Coupling of quinone dynamics to proton pumping in respiratory complex I

Respiratory complex I (NADH:quinone oxidoreductase) plays a central role in generating the proton electrochemical gradient in mitochondrial and bacterial membranes, which is needed to generate ATP. Several high-resolution structures of complex I have been determined, revealing its intricate architecture and complementing the biochemical and biophysical studies. However, the molecular mechanism of long-range coupling between ubiquinone (Q) reduction and proton pumping is not known. Computer simulations have been applied to decipher the dynamics of Q molecule in the ~30 Å long Q tunnel. In this short report, we discuss the binding and dynamics of Q at computationally predicted Q binding sites, many of which are supported by structural data on complex I. We suggest that the binding of Q at these sites is coupled to proton pumping by means of conformational rearrangements in the conserved loops of core subunits.     

The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction

Iron–sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only ～ 7 Å in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q₁ and Q₂ that were coupled to proton pumping. Apparent K_m values for Q₁ and Q₂ were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron–sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.     

Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex

In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y. lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second β-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y. lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function.     

How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics

The respiratory complex I is a redox-driven proton pump that employs the free energy released from quinone reduction to pump protons across its complete ca. 200?Å wide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear. Here we combine large-scale molecular simulations with quantum chemical density functional theory (DFT) models to study how contacts between neighboring antiporter-like subunits in the membrane domain of complex I affect the proton transfer energetics. Our combined results suggest that opening of conserved Lys/Glu ion pairs within each antiporter-like subunit modulates the barrier for the lateral proton transfer reactions. Our work provides a mechanistic suggestion for key coupling effects in the long-range force propagation process of complex I.     

The structure of eukaryotic and prokaryotic complex I

The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.     

Tracing the tail of ubiquinone in mitochondrial complex I

Mitochondrial complex I (proton pumping NADH:ubiquinone oxidoreductase) is the largest and most complicated component of the respiratory electron transfer chain. Despite its central role in biological energy conversion the structure and function of this membrane integral multiprotein complex is still poorly understood. Recent insights into the structure of complex I by X-ray crystallography have shown that iron–sulfur cluster N2, the immediate electron donor for ubiquinone, resides about 30 Å above the membrane domain and mutagenesis studies suggested that the active site for the hydrophobic substrate is located next to this redox-center. To trace the path for the hydrophobic tail of ubiquinone when it enters the peripheral arm of complex I, we performed an extensive structure/function analysis of complex I from Yarrowia lipolytica monitoring the interaction of site-directed mutants with five ubiquinone derivatives carrying different tails. The catalytic activity of a subset of mutants was strictly dependent on the presence of intact isoprenoid moieties in the tail. Overall a consistent picture emerged suggesting that the tail of ubiquinone enters through a narrow path at the interface between the 49-kDa and PSST subunits. Most notably we identified a set of methionines that seems to form a hydrophobic gate to the active site reminiscent to the M-domains involved in the interaction with hydrophobic targeting sequences with the signal recognition particle of the endoplasmic reticulum. Interestingly, two of the amino acids critical for the interaction with the ubiquinone tail are different in bovine complex I and we could show that one of these exchanges is responsible for the lower sensitivity of Y. lipolytica complex I towards the inhibitor rotenone. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

How an Inhibitor Bound to Subunit Interface Alters Triosephosphate Isomerase Dynamics

The tunnel region at triosephosphate isomerase (TIM)’s dimer interface, distant from its catalytic site, is a target site for certain benzothiazole derivatives that inhibit TIM’s catalytic activity in Trypanosoma cruzi, the parasite that causes Chagas disease. We performed multiple 100-ns molecular-dynamics (MD) simulations and elastic network modeling (ENM) on both apo and complex structures to shed light on the still unclear inhibitory mechanism of one such inhibitor, named bt10. Within the time frame of our MD simulations, we observed stabilization of aromatic clusters at the dimer interface and enhancement of intersubunit hydrogen bonds in the presence of bt10, which point to an allosteric effect rather than destabilization of the dimeric structure. The collective dynamics dictated by the topology of TIM is known to facilitate the closure of its catalytic loop over the active site that is critical for substrate entrance and product release. We incorporated the ligand’s effect on vibrational dynamics by applying mixed coarse-grained ENM to each one of 54,000 MD snapshots. Using this computationally efficient technique, we observed altered collective modes and positive shifts in eigenvalues due to the constraining effect of bt10 binding. Accordingly, we observed allosteric changes in the catalytic loop’s dynamics, flexibility, and correlations, as well as the solvent exposure of catalytic residues. A newly (to our knowledge) introduced technique that performs residue-based ENM scanning of TIM revealed the tunnel region as a key binding site that can alter global dynamics of the enzyme.     

