A cellular automata model of ligand passage over a protein hydrodynamic landscape

The subject of ligand passage to an active site on a protein is addressed. Current views on the mechanism and the possible role of surface water are discussed. A theory is presented in which the pattern of hydropathic states of protein surface amino acid side chains is invoked as the influence on the relative hydrophobic effects of nearby water. The theory describes a ligand passage through the hydrodynamic near-surface water which exhibits temporary organized cavities resembling the chreodes introduced by Waddington. The passage of the ligand to the active site is facilitated by this dynamic mechanism. Cellular automata models of preferential directional diffusion through these chreodes support the theory. The theory may be invoked to explain a number of ligand-active site observations and serves as an idea for further studies.     

Dynamical persistence of active sites identified in maltose-binding protein

This study identifies dynamical properties of maltose-binding protein (MBP) useful in unveiling active site residues susceptible to ligand binding. The described methodology has been previously used in support of novel topological techniques of persistent homology and statistical inference in complex, multi-scale, high-dimensional data often encountered in computational biophysics. Here we outline a computational protocol that is based on the anisotropic elastic network models of 14 all-atom three-dimensional protein structures. We introduce the notion of dynamical distance matrices as a measure of correlated interactions among 370 amino acid residues that constitute a single protein. The dynamical distance matrices serve as an input for a persistent homology suite of codes to further distinguish a small subset of residues with high affinity for ligand binding and allosteric activity. In addition, we show that ligand-free closed MBP structures require lower deformation energies than open MBP structures, which may be used in categorization of time-evolving molecular dynamics structures. Analysis of the most probable allosteric coupling pathways between active site residues and the protein exterior is also presented.     

On the Dynamical Behavior of the Cysteine Dioxygenase‐l‐Cysteine Complex in the Presence of Free Dioxygen and l‐Cysteine

In this work, viable models of cysteine dioxygenase (CDO) and its complex with l ‐cysteine dianion were built for the first time, under strict adherence to the crystal structure from X‐ray diffraction studies, for all atom molecular dynamics (MD). Based on the CHARMM36 FF, the active site, featuring an octahedral dummy Fe(II) model, allowed us observing water exchange, which would have escaped attention with the more popular bonded models. Free dioxygen (O₂) and l ‐cysteine, added at the active site, could be observed being expelled toward the solvating medium under Random Accelerated Molecular Dynamics (RAMD) along major and minor pathways. Correspondingly, free dioxygen (O₂), added to the solvating medium, could be observed to follow the same above pathways in getting to the active site under unbiased MD. For the bulky l ‐cysteine, 600 ns of trajectory were insufficient for protein penetration, and the molecule was stuck at the protein borders. These models pave the way to free energy studies of ligand associations, devised to better clarify how this cardinal enzyme behaves in human metabolism.     

Density-functional investigation on the mechanism of H-atom abstraction by lipoxygenase

Using experimentally calibrated density functional calculations on models of the active site of soybean lipoxygenase 1 (SLO-1), insight has been obtained into the coordination flexibility of the iron active site and its molecular mechanism of catalysis. The ferrous form of SLO-1 shows a variation in coordination number in solution that is related to a weakly coordinating Asn694 ligand. From the calculations it is determined that the weak Fe-O(694) bond associated with this coordination flexibility is due to a sideways tilted geometry of Asn694 that is imposed on the site by the protein. Release of this constraint (by altering the hydrogen bonding network) leads to a pure six-coordinate site. In contrast, the ferric form of the enzyme stays five-coordinate. In this case, deprotonation of a coordinated water gives a strong hydroxo donor in the cis position to Asn694, weakening the Fe-O(694) bond. Hence, Asn694 is a stronger ligand to the reduced relative to the oxidized site. Using these experimentally calibrated models, the reaction energy for H-atom transfer in SLO-1 has been calculated to be about -18 kcal/mol. The observed change in coordination number going from five-coordinate in ferric to six-coordinate in ferrous SLO-1 increases the reduction potential of the iron active site. Hence, the protein adjusts the active site for optimal reactivity. Analysis of the electronic structure along the reaction coordinate shows that the H-atom transfer in SLO-1 actually corresponds to a proton-coupled electron transfer (PCET). The transferred electron does not localize on the proton, but tunnels directly from the substrate to the ferric active site in a concerted proton tunneling-electron tunneling (PTET) process. The covalently linked Fe-O-H-C bridge in the transition state lowers the energy barrier and provides an efficient superexchange pathway for this tunneling. The thermal barrier for the PTET process is estimated from the calculations to be about +15 kcal/mol including zero-point energy corrections. This corresponds to a thermal reaction rate of k(therm) approximately 1 s(-1). In comparison, the rate of proton tunneling can be as high as 2 x 10(9) s(-1) under these conditions.     

Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design.

Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et al., 1996, Science 274:1531-1534), indicates that CAST pocket computation may provide a priori identification of target proteins for linked-fragment design. CAST analysis of 30 HIV-1 protease-inhibitor complexes shows that the flexible active site pocket can vary over a range of 853-1,566 A3, and that there are two pockets near or adjoining the active site that may be recruited for ligand design.     

Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site

The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed to be involved in heterologous protein associations occurring during synaptogenesis or upon neurodegeneration. A novel crystal form of mouse AChE, combined with spectrophotometric analyses of the crystals, enabled us to solve unique structures of AChE with a free peripheral site, and as three complexes with peripheral site inhibitors: the phenylphenanthridinium ligands, decidium and propidium, and the pyrogallol ligand, gallamine, at 2.20-2.35 A resolution. Comparison with structures of AChE complexes with the peptide fasciculin or with organic bifunctional inhibitors unveils new structural determinants contributing to ligand interactions at the peripheral site, and permits a detailed topographic delineation of this site. Hence, these structures provide templates for designing compounds directed to the enzyme surface that modulate specific surface interactions controlling catalytic activity and non-catalytic heterologous protein associations.     

Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.     

Selective redox regulation of cytokine receptor signaling by extracellular thioredoxin-1

The thiol-disulfide oxidoreductase thioredoxin-1 (Trx1) is known to be secreted by leukocytes and to exhibit cytokine-like properties. Extracellular effects of Trx1 require a functional active site, suggesting a redox-based mechanism of action. However, specific cell surface proteins and pathways coupling extracellular Trx1 redox activity to cellular responses have not been identified so far. Using a mechanism-based kinetic trapping technique to identify disulfide exchange interactions on the intact surface of living lymphocytes, we found that Trx1 catalytically interacts with a single principal target protein. This target protein was identified as the tumor necrosis factor receptor superfamily member 8 (TNFRSF8/CD30). We demonstrate that the redox interaction is highly specific for both Trx1 and CD30 and that the redox state of CD30 determines its ability to engage the cognate ligand and transduce signals. Furthermore, we confirm that Trx1 affects CD30-dependent changes in lymphocyte effector function. Thus, we conclude that receptor-ligand signaling interactions can be selectively regulated by an extracellular redox catalyst.     

A QM/MM study of proton transport pathways in a [NiFe] hydrogenase

A theoretical QM/MM study of the [NiFe] hydrogenase from Desulfovibrio fructosovorans has been performed to investigate possible routes of proton transfer between the active site and the protein surface. We obtained the minimum energy paths, with a modified version of the nudged elastic band method, for a set of proposed pathways. The calculations were carried out for the crystallographic structure and for several structures of the protein obtained from a molecular dynamics simulation. The results show one of the studied pathways to be preferred for transport from the active site to the surface, but the preference is not so strong when transport occurs in the opposite direction.     

A pseudo-particle approach for studying protein-ligand models truncated to their active sites

A molecular dynamics method has been developed to describe the structural and dynamic properties of protein-ligand complexes that are truncated to their active sites. The active site is comprised of the ligand and discontinuous, positionally unrestrained peptide chains. This truncated active-site complex is surrounded by big unspecific pseudo-particles representing the complete protein and the solvent. Thus, knowledge of the folding of the outer parts of the protein is not required, and the method can be applied to protein models, derived from homology modeling. The method has been tested using ligand complexes of adenylate kinase, retinol binding protein, HIV-1 protease, and human leucocyte antigen. Comparisons with their crystal structures and with results from time-demanding simulations of the whole complexes in explicit water solvent show that the ligand binding properties are conserved. Most of the hydrogen bonds between the ligand and the active-site residues are reproduced and, furthermore, the simulation time is reduced. © 1996 John Wiley & Sons, Inc.     

