Molecular dynamics of a thermostable multicopper oxidase from Thermus thermophilus HB27: structural differences between the apo and holo forms

Molecular dynamic (MD) simulations have been performed on Tth-MCO, a hyperthermophilic multicopper oxidase from thermus thermophilus HB27, in the apo as well as the holo form, with the aim of exploring the structural dynamic properties common to the two conformational states. According to structural comparison between this enzyme and other MCOs, the substrate in process to electron transfer in an outer-sphere event seems to transiently occupy a shallow and overall hydrophobic cavity near the Cu type 1 (T1Cu). The linker connecting the β-strands 21 and 24 of the second domain (loop (β21-β24)(D2)) has the same conformation in both states, forming a flexible lid at the entrance of the electron-transfer cavity. Loop (β21-β24)(D2) has been tentatively assigned a role occluding the access to the electron-transfer site. The dynamic of the loop (β21-β24)(D2) has been investigated by MD simulation, and results show that the structures of both species have the same secondary and tertiary structure during almost all the MD simulations. In the simulation, loop (β21-β24)(D2) of the holo form undergoes a higher mobility than in the apo form. In fact, loop (β21-β24)(D2) of the holo form experiences a conformational change which enables exposure to the electron-transfer site (open conformation), while in the apo form the opposite effect takes place (closed conformation). To confirm the hypothesis that the open conformation might facilitate the transient electron-donor molecule occupation of the site, the simulation was extended another 40 ns with the electron-donor molecule docked into the protein cavity. Upon electron-donor molecule stabilization, loops near the cavity reduce their mobility. These findings show that coordination between the copper and the protein might play an important role in the general mobility of the enzyme, and that the open conformation seems to be required for the electron transfer process to T1Cu.     

Homology modeling, simulation and molecular docking studies of catechol-2, 3-Dioxygenase from Burkholderia cepacia: Involved in degradation of Petroleum hydrocarbons

Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene, Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å. All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of catalyzing the above-mentioned substrate.     

Hsc70-interacting HPD loop of the J domain of polyomavirus T antigens fluctuates in ps to ns and micros to ms

The backbone dynamics of the J domain from polyomavirus T antigens have been investigated using 15N NMR relaxation and molecular dynamics simulation. Model-free relaxation analysis revealed picosecond to nanosecond motions in the N terminus, the I-II loop, the C-terminal end of helix II through the HPD loop to the beginning of helix III, and the C-terminal end of helix III to the C terminus. The backbone dynamics of the HPD loop and termini are dominated by motions with moderately large amplitudes and correlation times of the order of a nanosecond or longer. Conformational exchange on the microsecond to millisecond timescale was identified in the HPD loop, the N and C termini, and the I-II loop. A 9.7ns MD trajectory manifested concerted swings of the HPD loop. Transitions between major and minor conformations of the HPD loop featured distinct patterns of change in backbone dihedral angles and hydrogen bonds. Fraying of the C-terminal end of helix II and the N-terminal end of helix III correlated with displacements of the HPD loop. Correlation of crankshaft motions of Gly46 and Gly47 with the collective motions of the HPD loop suggested an important role of the two glycine residues in the mobility of the loop. Fluctuations of the HPD loop correlated with relative reorientation of side-chains of Lys35 and Asp44 that interact with Hsc70.     

Molecular basis for the substrate stereoselectivity in tryptophan dioxygenase

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are the only two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine. While human IDO is able to oxidize both L- and D-Trp, human TDO (hTDO) displays major specificity for L-Trp. In this work, we aim to interrogate the molecular basis for the substrate stereoselectivity of hTDO. Our previous molecular dynamics simulation studies of Xanthomonas campestris TDO (xcTDO) showed that a hydrogen bond between T254 (T342 in hTDO) and the ammonium group of the substrate is present in the L-Trp-bound enzyme, but not in the D-Trp-bound enzyme. The fact that this is the only notable structural alteration induced by the change in the stereo structure of the substrate prompted us to produce and characterize the T342A mutant of hTDO to evaluate the structural role of T342 in controlling the substrate stereoselectivity of the enzyme. The experimental results indicate that the mutation only slightly perturbs the global structural properties of the enzyme but totally abolishes the substrate stereoselectivity. Molecular dynamics simulations of xcTDO show that T254 controls the substrate stereoselectivity of the enzyme by (i) modulating the hydrogen bonding interaction between the NH(3)(+) group and epoxide oxygen of the ferryl-indole 2,3-epoxide intermediate of the enzyme and (ii) regulating the dynamics of two active site loops, loop(250-260) and loop(117-130), critical for substrate binding.     

