Nitric Oxide Dynamics in Truncated Hemoglobin: Docking Sites, Migration Pathways, and Vibrational Spectroscopy from Molecular Dynamics Simulations

Atomistic simulations of nitric oxide (NO) dynamics and migration in the trHbN of Mycobacterium tuberculosis are reported. From extensive molecular dynamics simulations (48 ns in total), the structural and energetic properties of the ligand docking sites in the protein have been characterized and a connectivity network between the ligand docking sites has been built. Several novel migration and exit pathways are found and are analyzed in detail. The interplay between a hydrogen-bonding network involving residues Tyr³³ and Gln⁵⁸ and the bound O₂ ligand is discussed and the role of Phe⁶² residue in ligand migration is examined. It is found that Phe⁶² is directly involved in controlling ligand migration. This is reminiscent of His⁶⁴ in myoglobin, which also plays a central role in CO migration pathways. Finally, infrared spectra of the NO molecule in different ligand docking sites of the protein are calculated. The pocket-specific spectra are typically blue-shifted by 5-10 cm⁻¹, which should be detectable in future spectroscopic experiments.     

Theoretical Investigations of Nitric Oxide Channeling in Mycobacterium tuberculosis Truncated Hemoglobin N

Mycobacterium tuberculosis group I truncated hemoglobin trHbN catalyzes the oxidation of nitric oxide (•NO) to nitrate with a second-order rate constant k ≈ 745 μM⁻¹ s⁻¹ at 23°C (nitric oxide dioxygenase reaction). It was proposed that this high efficiency is associated with the presence of hydrophobic tunnels inside trHbN structure that allow substrate diffusion to the distal heme pocket. In this work, we investigated the mechanisms of •NO diffusion within trHbN tunnels in the context of the nitric oxide dioxygenase reaction using two independent approaches. Molecular dynamics simulations of trHbN were performed in the presence of explicit •NO molecules. Successful •NO diffusion from the bulk solvent to the distal heme pocket was observed in all simulations performed. The simulations revealed that •NO interacts with trHbN at specific surface sites, composed of hydrophobic residues located at tunnel entrances. The entry and the internal diffusion of •NO inside trHbN were performed using the Long, Short, and EH tunnels reported earlier. The Short tunnel was preferentially used by •NO to reach the distal heme pocket. This preference is ascribed to its hydrophobic funnel-shape entrance, covering a large area extending far from the tunnel entrance. This funnel-shape entrance triggers the frequent formation of solvent-excluded cavities capable of hosting up to three •NO molecules, thereby accelerating •NO capture and entry. The importance of hydrophobicity of entrances for •NO capture is highlighted by a comparison with a polar mutant for which residues at entrances were mutated with polar residues. A complete map of •NO diffusion pathways inside trHbN matrix was calculated, and •NO molecules were found to diffuse from Xe cavity to Xe cavity. This scheme was in perfect agreement with the three-dimensional free-energy distribution calculated using implicit ligand sampling. The trajectories showed that •NO significantly alters the dynamics of the key amino acids of Phe⁶²(E15), a residue proposed to act as a gate controlling ligand traffic inside the Long tunnel, and also of Ile¹¹⁹(H11), at the entrance of the Short tunnel. It is noteworthy that •NO diffusion inside trHbN tunnels is much faster than that reported previously for myoglobin. The results presented in this work shed light on the diffusion mechanism of apolar gaseous substrates inside protein matrix.     

Structural characterization of the tunnels of Mycobacterium tuberculosis truncated hemoglobin N from molecular dynamics simulations

