Unexpected tricovalent binding mode of boronic acids within the active site of a penicillin-binding protein

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cβ bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.     

Coarse-graining of protein structures for the normal mode studies

The coarse-grained structural model such as Gaussian network has played a vital role in the normal mode studies for understanding protein dynamics related to biological functions. However, for the large proteins, the Gaussian network model is computationally unfavorable for diagonalization of Hessian (stiffness) matrix for the normal mode studies. In this article, we provide the coarse-graining method, referred to as "dynamic model condensation," which enables the further coarse-graining of protein structures consisting of small number of residues. It is shown that the coarser-grained structures reconstructed by dynamic model condensation exhibit the dynamic characteristics, such as low-frequency normal modes, qualitatively comparable to original structures. This sheds light on that dynamic model condensation and may enable one to study the large protein dynamics for gaining insight into biological functions of proteins.     

Copper redox cycling in the prion protein depends critically on binding mode

The prion protein (PrP) takes up 4-6 equiv of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60-91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc. 2005, 127, 12647-12656). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens, et al. PLoS Pathog.2009, 5 (4), e1000390). A current working hypothesis is that the high occupancy binding mode quenches copper's inherent redox cycling, thus, protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrP's putative role in cellular signaling.     

Unbinding of nicotine from the acetylcholine binding protein: steered molecular dynamics simulations

The ligand binding/unbinding process is critical to our understanding of the pharmacology of both the nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP). Steered molecular dynamics simulations were performed to learn about the unbinding process of the full agonist nicotine. Three different pulling models were designed to investigate the possible binding/unbinding pathways: radial and tangent models, and also a mixed model. Of the three, the tangent pulling model finally failed to dissociate nicotine from the ligand binding pocket. The efficiency of the pulling force profiles was superior, and the opening of the C-loop was smaller in the mixed pulling model than that in the radial model. The most favorable pathway for the cholinergic agonist nicotine to enter or leave the binding pocket is through the principal binding side, following a curvilinear track. Noticeably, it has been seen that the unbinding of the nicotine is concomitant with a global rotation of the protein-ligand complex which could be caused by the interactions of the ligand with protein at the tangent direction.     

The subspace iteration method in protein normal mode analysis

Normal mode analysis plays an important role in relating the conformational dynamics of proteins to their biological function. The subspace iteration method is a numerical procedure for normal mode analysis that has enjoyed widespread success in the structural mechanics community due to its numerical stability and computational efficiency in calculating the lowest normal modes of large systems. Here, we apply the subspace iteration method to proteins to demonstrate its advantageous properties in this area of computational protein science. An effective algorithm for choosing the number of iteration vectors in the method is established, offering a considerable improvement over the original implementation. In the present application, computational time scales linearly with the number of normal modes computed. Additionally, the method lends itself naturally to normal mode analyses of multiple neighboring macromolecular conformations, as demonstrated in a conformational change pathway analysis of adenylate kinase. These properties, together with its computational robustness and intrinsic scalability to multiple processors, render the subspace iteration method an effective and reliable computational approach to protein normal mode analysis. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nAChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole-AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.     

Vibrational study on the cobalt binding mode of Carnosine

The Co(II)-l-Carnosine (Carnos) system was investigated at different pH and metal/ligand molar ratios by Raman and IR spectroscopy. Raman spectra present some marker bands yielding information on the ability of the Co(II)/Carnos system to bind molecular oxygen and to identify the metal co-ordination site of the imidazole ring (N_π or N_τ atom) of Carnos.The existence of different oxygenated species is greatly affected by pH and the structure of the predominant complexes depends on the available nitrogen atoms. Under basic conditions, binuclear complexes binding molecular oxygen are the predominant species and two forms (monobridged and dibridged) were identified by the Raman νO-O band (750-850 cm⁻¹).Decreasing pH to 7, the species present in the system are less able to bind oxygen. Hydrogen peroxide and a Co(III) chelate not binding O₂, were formed with a significant conversion of peroxo into superoxo complexes. A slight excess of Carnos does not enhance metal chelation.In slightly acidic conditions, the formation of H₂O₂ and superoxo species is more enhanced than at pH 7 and another Co(III) chelate is probably formed.     

A computational model of the nicotinic acetylcholine binding site

We have derived a model of the nicotinic acetylcholine binding site. This was accomplished by using three known agonists (acetylcholine, nicotine and epibatidine) as templates around which polypeptide side chains, found to be part of the receptor cavity from published molecular biology studies, are allowed to flow freely in molecular dynamics simulations and mold themselves around these templates. The resulting supramolecular complex should thus be a complement, both in terms of steric effects as well as electronic effects, to the agonists and it should be a good estimation of the true receptor cavity structure. The shapes of those minireceptor cavities equilibrated rapidly on the simulation time scale and their structural congruence is very high, implying that a satisfactory model of the nicotinic acetylcholine binding site has been achieved. The computational methodology was internally tested against two rigid and specific antagonists (dihydro--erytroidine and erysoidine), that are expected to give rise to a somewhat differently shaped binding site compared to that derived from the agonists. Using these antagonists as templates there were structural reorganizations of the initial receptor cavities leading to distinctly different cavities compared to agonists. This indicates that adequate times and temperatures were used in our computational protocols to achieve equilibrium structures for the agonists. Overall, both minireceptor geometries for agonists and antagonists are similar with the exception of one amino acid (ARG209).     