Flexibility of prolyl oligopeptidase: molecular dynamics and molecular framework analysis of the potential substrate pathways

The flexibility of prolyl oligopeptidase has been investigated using molecular dynamics (MD) and molecular framework approaches to delineate the route of the substrate to the active site. The selectivity of the enzyme is mediated by a seven-bladed beta-propeller that in the crystal structure does not indicate the possible passage for the substrate to the catalytic center. Its open topology however, could allow the blades to move apart and let the substrate into the large central cavity. Flexibility analysis of prolyl oligopeptidase structure using the FIRST (Floppy Inclusion and Rigid Substructure Topology) approach and the atomic fluctuations derived from MD simulations demonstrated the rigidity of the propeller domain, which does not permit the substrate to approach the active site through this domain. Instead, a smaller tunnel at the inter-domain region comprising the highly flexible N-terminal segment of the peptidase domain and a facing hydrophilic loop from the propeller (residues 192-205) was identified by cross-correlation analysis and essential dynamics as the only potential pathway for the substrate. The functional importance of the flexible loop has been also verified by kinetic analysis of the enzyme with a split loop. Catalytic effect of engineered disulfide bridges was rationalized by characterizing the concerted motions of the two domains.     

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.     

Multiple Molecular Dynamics Simulations of TEM β-Lactamase: Dynamics and Water Binding of the Ω-Loop

The Ω-loop of TEM β-lactamase is involved in substrate recognition and catalysis. Its dynamical properties and interaction with water molecules were investigated by performing multiple molecular dynamics simulations of up to 50 ns. Protein flexibility was assessed by calculating the root mean-square fluctuations and the generalized order parameter, S². The residues in secondary structure elements are highly ordered, whereas loop regions are more flexible, which is in agreement with previous experimental observations. Interestingly, the Ω-loop (residues 161-179) is rigid with order parameters similar to secondary structure elements, with the exception of the tip of the loop (residues 173-177) that has a considerably higher flexibility and performs an opening and closing motion on the 50-ns timescale. The rigidity of the main part of the Ω-loop is mediated by stabilizing and highly conserved water bridges inside a cavity lined by the Ω-loop and residues 65-69 of the protein core. In contrast, the flexible tip of the Ω-loop lacks these interactions. Hydration of the cavity and exchange of the water molecules with the bulk solvent occurs via two pathways: the flexible tip that serves as a door to the cavity, and a temporary water channel involving the side chain of Arg¹⁶⁴.     

Molecular dynamics studies of the full-length integrase-DNA complex

We have carried out a molecular dynamics (MD) simulation of full-length HIV-1 integrase (IN) dimer complexed with viral DNA with the aim of gaining information about the enzyme motion and investigating the movement of the catalytic flexible loop (residues 140-149) thought to be essential in the catalytic mechanism of IN. During the simulation, we observed quite a different behavior of this region in the presence or absence of the viral DNA. In particular, the MD results underline the crucial role of the residue Tyr143 in the mechanism of integration of viral DNA into the host chromosome. The present findings confirm the experimental data (e.g., site-directed mutagenesis experiments) showing that the loop is involved in the integration reactions and its mobility is correlated with the catalytic activity of HIV-1 integrase.     

Molecular dynamics simulation of class 3 aldehyde dehydrogenase

Molecular dynamics (MD) simulation of the rat class 3 aldehyde dehydrogenase (ALDH) with nicotinamide dinucleotide (NAD) cofactors and explicit water molecules are reported. Our results demonstrate that MD simulation using the latest methodologies can maintain the crystal structure of the enzyme, as well as closely reproduce the short timescale dynamics of the enzyme. Furthermore, the examination of the distance between the nucleophilic Cys-243 and the NAD cofactor reveal important fluctuations that could be linked to ALDH catalysis. Finally, our quantum mechanical model of benzaldehyde in the active site of ALDH demonstrates that the enzyme requires only minor conformational changes to be poised for nucleophilic attack on the substrate.     

Molecular dynamics simulation of class 3 aldehyde dehydrogenase

Molecular dynamics (MD) simulation of the rat class 3 aldehyde dehydrogenase (ALDH) with nicotinamide dinucleotide (NAD) cofactors and explicit water molecules are reported. Our results demonstrate that MD simulation using the latest methodologies can maintain the crystal structure of the enzyme, as well as closely reproduce the short timescale dynamics of the enzyme. Furthermore, the examination of the distance between the nucleophilic Cys-243 and the NAD cofactor reveal important fluctuations that could be linked to ALDH catalysis. Finally, our quantum mechanical model of benzaldehyde in the active site of ALDH demonstrates that the enzyme requires only minor conformational changes to be poised for nucleophilic attack on the substrate.     

Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex

The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.     

Functional analysis of the copper-dependent quercetin 2,3-dioxygenase. 2. X-ray absorption studies of native enzyme and anaerobic complexes with the substrates quercetin and myricetin

Quercetin 2,3-dioxygenase (2,3QD) is a mononuclear copper-dependent dioxygenase which catalyzes the cleavage of the heterocyclic ring of the flavonol quercetin (5,7,3',4'-tetrahydroxy flavonol) to produce 2-protocatechuoyl-phloroglucinol carboxylic acid and carbon monoxide. In this study, X-ray absorption spectroscopy has been used to characterize the local structural environment of the Cu(2+) center of Aspergillus japonicus 2,3QD. Analysis of the EXAFS region of native 2,3QD at functionally relevant pH (pH 6.0) indicates an active site equally well-described by either four or five ligands (3N(His) + 1-2O) at an average distance of 2.00 A. Bond valence sum analysis confirms that the best model is somewhere between the two. When, however, 2,3QD is anaerobically complexed with its natural substrate quercetin, the copper environment undergoes a transition to a five-coordinated cage, which is also best modeled by a single shell of N/O scatterers at the average distance of 2.00 A. This coordination is independently confirmed by the anaerobic complex with myricetin (5'-hydroxy quercetin). XANES analysis confirms that substrate binding does not reduce the Cu(2+) ion. The present study gives the first direct insights into the coordination chemistry of the enzyme complexed with its substrates. It suggests that activation for O(2) attack is achieved by monodentate substrate complexation to the copper ion through the 3-hydroxyl group. In addition, monodentate carboxylate ligation by the Glu73 side chain is likely to play a role in the fine-tuning of the equilibrium leading to the formation of the activated E.S complex.     

Brownian and essential dynamics studies of the HIV-1 integrase catalytic domain

The three-dimensional structure of the active site region of the enzyme HIV-1 integrase is not unambiguously known. This region includes a flexible peptide loop that cannot be well resolved in crystallographic determinations. Here we present two different computational approaches with different levels of resolution and on different time-scales to understand this flexibility and to analyze the dynamics of this part of the protein. We have used molecular dynamics simulations with an atomic model to simulate the region in a realistic and reliable way for 1 ns. It is found that parts of the loop wind up after 300 ps to extend an existing helix. This indicates that the helix is longer than in the earlier crystal structures that were used as basis for this study. Very recent crystal data confirms this finding, underlining the predictive value of accurate MD simulations. Essential dynamics analysis of the MD trajectory yields an anharmonic motion of this loop. We have supplemented the MD data with a much lower resolution Brownian dynamics simulation of 600 ns length. It provides ideas about the slow-motion dynamics of the loop. It is found that the loop explores a conformational space much larger than in the MD trajectory, leading to a "gating"-like motion with respect to the active site.     

A molecular dynamics analysis of protein structural elements

The relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, beta-sheet, and loop structural elements in free and substrate-bound hen egg-white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and beta-sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of the loop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate-bound simulation. A loop region (residues 101-107) of the active site cleft had significantly reduced motion due to interactions with the substrate. Moreover, part of a loop and a 3(10) helix (residues of 67-88) not in contact with the substrate showed a marked increase in fluctuations. That these differences in dynamics of free and substrate-bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuations of the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain phi and psi angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place either abruptly, in 1 psec or less, or with a diffusive character over 5-10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 A rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface area and the dynamics of the different structural elements.     

Glycine-Rich Loop of Mitochondrial Processing Peptidase α-Subunit Is Responsible for Substrate Recognition by a Mechanism Analogous to Mitochondrial Receptor Tom20

