Conformational Behavior and Flexibility of Terminally Blocked Cysteine and Cystine

Conformational potential energy hypersurfaces, PES, for the terminally blocked L-Cysteine, L,L-Cystine and D,L-Cystine have been analyzed by means of molecular mechanics in combination with the programs ROSE, CICADA, PANIC and COMBINE. Low energy conformations and conformational transitions, conformational channels, have been located. Global and fragmental flexibility and conformational softness have been calculated for each conformer as well as for the entire molecule. The PES analyses were used for simulation of conformational movement based on Boltzmann probability of the points obtained on the PES. Boltzmann travelling revealed interesting correlated conformational movement where three or even more dihedral angles changed simultaneously. It could be shown that conformational behavior and flexibility were strongly influenced by the absolute configurations of the amino acids in the peptides.     

How much can physics do for protein design?

Physics and physical chemistry are an important thread in computational protein design, complementary to knowledge-based tools. They provide molecular mechanics scoring functions that need little or no ad hoc parameter readjustment, methods to thoroughly sample equilibrium ensembles, and different levels of approximation for conformational flexibility. They led recently to the successful redesign of a small protein using a physics-based folded state energy. Adaptive Monte Carlo or molecular dynamics schemes were discovered where protein variants are populated as per their ligand-binding free energy or catalytic efficiency. Molecular dynamics have been used for backbone flexibility. Implicit solvent models have been refined, polarizable force fields applied, and many physical insights obtained.     

Conformational flexibility and binding energy profile of c-Abl tyrosine kinase complexed with Imatinib: an insight from MD study

Structural biology of kinase and in particular of tyrosine kinase has given detailed insights into the intrinsic flexibility of the catalytic domain and has provided a rational basis for obtaining selective inhibitors. In this paper, we have studied the conformational flexibility of c-Abl tyrosine kinase complexed with Imatinib (STI), in the presence of TIP3P water in physiological conditions at neutral pH. The conformational studies suggest that the flexibility of activation loop is responsible to facilitate the nucleotide binding and release. Owing to the conformational adaptability, adenosine triphosphate (ATP) binds at a particular site in the loop region of the tyrosine kinase. The molecular mechanics Poisson–Boltzmann surface area methods are analysed, as is a free-energy pathways method, which shows the stable binding with free energy ? 6.04 kcal/mol for STI. The binding energy calculated by the Sietraj method is approximately the same as the experimental binding energy of STI with c-Abl kinase. It is suggested that the conserved glutamic acid and lysine residues are necessary for the stability and optimum activity of inhibitor. This study may be helpful in rational drug designing of new kinase inhibitors.     

Differences in Conformational Behavior of ATA and TAT Sequences in Single Strand DNA Trimer

Abstract

The conformational behavior of single strand (ss) TAT and ATA trimers of DNA have been studied by computational chemistry tools including CICADA software interfaced with AMBER molecular mechanics and dynamics. The Single-Coordinate-Driving (SCD) method has been used in conjunction with molecular dynamics simulated annealing. It has been revealed that the conformational flexibility of each sequence differs substantially from the other one. Four common conformational families have been found for both trimers. These are: helical, reverse-stacked (base 3), half-stacked (base 3), reverse-stacked (base 1). However, the energies of conformers representing the families are different for both the studied systems. An additional conformational family, bulged, has been found for ss(ATA), while ss(TAT) has been found also in half-stacked (base 1) conformation. In general, ss(TAT) exhibits a higher number of low energy conformations while ss(ATA) shows one interesting low energy conformational interconversion between reverse-stacked (A3) family and half-stacked (A3) family. The high conformational variability of the trimers has been confirmed by flexibility analysis and by molecular dynamics simulations, which have also shown the conformational stability of single conformational families. It has been concluded that the methodology used is able to provide a very detailed picture of the conformational space of these molecules.     