The structure of oxygenated trHbN from Mycobacterium tuberculosis shows an extended heme distal hydrogen‐bond network that includes Tyr33(B10), Gln58(E11), and the bound O₂. In addition, trHbN structure shows a network of hydrophobic cavities organized in two orthogonal branches. In the present work, the structure and the dynamics of oxygenated and deoxygenated trHbN in explicit water was investigated from 100 ns molecular dynamics (MD) simulations. Results show that, depending on the presence or the absence of a coordinated O₂, the Tyr33(B10) and Gln58(E11) side chains adopt two different configurations in concert with hydrogen bond network rearrangement. In addition, our data indicate that Tyr33(B10) and Gln58(E11) control the dynamics of Phe62(E15). In deoxy‐trHbN, Phe62(E15) is restricted to one conformation. Upon O₂ binding, the conformation of Gln58(E11) changes and residue Phe62(E15) fluctuates between two conformations. We also conducted a systematic study of trHbN tunnels by analyzing thousands of MD snapshots with CAVER. The results show that tunnel formation is the result of the dynamic reshaping of short‐lived hydrophobic cavities. The analyses indicate that the presence of these cavities is likely linked to the rigid structure of trHbN and also reveal two tunnels, EH and BE, that link the protein surface to the buried distal heme pocket and not present in the crystallographic structure. The cavities are sufficiently large to accomodate and store ligands. Tunnel dynamics in trHbN was found to be controlled by the side‐chain conformation of the Tyr33(B10), Gln58(E11), and Phe62(E15) residues. Importantly, in contrast to recently published works, our extensive systematic studies show that the presence or absence of a coordinated dioxygen does not control the opening of the long tunnel but rather the opening of the EH tunnel. In addition, the data lead to new and distinctly different conclusion on the impact of the Phe62(E15) residue on trHbN tunnels. We propose that the EH and the long tunnels are used for apolar ligands storage. The trajectories bring important new structural insights related to trHbN function and to ligand diffusion in proteins. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Ligand-induced dynamical regulation of NO conversion in Mycobacterium tuberculosis truncated hemoglobin-N

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is forced into latency by nitric oxide produced by macrophages during infection. In response to nitrosative stress M. tuberculosis has evolved a defense mechanism that relies on the oxygenated form of "truncated hemoglobin" N (trHbN), formally acting as NO-dioxygenase, yielding the harmless nitrate ion. X-ray crystal structures have shown that trHbN hosts a two-branched protein matrix tunnel system, proposed to control diatomic ligand migration to the heme, as the rate-limiting step in NO conversion to nitrate. Extended molecular dynamics simulations (0.1 micros), employed here to characterize the factors controlling diatomic ligand diffusion through the apolar tunnel system, suggest that O2 migration in deoxy-trHbN is restricted to a short branch of the tunnel, and that O2 binding to the heme drives conformational and dynamical fluctuations promoting NO migration through the long tunnel branch. The simulation results suggest that trHbN has evolved a dual-path mechanism for migration of O2 and NO to the heme, to achieve the most efficient NO detoxification.     

Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme

Macrophage-generated oxygen- and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis 'truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for approximately 28 A through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.     

Structure and dynamics of Mycobacterium tuberculosis truncated hemoglobin N: insights from NMR spectroscopy and molecular dynamics simulations

The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe2?-O? + (?)NO → trHbN-Fe3?-OH? + NO??) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (?)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (?)NO and O? to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.     

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.     

Influence of Distal Residue B10 on CO Dynamics in Myoglobin and Neuroglobin

For many years, myoglobin has served as a paradigm for structure–function studies in proteins. Ligand binding and migration within myoglobin has been studied in great detail by crystallography and spectroscopy, showing that gaseous ligands such as O₂, CO, and NO not only bind to the heme iron but may also reside transiently in three internal ligand docking sites, the primary docking site B and secondary sites C and D. These sites affect ligand association and dissociation in specific ways. Neuroglobin is another vertebrate heme protein that also binds small ligands. Ligand migration pathways in neuroglobin have not yet been elucidated. Here, we have used Fourier transform infrared temperature derivative spectroscopy at cryogenic temperatures to compare the influence of the side chain volume of amino acid residue B10 on ligand migration to and rebinding from docking sites in myoglobin and neuroglobin.     

Exploring a potential palonosetron allosteric binding site in the 5-HT3 receptor

Palonosetron (Aloxi) is a potent second generation 5-HT₃ receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT₃ receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr⁷³, Phe¹³⁰, Ser¹⁶³, and Asp¹⁶⁵) and in the 5-HT3B receptor subunit (His⁷³, Phe¹³⁰, Glu¹⁷⁰, and Tyr¹⁴³) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT₃A) and heteromeric (5-HT₃AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT₃A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking.     

