar amino acid residues, similar to enzymatic reactions. The results emphasize the nature of HSA as a versatile and indiscriminate receptor, capable of binding a variety of ligands by adapting its binding pockets. In this regard, binding of HBXs, and other structurally similar ligands, in subdomain IIA is best described by the induced-fit model in which considerable flexibility of the binding site is necessary for molecular recognition. The results also point to the high affinity of the warfarin binding pocket (within subdomain IIA) for binding versatile molecular structures including several drugs. This affinity stems from the flexibility of the amino acids forming the binding pocket.

Multidrug Binding Properties of the AcrB Efflux Pump Characterized by Molecular Dynamics Simulations

Multidrug resistance in Gram-negative bacteria, to which multidrug efflux pumps such as AcrB makes a major contribution, is becoming a major public health problem. Unfortunately only a few compounds have been cocrystallized with AcrB, and thus computational approaches are essential in elucidating the interaction between diverse ligands and the pump protein. We used molecular dynamics simulation to examine the binding of 9 substrates, 2 inhibitors, and 2 non-substrates to the distal binding pocket of AcrB, identified earlier by X-ray crystallography. This approach gave us more realistic views of the binding than the previously-used docking approach, as the explicit water molecules contributed to the process and the flexible binding site was often seen to undergo large structural changes. We analyzed the interaction in detail in terms of the binding energy, hydrophobic surface matching, and the residues mostly stabilizing the complex. We found that all substrates tested bound to the pocket, whereas the binding to this site was not preferred for the non-substrates. Interestingly, both inhibitors (Phe-Arg-β-naphthylamide and 1-(1-naphtylmethyl)-piperazine) tended to move out of the pocket at least partially, getting into contact with a glycine-rich loop that separates the distal pocket from the more proximal region of the protein and is thought to control the access of substrates to the distal pocket.

Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations

Multidrug resistance in Gram-negative bacteria, to which multidrug efflux pumps such as the AcrB transporter makes a major contribution, is becoming a major public health problem. Unfortunately only a few compounds have been cocrystallized with AcrB, and thus computational approaches are essential in elucidating the interaction between diverse ligands and the pump protein. We used molecular dynamics simulation to examine the binding of nine substrates, two inhibitors, and two nonsubstrates to the distal binding pocket of AcrB, identified earlier by X-ray crystallography. This approach gave us more realistic views of the binding than the previously used docking approach, as the explicit water molecules contributed to the process and the flexible binding site was often seen to undergo large structural changes. We analyzed the interaction in detail in terms of the binding energy, hydrophobic surface-matching, and the residues involved in the process. We found that all substrates tested bound to the pocket, whereas the binding to this site was not preferred for the nonsubstrates. Interestingly, both inhibitors [Phe-Arg-β-naphthylamide and 1-(1-naphtylmethyl)-piperazine] tended to move out of the pocket at least partially, getting into contact with a glycine-rich loop that separates the distal pocket from the more proximal region of the protein and is thought to control the access of substrates to the distal pocket.

Side-chain rotamer changes upon ligand binding: common, crucial, correlate with entropy and rearrange hydrogen bonding

Motivation: Protein movements form a continuum from large domain rearrangements (including folding and restructuring) to side-chain rotamer changes and small rearrangements. Understanding side-chain flexibility upon binding is important to understand molecular recognition events and predict ligand binding.Methods: In the present work, we developed a well-curated non-redundant dataset of 188 proteins in pairs of structures in the Apo (unbound) and Holo (bound) forms to study the extent and the factors that guide side-chain rotamer changes upon binding.Results: Our analysis shows that side-chain rotamer changes are widespread with only 10% of binding sites displaying no conformational changes. Overall, at most five rotamer changes account for the observed movements in 90% of the cases. Furthermore, rotamer changes are essential in 32% of flexible binding sites. The different amino acids have a 11-fold difference in their probability to undergo changes. Side-chain flexibility represents an intrinsic property of amino acids as it correlates well with configurational entropy differences. Furthermore, on average b-factors and solvent accessible surface areas can discriminate flexible side-chains in the Apo form. Finally, there is a rearrangement of the hydrogen-bonding network upon binding primarily with a loss of H-bonds with water molecules and a gain of H-bonds with protein residues for flexible residues. Interestingly, only 25% of side chains capable of forming H-bonds do so with the ligand upon binding. In terms of drug design, this last result shows that there is a large number of potential interactions that may be exploited to modulate the specificity and sensitivity of inhibitors.Contact: rafael.najmanovich@usherbrooke.ca

Compound Activity Prediction Using Models of Binding Pockets or Ligand Properties in 3D

Transient interactions of endogenous and exogenous small molecules with flexible binding sites in proteins or macromolecular assemblies play a critical role in all biological processes. Current advances in high-resolution protein structure determination, database development, and docking methodology make it possible to design three-dimensional models for prediction of such interactions with increasing accuracy and specificity. Using the data collected in the Pocketome encyclopedia, we here provide an overview of two types of the three-dimensional ligand activity models, pocketbased and ligand property-based, for two important classes of proteins, nuclear and G-protein coupled receptors. For half the targets, the pocket models discriminate actives from property matched decoys with acceptable accuracy (the area under ROC curve, AUC, exceeding 84%) and for about one fifth of the targets with high accuracy (AUC > 95%). The 3D ligand property field models performed better than 95% in half of the cases. The high performance models can already become a basis of activity predictions for new chemicals. Family-wide benchmarking of the models highlights strengths of both approaches and helps identify their inherent bottlenecks and challenges.