Conformational Flexibility of Two RNA Trimers Explored by Computational Tools and Database Search

Abstract

Two RNA sequences, AAA and AUG, were studied by the conformational search program CICADA and by molecular dynamics (MD) in the framework of the AMBER force field, and also via thorough PDB database search. CICADA was used to provide detailed information about conformers and conformational interconversions on the energy surfaces of the above molecules. Several conformational families were found for both sequences. Analysis of the results shows differences, especially between the energy of the single families, and also in flexibility and concerted conformational movement. Therefore, several MD trajectories (altogether 16 ns) were run to obtain more details about both the stability of conformers belonging to different conformational families and about the dynamics of the two systems. Results show that the trajectories strongly depend on the starting structure. When the MD start from the global minimum found by CICADA, they provide a stable run, while MD starting from another conformational family generates a trajectory where several different conformational families are visited. The results obtained by theoretical methods are compared with the thorough database search data. It is concluded that all except for the highest energy conformational families found in theoretical result also appear in experimental data.

Registry numbers:

adenylyl-(3′ →5′)-adenylyl-(3′ →5′)-adenosine [917-44-2]

adenylyl-(3′ →5′)-uridylyl-(3′ →5′)-guanosine [3494-35-7]     

Structure,Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.     

Conformational plasticity and dynamics in the generic protein folding catalyst SlyD unraveled by single-molecule FRET

The relation between conformational dynamics and chemistry in enzyme catalysis recently has received increasing attention. While, in the past, the mechanochemical coupling was mainly attributed to molecular motors, nowadays, it seems that this linkage is far more general. Single-molecule fluorescence methods are perfectly suited to directly evidence conformational flexibility and dynamics. By labeling the enzyme SlyD, a member of peptidyl-prolyl cis-trans isomerases of the FK506 binding protein type with an inserted chaperone domain, with donor and acceptor fluorophores for single-molecule fluorescence resonance energy transfer, we directly monitor conformational flexibility and conformational dynamics between the chaperone domain and the FK506 binding protein domain. We find a broad distribution of distances between the labels with two main maxima, which we attribute to an open conformation and to a closed conformation of the enzyme. Correlation analysis demonstrates that the conformations exchange on a rate in the 100 Hz range. With the aid from Monte Carlo simulations, we show that there must be conformational flexibility beyond the two main conformational states. Interestingly, neither the conformational distribution nor the dynamics is significantly altered upon binding of substrates or other known binding partners. Based on these experimental findings, we propose a model where the conformational dynamics is used to search the conformation enabling the chemical step, which also explains the remarkable substrate promiscuity connected with a high efficiency of this class of peptidyl-prolyl cis-trans isomerases.     

Biased coarse-grained molecular dynamics simulation approach for flexible fitting of X-ray structure into cryo electron microscopy maps

Several approaches have been introduced to interpret, in terms of high-resolution structure, low-resolution structural data as obtained from cryo-EM. As conformational changes are often observed in biological molecules, these techniques need to take into account the flexibility of proteins. Flexibility has been described in terms of movement between rigid domains and between rigid secondary structure elements, which present some limitations for studying dynamical properties. Normal mode analysis has also been used, but is limited to medium resolution data. All-atom molecular dynamics fitting techniques are more appropriate to fit structures into higher-resolution data as full protein flexibility is considered, but are cumbersome in terms of computational time. Here, we introduce a coarse-grained approach; a Go-model was used to represent biological molecules, combined with biased molecular dynamics to reproduce accurately conformational transitions. Illustrative examples on simulated data are shown. Accurate fittings can be obtained for resolution ranging from 5 to 20 Å. The approach was also tested on experimental data of Elongation Factor G and Escherichia coli RNA polymerase, where its validity is compared to previous models obtained from different techniques. This comparison demonstrates that quantitative flexible techniques, as opposed to manual docking, need to be considered to interpret low-resolution data.     