Tryptophan fluorescence measurements were used to characterize the local dynamics of the highly conserved glycine-rich loop (GRL) of the mitochondrial processing peptidase (MPP) α-subunit in the presence of the substrate precursor. Reporter tryptophan residue was introduced into the GRL of the yeast α-MPP (Y299W) or at a proximal site (Y303W). Time-resolved and steady-state fluorescence spectroscopy demonstrated that for Trp299, the primary contact with the yeast malate dehydrogenase precursor evokes a change of the local GRL mobility. Moreover, time-resolved measurements showed that a functionless α-MPP with a single-residue deletion in the loop (Y303W/ΔG292) is defective particularly in the primary contact with substrate. Thus, the GRL was proved to be part of a contact site of the enzyme specifically recognizing the substrate. Regarding the surface exposure and presence of the hydrophobic patches within the GRL, we proposed a functional analogy between the presequence recognition by the hydrophobic binding groove of the Tom20 mitochondrial import receptor and the GRL of the α-MPP. A molecular dynamics (MD) simulation of the MPP-substrate peptide complex model was employed to test this hypothesis. The initial positioning and conformation of the substrate peptide in the model fitting were chosen based on the analogy of its interaction with the Tom20 binding groove. MD simulation confirmed the stability of the proposed interaction and showed also a decrease in GRL flexibility in the presence of substrate, in agreement with fluorescence measurements. Moreover, conserved substrate hydrophobic residues in positions + 1 and − 4 to the cleavage site remain in close contact with the side chains of the GRL during the entire production part of MD simulation as stabilizing points of the hydrophobic interaction. We conclude that the GRL of the MPP α-subunit is the crucial evolutional outcome of the presequence recognition by MPP and represents a functional parallel with Tom20 import receptor.     

A molecular dynamics study of Beta-Glucosidase B upon small substrate binding

Paenibacillus polymyxa β-glucosidase B (BglB), belongs to a GH family 1, is a monomeric enzyme that acts as an exo-β-glucosidase hydrolysing cellobiose and cellodextrins of higher degree of polymerization using retaining mechanism. A molecular dynamics (MD) simulation was performed at 300 K under periodic boundary condition for 5 ns using the complexes structure obtained from previous docking study, namely BglB-Beta-d-glucose and BglB-Cellobiose. From the root-mean-square deviation analysis, both enzyme complexes were reported to deviate from the initial structure in the early part of the simulation but it was stable afterwards. The root-mean-square fluctuation analysis revealed that the most flexible regions comprised of the residues from 26 to 29, 43 to 53, 272 to 276, 306 to 325 and 364 to 367. The radius of gyration analysis had shown the structure of BglB without substrate became more compact towards the end of the simulation compare to other two complexes. The residues His122 and Trp410 were observed to form stable hydrogen bond with occupancy higher than 10%. In conclusion, the behaviour of BglB enzyme towards the substrate binding was successfully explored via MD simulation approaches.     

Brownian and Essential Dynamics Studies of the HIV-1 Integrase Catalytic Domain

Abstract

The three-dimensional structure of the active site region of the enzyme HIV-1 integrase is not unambiguously known. This region includes a flexible peptide loop that cannot be well resolved in crystallographic determinations. Here we present two different computional approaches with different levels of resolution and on different time-scales to understand this flexibility and to analyze the dynamics of this part of the protein. We have used molecular dynamics simulations with an atomic model to simulate the region in a realistic and reliable way for 1 ns. It is found that parts of the loop wind up after 300 ps to extend an existing helix. This indicates that the helix is longer than in the earlier crystal structures that were used as basis for this study. Very recent crystal data confirms this finding, underlining the predictive value of accurate MD simulations. Essential dynamics analysis of the MD trajectory yields an anharmonic motion of this loop. We have supplemented the MD data with a much lower resolution Brownian dynamics simulation of 600 ns length. It provides ideas about the slow-motion dynamics of the loop. It is found that the loop explores a conformational space much larger than in the MD trajectory, leading to a “gating”-like motion with respect to the active site.     

Inter-domain movements in polyketide synthases: a molecular dynamics study

Insights into the structure and dynamics of modular polyketide synthases (PKS) are essential for understanding the mechanistic details of the biosynthesis of a large number of pharmaceutically important secondary metabolites. The crystal structures of the KS-AT di-domain from erythromycin synthase have revealed the relative orientation of various catalytic domains in a minimal PKS module. However, the relatively large distance between catalytic centers of KS and AT domains in the static structure has posed certain intriguing questions regarding mechanistic details of substrate transfer during polyketide biosynthesis. In order to investigate the role of inter-domain movements in substrate channeling, we have carried out a series of explicit solvent MD simulations for time periods ranging from 10 to 15 ns on the KS-AT di-domain and its sub-fragments. Analyses of these MD trajectories have revealed that both the catalytic domains and the structured inter-domain linker region remain close to their starting structures. Inter-domain movements at KS-linker and linker-AT interfaces occur around hinge regions which connect the structured linker region to the catalytic domains. The KS-linker interface was found to be more flexible compared to the linker-AT interface. However, inter-domain movements observed during the timescale of our simulations do not significantly reduce the distance between catalytic centers of KS and AT domains for facilitating substrate channeling. Based on these studies and prediction of intrinsic disorder we propose that the intrinsically unstructured linker stretch preceding the ACP domain might be facilitating movement of ACP domains to various catalytic centers.