Effect of interprotein polarization on protein–protein binding energy

Molecular dynamics simulation in explicit water for the binding of the benchmark barnase‐barstar complex was carried out to investigate the effect polarization of interprotein hydrogen bonds on its binding free energy. Our study is based on the AMBER force field but with polarized atomic charges derived from fragment quantum mechanical calculation for the protein complex. The quantum‐derived atomic charges include the effect of polarization of interprotein hydrogen bonds, which was absent in the standard force fields that were used in previous theoretical calculations of barnase‐barstar binding energy. This study shows that this polarization effect impacts both the static (electronic) and dynamic interprotein electrostatic interactions and significantly lowers the free energy of the barnase‐barstar complex. © 2012 Wiley Periodicals, Inc.     

The mode of binding ACMA-DNA relies on the base-pair nature

A thermodynamic and kinetic study on the mode of binding of 9-amino-6-chloro-2-methoxi-acridine (ACMA) to poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) has been undertaken at pH = 7.0 and I = 0.1 M. The spectrophotometric, kinetic (T-jump), circular dichroism, viscometric and calorimetric information gathered point to formation of a fully intercalated ACMA complex with poly(dA-dT)·poly(dA-dT) and another one only partially intercalated (7%) with poly(dG-dC)·poly(dG-dC). The ACMA affinity with the A-T bases was higher than with the G-C bases. The two polynucleotide sequences give rise to external complexes when the ACMA concentration is raised, namely, the electrostatic complex poly(dA-dT)·poly(dA-dT)-ACMA and the major groove binding complex poly(dG-dC)·poly(dG-dC)-ACMA. A considerable quenching effect of the ACMA fluorescence is observed with poly(dA-dT)·poly(dA-dT), ascribable to face-to-face location in the intercalated A-T-ACMA base-pairs. The even stronger effect observed in the presence of poly(dG-dC)·poly(dG-dC) is related to the guanine residue from on- and off-slot ACMA positions.     

Normal mode analysis of Trp RNA binding attenuation protein: structure and collective motions

The Trp RNA-binding protein (TRAP) has a toroidal topology and a perfect 11-fold symmetry, which makes it an excellent candidate for a vibrational study of elastic properties. Normal mode analysis in combination with correlation matrix calculations was used to detect collective low-frequency motions in TRAP. The results reveal the presence of highly correlated modes at the lower end of the spectrum, which directly reflect the annular and toroidal topology. The integral of the correlations over the low-frequency torsional part of the vibrational spectrum further demonstrates the relative rigidity of the 11 monomer building blocks of TRAP. The internal flexibility of each monomer and the effects of Trp-binding were also examined. The study clearly shows the determining influence of symmetry and topology on the elastic properties and also offers a detailed view on the Trp affinity of TRAP.     

Identifying the binding mode of a molecular scaffold

We describe a method for docking of a scaffold-based series and present its advantages over docking of individual ligands, for determining the binding mode of a molecular scaffold in a binding site. The method has been applied to eight different scaffolds of protein kinase inhibitors (PKI). A single analog of each of these eight scaffolds was previously crystallized with different protein kinases. We have used FlexX to dock a set of molecules that share the same scaffold, rather than docking a single molecule. The main mode of binding is determined by the mode of binding of the largest cluster among the docked molecules that share a scaffold. Clustering is based on our 'nearest single neighbor' method [J. Chem. Inf. Comput. Sci., 43 (2003) 208-217]. Additional criteria are applied in those cases in which more than one significant binding mode is found. Using the proposed method, most of the crystallographic binding modes of these scaffolds were reconstructed. Alternative modes, that have not been detected yet by experiments, could also be identified. The method was applied to predict the binding mode of an additional molecular scaffold that was not yet reported and the predicted binding mode has been found to be very similar to experimental results for a closely related scaffold. We suggest that this approach be used as a virtual screening tool for scaffold-based design processes.     

On the effect of molecular structure on the superposition of normal modes of vibration

The normal modes of vibration in cartesian coordinates were calculated for ethylene, C₂H₄, and an ethylene complex, C₂H₄-Tl³⁺-H₂O, which is presumably formed during the catalytic oxidation of C₂H₄. For the CC bond of C₂H₄ as the critical coordinate of this reaction the distortions were then calculated which are caused by superimposing the normal modes. These calculations indicate that the maximum distortion of the CC bond which is attainable by superimposing normal modes in their ground state is larger in some conformations of the complex than in the free molecule. This indicates the general possibility that, depending on proper symmetry, complex formation may increase the reactivity of a compound because, compared to the free molecule, the superposition of a greater number of 3N-6 normal modes can produce greater momentary distortions of internal coordinates. The effect could be of considerable importance for the reactivity of very large systems, like, e.g., enzyme-substrate complexes.     