A salt-bridge switch in the molecular recognition between RS receptor and RGD ligand from the ABEEM σπ molecular dynamics simulations

Abstract

How the receptor and ligand recognise each other is a challenging subject in explaining the mechanism of recognition at the molecular level. As a starting point, here, a synthesised RS receptor and its RGD ligand were investigated as a proper model to simulate their recognition process in terms of ABEEMσπ/MM polarisable force field. It is found that a switch of forming up a salt bridge in the ligand triggers the recognition of the receptor and ligand. Through the salt-bridge switch that undergoes several cycles from on-state with parallel hydrogen bonds to off-state with bifurcated hydrogen bonds, the active site of ligand can flex easily to interact with the active site of the receptor. In addition, the water molecules form a decisive bridge connecting the active sites of the bound system. The salt-bridge switch and water-mediated movement are cooperative as the important factors for the receptor-ligand recognition. In addition, the properties, such as binding free energy, conformational flexibility and solvent accessible surface area have been calculated to provide adequate evidence for the whole recognition process. According to the simulation, a detailed mechanism was derived involving diffusion, a switch triggered cooperative water-mediated movement, and conformational folding, for the flexible recognition.     

Conformational behaviour of the architectural units of peptides and proteins. Assessment of current understanding by ab initio quantum mechanical methods and refinement of the dipeptide energy surface.

The conformational energy surfaces of analogues of the dipeptide unit of polypeptides and proteins are calculated by ab initio methods using extended basis sets.The calculations are not particularly sensitive to the choice of (extended) basis set.The calculations are shown to support a particular empirical method parameterized with respect to crystal data. Non-hydrogen bonded conformations agree to within 3 kcal mol^?1, even for conformations in which quite considerable degrees of atomic overlap occur.Hydrogen bonded conformations, are, however, in less satisfactory agreement and it is the ab initio calculations which appear to be at fault.A simple correction is applied to the ab initio energy for hydrogen bonded conformations, and with the use of the empirical energy surface a full quantum mechanical conformational energy map is interpolated for the alanyl dipeptide.The effect of flexibility in the peptide backbone is taken into account, and supports recent empirical findings that distortions in valence angles must be considered in calculations of the conformational behaviour of peptides.     

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been performed to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol.     

Extracellular-Regulated Kinase 2 Is Activated by the Enhancement of Hinge Flexibility

Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.     

Triple mutated antibody scFv2F3 with high GPx activity: insights from MD, docking, MDFE, and MM-PBSA simulation

By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp²⁹–Sec⁵²–Gln⁷²). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser⁵²/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe²⁹/Trp–Val⁷²/Gln mutations enhance the reaction rate via functional cooperation with Sec⁵². Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr¹⁰¹ occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr⁵⁴, Asn⁵⁵, and Gly⁵⁶ to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pK _a shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole–dipole interactions among Trp²⁹–Sec⁵²–Gln⁷² lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.     

Conformational basis of N-glycosylation of proteins: conformational analysis of Ac-Asn-Ala-Thr-NH2

The structural requirements of the Asn-X-Thr(Ser) sequence for the N-glycosylation of proteins has been traced to a local conformation acting as a signal for the enzymatic process. The conformational space of the smallest in vivoN-glycosylation substrate, Ac-Asn-Ala-Thr-NH₂, has been thoroughly explored using energy calculations. All the lowest energy conformers have been characterized as bended structures.     

A molecular dynamics simulation investigation of the relative stability of the cyclic peptide octreotide and its deprotonated and its (CF3)-Trp substituted analogs in different solvents

The cyclic octa-peptide octreotide and its derivatives are used as diagnostics and therapeutics in relation to particular types of cancers. This led to investigations of their conformational properties using spectroscopic, NMR and CD, methods. A CF₃-substituted derivative, that was designed to stabilize the dominant octreotide conformer responsible for receptor binding, turned out to have a lower affinity. The obtained spectroscopic data were interpreted as to show an increased flexibility of the CF₃ derivative compared to the unsubstituted octreotide, which could then explain the lower affinity.In this article, we use MD simulation without and with time-averaged NOE distance and time-averaged local-elevation ³J-coupling restraining representing experimental NMR data to determine the conformational properties of the different peptides in the different solvents for which experimental data are available, that are compatible with the NOE atom–atom distance bounds and the ³J_HNHα-couplings as derived from the NMR measurements. The conformational ensembles show that the CF₃ substitution in combination with the change of solvent from water to methanol leads to a decrease in flexibility and a shift in the populations of the dominant conformers that are compatible with the experimental data.     