Multi‐spectroscopic and molecular modeling investigation of the interactions between prantschimgin and matrix metalloproteinase 9 (MMP9)

The binding of prantschimgin (PRAN) to matrix metalloproteinase 9 (MMP9) was investigated using multiple techniques. Fluorescence spectroscopy showed that PRAN could quench the MMP9 fluorescence spectra. Changes in the UV/vis and Fourier transform infrared (FTIR) spectra were observed upon ligand binding, along with a significant degree of tryptophan fluorescence quenching on complex formation. The interaction of PRAN with MMP9 has also been studied using molecular docking and molecular dynamics (MD) simulation. The binding models demonstrated aspects of PRAN's conformation, active site interaction, important amino acids and hydrogen bonding. Computational mapping of the possible binding site of PRAN revealed that the ligand is bound in a large hydrophobic cavity of MMP9. The MD simulation results suggested that this ligand can interact with the protein, with little affecting the secondary structure. The results not only lead to a better understanding of interactions between PRAN and MMP9, but also provide useful data about the influence of PRAN on the structural conformation. The data provided in this study will be useful for designing a new agonist of MMP9 with the desired activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Co-evolution of proteins with their interaction partners

The divergent evolution of proteins in cellular signaling pathways requires ligands and their receptors to co-evolve, creating new pathways when a new receptor is activated by a new ligand. However, information about the evolution of binding specificity in ligand-receptor systems is difficult to glean from sequences alone. We have used phosphoglycerate kinase (PGK), an enzyme that forms its active site between its two domains, to develop a standard for measuring the co-evolution of interacting proteins. The N-terminal and C-terminal domains of PGK form the active site at their interface and are covalently linked. Therefore, they must have co-evolved to preserve enzyme function. By building two phylogenetic trees from multiple sequence alignments of each of the two domains of PGK, we have calculated a correlation coefficient for the two trees that quantifies the co-evolution of the two domains. The correlation coefficient for the trees of the two domains of PGK is 0. 79, which establishes an upper bound for the co-evolution of a protein domain with its binding partner. The analysis is extended to ligands and their receptors, using the chemokines as a model. We show that the correlation between the chemokine ligand and receptor trees' distances is 0.57. The chemokine family of protein ligands and their G-protein coupled receptors have co-evolved so that each subgroup of chemokine ligands has a matching subgroup of chemokine receptors. The matching subfamilies of ligands and their receptors create a framework within which the ligands of orphan chemokine receptors can be more easily determined. This approach can be applied to a variety of ligand and receptor systems.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Rapid binding of a cationic active site inhibitor to wild type and mutant mouse acetylcholinesterase: Brownian dynamics simulation including diffusion in the active site gorge

It is known that anionic surface residues play a role in the long-range electrostatic attraction between acetylcholinesterase and cationic ligands. In our current investigation, we show that anionic residues also play an important role in the behavior of the ligand within the active site gorge of acetylcholinesterase. Negatively charged residues near the gorge opening not only attract positively charged ligands from solution to the enzyme, but can also restrict the motion of the ligand once it is inside of the gorge. We use Brownian dynamics techniques to calculate the rate constant k_on for wild type and mutant acetylcholinesterase with a positively charged ligand. These calculations are performed by allowing the ligand to diffuse within the active site gorge. This is an extension of previously reported work in which a ligand was allowed to diffuse only to the enzyme surface. By setting the reaction criteria for the ligand closer to the active site, better agreement with experimental data is obtained. Although a number of residues influence the movement of the ligand within the gorge, Asp74 is shown to play a particularly important role in this function. Asp74 traps the ligand within the gorge, and in this way helps to ensure a reaction. © 1998 John Wiley & Sons, Inc. Biopoly 46: 465–474, 1998     

Investigations of the alkaline and acid transitions of umecyanin, a stellacyanin from horseradish roots