Effect of surface viscosity on the vibration of microcantilevers

Using the theory of beam bending and the theory of surface rheology, the equation describing the effect of a surface viscous film on the vibration of an elastic microcantilever is derived. A generalized solution for the vibration of the microcantilever is obtained, which depends on the boundary conditions and the surface viscosity. There is a phase lag between the externally harmonic excitation and the microcantilever response due to the energy loss from surface flow. For small energy dissipation with negligible surface energy, the maximum energy loss occurs approximately at the natural frequencies of the microcantilever.     

顺铂与F-肌动蛋白的结合方式的LMCT谱研究

本文根据顺铂及顺铂与F-肌动蛋白电子光谱中电荷转移谱带研究了顺二氨合铂(II)离子与F-肌动蛋白的结合方式及构型.将所得光谱解极为个别吸收谱带,并与按不同构型、不同配位原子计算结果相比较,结果表明,铂以平面四方形构型主要与两个氨分子、一个硫和一个氨基氮配位.     

乙硼烷的简正振动频率和光谱熵

本文用群论方法讨论了Coriolis微扰,认为950cm~(-1)为ν_9是合理的.按非刚性转子-谐振子模型,采用直接加和法计算转动、振动配分函数.应用Cromemco微处理机计算了100～2500K范围内~(10)B_2H_6和~(11)B_2H_6的光谱熵.     

双原子分子振动对热力学性质的影响

本文在分析了双原子分子振动能级的完备性和有限性及其对统计计算带来的影响的基础上,借助代数（AM）方法得到的双原子分子振动能级完全集合,采用量子力学统计系综方法,讨论了双原子分子振动能量对宏观热力学性质的统计贡献,并以氮气为例计算了相应的热力学函数和振动热容量．结果表明,真实的双原子分子振动能级是有限的;确定最高振动量子数和振动能级完全集合是正确进行统计分析的基础和关键;考虑振动能级的完备性和有限性后,只能导致数值解而不是解析解,所得的结果优于谐振子模型的解析结果,与实验数据吻合得很好．     

Molecular vibration mode assignment of nematic liquid crystal 5CB on Terahertz spectra

Using DFT/B3LYP/6-311++G** method, the molecular structure and absorption spectra in terahertz (THz) range of liquid crystal 5CB are investigated. In a frequency range 0–15 THz, an assignment of the vibrational modes corresponding to absorption frequencies is performed using potential energy distribution for the first time. It is found that the cyano group radical (–CN) do actively take part in the strongest THz absorption of 1.743, 3.942, 5.169 and 14.769 THz in different vibration modes. The results suggest that the strong polar group should be avoided in designing liquid crystal molecule and mixtures in order to reduce the absorption intensity in THz range.     

A perturbed pK(a) at the binding site of the nicotinic acetylcholine receptor: implications for nicotine binding

A series of tethered quaternary ammonium derivatives of Tyr have been incorporated into the binding site of the nicotinic acetylcholine receptor (nAChR) using the in vivo nonsense suppression method, producing constitutively active (self-gating) receptors. We have incorporated primary, secondary, and tertiary amine tethered agonists to give receptors whose constitutive activity can be modulated by pH. Lowering the pH protonates the tethered amine, giving it a positive charge and allowing it to reversibly activate the receptor. Tertiary and secondary tethered amines, TyrO3T and TyrO3S, have been successfully incorporated at alpha149 in the nAChR. Constitutive currents at pH 5.5 are 6 times those at pH 9.0. The pKa of TyrO3T in the binding site appears to be 6 or lower, differing substantially from its pKa in solution ( approximately 9.3). This local pKa perturbation has substantial implications for pharmacological research on the nAChR: of the tertiary agonists considered, noracetylcholine experiences this pKa perturbation, while nicotine does not.     

An activity-based protein profiling probe for the nicotinic acetylcholine receptor

Activity-based protein profiling (ABPP) has been used extensively to characterize the physiological functions of enzymes but has not yet been extended to ion channels. We have synthesized a state-dependent photoaffinity probe for the nicotinic acetylcholine receptor (nAChR) as a proof of concept for the development of ion channel directed ABPP probes. The candidate probe BPyneTEA comprises an nAChR binding moiety, a benzophenone moiety for photolabeling, and an alkyne moiety for biotinylation via "click chemistry". Single-molecule current measurements show that BPyneTEA blocks both the closed and open (i.e., nondesensitized) conformations of the nAChR with similar kinetics. In living cells, BPyneTEA photolabels the closed state selectively over the inactive desensitized state. BPyneTEA thus shows promise as a probe for nondesensitized nAChRs and may be useful in studying the molecular physiology of desensitization. The structure and reactivity of ion channel pores in general suggest that they will be a broadly useful target for ABPP probes.