Conformational Flexibility of Human Casein Kinase Catalytic Subunit Explored by Metadynamics

Casein kinase CK2 is an essential enzyme in higher organisms, catalyzing the transfer of the γ phosphate from ATP to serine and threonine residues on protein substrates. In a number of animal tumors, CK2 activity has been shown to escape normal cellular control, making it a potential target for cancer therapy. Several crystal structures of human CK2 have been published with different conformations for the CK2α catalytic subunit. This variability reflects a high flexibility for two regions of CK2α: the interdomain hinge region, and the glycine-rich loop (p-loop). Here, we present a computational study simulating the equilibrium between three conformations involving these regions. Simulations were performed using well-tempered metadynamics combined with a path collective variables approach. This provides a reference pathway describing the conformational changes being studied, based on analysis of free energy surfaces. The free energies of the three conformations were found to be close and the paths proposed had low activation barriers. Our results indicate that these conformations can exist in water. This information should be useful when designing inhibitors specific to one conformation.     

Incorporation of side chain flexibility into protein binding pockets using MTflex

The plasticity of active sites plays a significant role in drug recognition and binding, but the accurate incorporation of ‘receptor flexibility’ remains a significant computational challenge. Many approaches have been put forward to address receptor flexibility in docking studies by generating relevant ensembles on the energy surface. Herein, we describe the Movable Type with flexibility (MT_flex) method that generates ensembles on the more relevant free energy surface in a computationally tractable manner. This novel approach enumerates conformational states based on side chain flexibility and then estimates their relative free energies using the MT methodology. The resultant conformational states can then be used in subsequent docking and scoring exercises. In particular, we demonstrate that using the MT_flex ensembles improves MT’s ability to predict binding free energies over docking only to the crystal structure.     

Rotational mobility and domain flexibility of membrane-bound bacterial coupling factor as detected with the triplet probe eosin-isothiocyanate

The membrane-bound coupling factor (BF₁) of chromatophores from the photosynthetic bacerium Rhodopseudomonas sphaeroides was covalently labeled with the triplet probe eosin-isothiocyanate. The labeled enzyme was isolated and functionally reconstituted into depleted chromatophores from the same bacterium. ATPase and ATP synthase activities of the reconstituted vesicles were strongly dependent on the labeling conditions, decreasing at increasing load of eosin molecules per BF₁. When labeling was carried out in the dark and in the presence of ATP, one molecule of eosin isothiocyanate was bound per BF₁ and the activities catalyzed by the reconstituted and labeled enzyme were as high as in untreated chromatophores. Upon light energization of the chromatophore membrane, a large conformational change of BF₁ could be detected by using the triplet probe as a spectroscopic tool. The domain flexibility and rotational mobility of the reconstituted coupling enzyme were directly related to the enhancement of the ATPase activity induced by light. Both the light-stimulated ATPase activity and conformational changes could be prevented by addition of ADP or oligomycin and affected to the same extent by uncouplers and inhibitors of electron transport. Moreover, the detected conformational changes were reversible in time, appearing with a half-time of 10 ms upon illumination of the chromatophores, and disappearing with a half-time of 70 ms in the dark. The results obtained prove the feasibility of the spectroscopic technique in detecting conformational changes of the membrane-bound BF₁, similarly to what already has been observed for chloroplast coupling factor (Wagner, R. and Junge, W. (1980) FEBS Lett. 114, 327–333), and add to the possibility of characterizing, by this method, energy transduction at a molecular level.