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θD0~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c2, and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.

Protein Dynamics and the Diversity of an Antibody Response

The immune system is remarkable in its ability to produce antibodies (Abs) with virtually any specificity from a limited repertoire of germ line precursors. Although the contribution of sequence diversity to this molecular recognition has been studied for decades, recent models suggest that protein dynamics may also broaden the range of targets recognized. To characterize the contribution of protein dynamics to immunological molecular recognition, we report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight Abs elicited to the chromophoric antigen 8-methoxypyrene-1,3,6-trisulfonate (MPTS). Based on the sequence data, three of the Abs arose from unique germ line Abs, whereas the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor. The thermodynamic data indicate that the Abs recognize MPTS via a variety of mechanisms. Although the spectroscopic data reveal small differences in protein dynamics, the anti-MPTS Abs generally show similar levels of flexibility and conformational heterogeneity, possibly representing the convergent evolution of the dynamics necessary for function. However, one Ab is significantly more rigid and conformationally homogeneous than the others, including a sibling Ab from which it differs by only five somatic mutations. This example of divergent evolution demonstrates that point mutations are capable of fixing significant differences in protein dynamics. The results provide unique insight into how high affinity Abs may be produced that bind virtually any target and possibly, from a more general perspective, how new protein functions are evolved.

Application of Drug-Perturbed Essential Dynamics/Molecular Dynamics (ED/MD) to Virtual Screening and Rational Drug Design.

We present here the first application of a new algorithm, essential dynamics/molecular dynamics (ED/MD), to the field of small molecule docking. The method uses a previously existing molecular dynamics (MD) ensemble of a protein or protein-drug complex to generate, with a very small computational cost, perturbed ensembles which represent ligand-induced binding site flexibility in a more accurate way than the original trajectory. The use of these perturbed ensembles in a standard docking program leads to superior performance than the same docking procedure using the crystal structure or ensembles obtained from conventional MD simulations as templates. The simplicity and accuracy of the method opens up the possibility of introducing protein flexibility in high-throughput docking experiments.

Analysis of Flexibility and Hotspots in Bcl-xL and Mcl-1 Proteins for the Design of Selective Small-Molecule Inhibitors.

Although Bcl-xL and Mcl-1, two antideath Bcl-2 members, have similar, flexible binding sites, they can achieve high binding selectivity to endogenous binding partners and synthetic small-molecule inhibitors. Here, we employed molecular dynamic (MD) simulations and hotspot analysis to investigate the conformational flexibility of these proteins and their binding hotspots at the binding sites. Backbone flexibility analyses indicate that the highest degree of flexibility in Mcl-1 is the α4 helical segment as opposed to the α3 helix in Bcl-xL among four helical segments in their binding sites. Furthermore, common and unique binding hotspots at both proteins were identified using small-molecule probes. These analyses can aid the design of potent and specific small-molecule inhibitors for these proteins.

Comparative studies in series of cytochrome c oxidase models

This study compares the behavior as cytochrome c oxidase (CcO) functional and structural models of a series of reported and unreported ligands that provide either a binding site for copper without a built-in proximal base, or both a flexible binding site for copper and a built-in proximal base, or a fixed binding site for copper with a built-in proximal base. The comparisons of the models show that the relative position of the two metal sites is not only a crucial parameter in the control of the catalytic behavior but also essential in mimicking other features of the enzyme such as CO exchange between the ferrous heme a3 and the cuprous CuB center.

Molecular dynamics of Mycobacterium tuberculosis KasA: implications for inhibitor and substrate binding and consequences for drug design

Inhibition of the production of fatty acids as essential components of the mycobacterial cell wall has been an established way of fighting tuberculosis for decades. However, increasing resistances and an outdated medical treatment call for the validation of new targets involved in this crucial pathway. In this regard, the β-ketoacyl ACP synthase KasA is a promising enzyme. In this study, three molecular dynamics simulations based on the wildtype crystal structures of inhibitor bound and unbound KasA were performed in order to investigate the flexibility and conformational space of this target. We present an exhaustive analysis of the binding-site flexibility and representative pocket conformations that may serve as new starting points for structure-based drug design. We also revealed a mechanism which may account for the comparatively low binding affinity of thiolactomycin. Furthermore, we examined the behavior of water molecules within the binding pocket and provide recommendations how to handle them in the drug design process. Finally, we analyzed the dynamics of a channel that accommodates the long-chain fatty acid substrates and, thereby, propose a mechanism of substrate access to this channel and how products are most likely released.

Ion selectivity of proteins: lessons from molecular dynamics simulations on valinomycin

In the introduction to the recent JGP series Perspectives on: Ion selectivity, [Andersen (2011)][1] notes that present theoretical models do not yet deal fully with the energetic consequences of the known flexibility of ion binding sites in proteins (how changes in flexibility alter the ion

Solution structure and dynamics of ADF from Toxoplasma gondii

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹⁵N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed β-sheet comprised of six β-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.