Distinct Phosphotyrosine-dependent Functions of the ShcA Adaptor Protein Are Required for Transforming Growth Factor β (TGFβ)-induced Breast Cancer Cell Migration,Invasion, and Metastasis

The ErbB2 and TGFβ signaling pathways cooperate to promote the migratory, invasive, and metastatic behavior of breast cancer cells. We previously demonstrated that ShcA is necessary for these synergistic interactions. Through a structure/function approach, we now show that the phosphotyrosine-binding, but not the Src homology 2, domain of ShcA is required for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We further demonstrate that the tyrosine phosphorylation sites within ShcA (Tyr²³⁹/Tyr²⁴⁰ and Tyr³¹³) transduce distinct and non-redundant signals that promote these TGFβ-mediated effects. We demonstrate that Grb2 is required specifically downstream of Tyr³¹³, whereas the Tyr²³⁹/Tyr²⁴⁰ phosphorylation sites require the Crk adaptor proteins to augment TGFβ-induced migration and invasion. Furthermore, ShcA Tyr³¹³ phosphorylation enhances tumor cell survival, and ShcA Tyr²³⁹/Tyr²⁴⁰ signaling promotes endothelial cell recruitment into ErbB2-expressing breast tumors in vivo, whereas all three ShcA tyrosine residues are required for efficient breast cancer metastasis to the lungs. Our data uncover a novel ShcA-dependent signaling axis downstream of TGFβ and ErbB2 that requires both the Grb2 and Crk adaptor proteins to increase the migratory and invasive properties of breast cancer cells. In addition, signaling downstream of specific ShcA tyrosine residues facilitates the survival, vascularization, and metastatic spread of breast tumors.     

Kinetics and computational studies of ligand migration in nitrophorin 7 and its Δ1–3 mutant

Nitrophorins (NPs) are nitric oxide (NO)-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. Though NP7 exhibits a large sequence resemblance with other NPs, two major differential features are the ability to interact with negatively charged cell surfaces and the presence of a specific N-terminus composed of three extra residues (Leu1-Pro2-Gly3). The aim of this study is to examine the influence of the N-terminus on the ligand binding, and the topological features of inner cavities in closed and open states of NP7, which can be associated to the protein structure at low and high pH, respectively. Laser flash photolysis measurements of the CO rebinding kinetics to NP7 and its variant NP7(Δ1–3), which lacks the three extra residues at the N-terminus, exhibit a similar pattern and support the existence of a common kinetic mechanism for ligand migration and binding. This is supported by the existence of a common topology of inner cavities, which consists of two docking sites in the heme pocket and a secondary site at the back of the protein. The ligand exchange between these cavities is facilitated by an additional site, which can be transiently occupied by the ligand in NP7, although it is absent in NP4. These features provide a basis to explain the enhanced internal gas hosting capacity found experimentally in NP7 and the absence of ligand rebinding from secondary sites in NP4. The current data allow us to speculate that the processes of docking to cell surfaces and NO release may be interconnected in NP7, thereby efficiently releasing NO into a target cell. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Localization of a single transglutaminase-reactive glutamine in the third domain of RAP, the alpha2-macroglobulin receptor-associated protein

The 39-kDa receptor-associated protein (RAP) is an intracellular glycoprotein that interacts with hitherto unknown sites in several members of the low-density-lipoprotein receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. In the present study, the transglutaminase-catalyzed incorporation of radioactively labeled putrescine and a dansylated glutamine-containing peptide into human RAP has been studied. The results indicate the presence of both glutamine and lysine residues in RAP, accessible for transglutaminase cross-linking. Moreover, enzymatic digestion followed by sequence analysis of radiolabeled fractions demonstrated that Gln²⁶¹ acts as the amine acceptor site. This residue is located in the third domain of RAP and is conserved among the RAP interspecies homologues. Insertion of a reporter group into the protein could prove useful to assess ligand/receptor interactions.     

Truncated hemoglobins and nitric oxide action

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO3-.     

Ligand interactions in the distal heme pocket of Mycobacterium tuberculosis truncated hemoglobin N: roles of TyrB10 and GlnE11 residues

The crystallographic structure of oxygenated trHbN from Mycobacterium tuberculosis showed an extended heme distal site hydrogen-bonding network that includes Y(B10), Q(E11), and the bound O(2) (Milani, M., et al. (2001) EMBO J. 20, 3902-3909). In the present work, we analyze the effects that substitutions at the B10 and E11 positions exert on the heme and its coordinated ligands, using steady-state resonance Raman spectroscopy, absorption spectroscopy and X-ray crystallography. Our results show that (1) residues Y(B10) and Q(E11) control the binding and the ionization state of the heme-bound water molecules in ferric trHbN and are important in keeping the sixth coordination position vacant in deoxy trHbN; (2) residue Q(E11) plays a role in maintaining the integrity of the proximal Fe-His bond in deoxy trHbN; (3) in wild-type oxy-trHbN, the size and hydrogen-bonding capability of residue E11 is important to sustain proper interaction between Y(B10) and the heme-bound O(2); (4) CO-trHbN is in a conformational equilibrium, where either the Y(B10) or the Q(E11) residue interacts with the heme-bound CO; and (5) Y(B10) and Q(E11) residues control the conformation (and likely the dynamics) of the protein matrix tunnel gating residue F(E15). These findings suggest that the functional processes of ligand binding and diffusion are controlled in trHbN through the dynamic interaction of residues Y(B10), Q(E11), F(E15), and the heme ligand.     