The effect of pH on Cu(I) and Cu(II) umecyanin (UCu), a phytocyanin obtained from horseradish roots, has been studied by electronic and NMR spectroscopy and using direct electrochemical measurements. A pK(a) value of approximately 9.5-9.8 is observed for the alkaline transition in UCu(II), and this leads to a slightly altered active site structure, as indicated by the changes in the paramagnetic 1H NMR spectrum. Electrochemical studies show that the pK(a) value for this transition in UCu(I) is 9.9. The alkaline transition is caused by the deprotonation of a surface lysine residue, with Lys96 being the most likely candidate. The isotropically shifted resonances in the (1)H NMR spectrum of UCu(II) also shift upon lowering the pH (pK(a) 5.8), and this can be assigned to the protonation of the surface (noncoordinating) His65 residue. This histidine titrates in UCu(I) with a pK(a) of 6.3. The reduction potential of the protein in this range is also dependent on pH, and pK(a) values matching those from NMR, for the two oxidation states of the protein, are obtained. There is no evidence for either of the active site histidines (His44 and His90) titrating in UCu(I) in the pH range studied (down to pH 3.7). Also highlighted in these studies are the remarkable active site similarities between umecyanin and the other phytocyanins which possess an axial Gln ligand.     

X-ray structures of small ligand-FKBP complexes provide an estimate for hydrophobic interaction energies

A new crystal form of native FK506 binding protein (FKBP) has been obtained which has proved useful in ligand binding studies. Three different small molecule ligand complexes and the native enzyme have been determined at higher resolution than 2.0 A. Dissociation constants of the related small molecule ligands vary from 20 mM for dimethylsulphoxide to 200 microM for tetrahydrothiophene 1-oxide. Comparison of the four available crystal structures shows that the protein structures are identical to within experimental error, but there are differences in the water structure in the active site. Analysis of the calculated buried surface areas of these related ligands provides an estimated van der Waals contribution to the binding energy of -0.5 kJ/A(2) for non-polar interactions between ligand and protein.     

Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery.

The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl-5'-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformational changes in the protein concomitant with ligand binding. In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding. Results of these studies indicate that binding of biotin to the protein results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage. Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding. Binding of both bio-5'-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein.     

A multi‐resolution model to capture both global fluctuations of an enzyme and molecular recognition in the ligand‐binding site

In multi‐resolution simulations, different system components are simultaneously modeled at different levels of resolution, these being smoothly coupled together. In the case of enzyme systems, computationally expensive atomistic detail is needed in the active site to capture the chemistry of ligand binding. Global properties of the rest of the protein also play an essential role, determining the structure and fluctuations of the binding site; however, these can be modeled on a coarser level. Similarly, in the most computationally efficient scheme only the solvent hydrating the active site requires atomistic detail. We present a methodology to couple atomistic and coarse‐grained protein models, while solvating the atomistic part of the protein in atomistic water. This allows a free choice of which protein and solvent degrees of freedom to include atomistically. This multi‐resolution methodology can successfully model stable ligand binding, and we further confirm its validity by exploring the reproduction of system properties relevant to enzymatic function. In addition to a computational speedup, such an approach can allow the identification of the essential degrees of freedom playing a role in a given process, potentially yielding new insights into biomolecular function. Proteins 2016; 84:1902–1913. © 2016 Wiley Periodicals, Inc.     

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.     

Predicting structural effects in HIV-1 protease mutant complexes with flexible ligand docking and protein side-chain optimization

We present a computational approach for predicting structures of ligand-protein complexes and analyzing binding energy landscapes that combines Monte Carlo simulated annealing technique to determine the ligand bound conformation with the dead-end elimination algorithm for side-chain optimization of the protein active site residues. Flexible ligand docking and optimization of mobile protein side-chains have been performed to predict structural effects in the V32I/I47V/V82I HIV-1 protease mutant bound with the SB203386 ligand and in the V82A HIV-1 protease mutant bound with the A77003 ligand. The computational structure predictions are consistent with the crystal structures of these ligand-protein complexes. The emerging relationships between ligand docking and side-chain optimization of the active site residues are rationalized based on the analysis of the ligand-protein binding energy landscape. Proteins 33:295–310, 1998. © 1998 Wiley-Liss, Inc.