Localization of a Single Transglutaminase-Reactive Glutamine in the Third Domain of RAP, the α2-Macroglobulin Receptor-Associated Protein

The 39-kDa receptor-associated protein (RAP) is an intracellular glycoprotein that interacts with hitherto unknown sites in several members of the low-density-lipoprotein receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. In the present study, the transglutaminase-catalyzed incorporation of radioactively labeled putrescine and a dansylated glutamine-containing peptide into human RAP has been studied. The results indicate the presence of both glutamine and lysine residues in RAP, accessible for transglutaminase cross-linking. Moreover, enzymatic digestion followed by sequence analysis of radiolabeled fractions demonstrated that Gln²⁶¹ acts as the amine acceptor site. This residue is located in the third domain of RAP and is conserved among the RAP interspecies homologues. Insertion of a reporter group into the protein could prove useful to assess ligand/receptor interactions.     

Insights into binding modes of tumstatin peptide T7 with the active site of αvβ3 integrin

Through interaction with the active site of α_vβ₃ integrin, tumstatin T7 peptide inhibits both the angiogenesis and the proliferation of tumour cells. In this work, docking in conjunction with molecular dynamics simulation was used to explore the binding mode of T7 peptide and α_vβ₃ integrin. The binding mode analysis revealed that the residues Ser⁹⁰, Arg⁹¹, Asp⁹³ and Tyr⁹⁴ in T7 peptide, and (α)-Asp¹⁵⁰, (β)-Arg²¹⁴, (α)-Asp¹⁴⁸ (α)-Gln²¹⁴ and (α)-Glu¹²³ in the active site of α_vβ₃ integrin were most likely the key interaction sites. The hydroxyl of Tyr⁹⁴ coordinates α_vβ₃ via a Mn²⁺ ion, revealing that Mn²⁺ is also an important factor for the interaction. The insight into these key interaction sites not only suggests that the active site of α_vβ₃ integrin can bind to molecules through multiple binding mechanisms, but also provides some useful information for structure-based drug design.     

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA^Gln formation. To analyze the peculiarities of the structure–function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3Å and a docking model of the synthetase complexed to tRNA^Gln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA^Gln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA^Gln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA^Gln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.     

Overexpression of genes encoding tRNA<Superscript>Tyr</Superscript> and tRNA<Superscript>Gln</Superscript> increases the viability of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains with nonsense mutations in the <Emphasis Type="Italic">SUP45</Emphasis> gene

At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsense-mutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wild-type tRNA^Tyr and four genes encoding wild-type tRNA^Gln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the full-length eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNA^Tyr and tRNA^Gln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed.     

Genetic analysis of functional connectivity between substrate recognition domains ofEscherichia coli glutaminyl-tRNA synthetase

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA^Gln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3′ end of tRNA^Gln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNA^Tyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNA _CUA ^Gln from tRNA ₂ ^Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K_m for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.     

CO migration pathways in cytochrome P450cam studied by molecular dynamics simulations

Previous laser flash photolysis investigations between 100 and 300 K have shown that the kinetics of CO rebinding with cytochrome P450(cam)(camphor) consist of up to four different processes revealing a complex internal dynamics after ligand dissociation. In the present work, molecular dynamics simulations were undertaken on the ternary complex P450(cam)(cam)(CO) to explore the CO migration pathways, monitor the internal cavities of the protein, and localize the CO docking sites. One trajectory of 1 nsec with the protein in a water box and 36 trajectories of 1 nsec in the vacuum were calculated. In each trajectory, the protein contained only one CO ligand on which no constraints were applied. The simulations were performed at 200, 300, and 320 K. The results indicate the presence of seven CO docking sites, mainly hydrophobic, located in the same moiety of the protein. Two of them coincide with xenon binding sites identified by crystallography. The protein matrix exhibits eight persistent internal cavities, four of which corresponding to the ligand docking sites. In addition, it was observed that water molecules entering the protein were mainly attracted into the polar pockets, far away from the CO docking sites. Finally, the identified CO migration pathways provide a consistent interpretation of the experimental rebinding kinetics.