Proton motive function of the terminal antiporter-like subunit in respiratory complex I

In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.     

Global collective motions in the mammalian and bacterial respiratory complex I

The respiratory complex I is an enzyme responsible for the conversion of chemical energy into an electrochemical proton motive force across the membrane. Despite extensive studies, the mechanism by which the activity of this enormous, ca. 1?MDa, redox-coupled proton pump is regulated still remains unclear. Recent structural studies (Zhu et al., Nature 2016; Fiedorczuk et al., Nature 2016) resolved complex I in different conformations connected to the active-to-deactive (A/D) transition that regulate complex I activity in several species. Based on anisotropic network models (ANM) and principal component analysis (PCA), we identify here transitions between experimentally resolved structures of the mammalian complex I as low-frequency collective motions of the enzyme, highlighting similarities and differences between the bacterial and mammalian enzymes. Despite the reduced complexity of the smaller bacterial enzyme, our results suggest that the global dynamics of complex I is overall conserved. We further probe how the supernumerary subunits could be involved in the modulation of the A/D-transition, and show that in particular the 42?kDa and B13 subunits affect the global motions of the mammalian enzyme.     

Proton uptake associated with the reduction of the primary quinone Q(A) influences the binding site of the secondary quinone Q(B) in Rhodopseudomonas viridis photosynthetic reaction centers

Previously, two binding sites for the secondary quinone Q(B) in the photosynthetic reaction center (RC) from Rhodopseudomonas viridis were identified by X-ray crystallography, a 'proximal' binding site close to the non-heme iron, and a 'distal' site, displaced by 4.2 A along the path of the isoprenoid tail [C.R.D. Lancaster and H. Michel, Structure 5 (1997) 1339-1359]. The quinone ring planes in the two sites differ by roughly a 180 degrees rotation around the isoprenoid tail. Here we present molecular dynamics simulations, which support the theory of a spontaneous transfer of Q(B) between the distal site and the proximal site. In contrast to earlier computational studies on RCs, the molecular dynamics simulations of Q(B) migration resulted in a proximal Q(B) binding pattern identical to that of the crystallographic findings. Also, we demonstrate that the preference towards the proximal Q(B) location is not necessarily attributed to reduction of Q(B) to the semiquinone, but already to the preceding reduction of the primary quinone Q(A) and resulting protonation changes in the protein. Energy mapping of the Q(B) binding pocket indicates that the quinone ring rotation required for completion of the transfer between the two sites is improbable at the distal or proximal binding sites due to high potential barriers, but may be possible at a newly identified position near the distal binding site.     

Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation

The oleaginous yeast Yarrowia lipolytica is known to inhabit various lipid-containing environments. One of the most striking features in this yeast is the presence of several multigene families involved in the metabolic pathways of hydrophobic substrate utilization. The complexity and the multiplicity of these genes give Y. lipolytica a wide capability range towards hydrophobic substrate (HS) utilization and storage. The combination of the increasing knowledge of this yeast's metabolism and the development of more efficient genetic tools is offering new perspectives in using Y. lipolytica as a model organism to study the mechanisms involved in lipid metabolism associated to fat uptake, storage, deposition, mobilization and regulation. Nutrient status and culture conditions seem to play a major role in obesity.     

Unidirectional peristaltic movement in multisite drug binding pockets of AcrB from molecular dynamics simulations

Tripartite complex AcrB-ToIC, the major efflux system in Escherichia coli, is the principal multidrug transporter in Gram-negative bacteria, which is important in antibiotic drug tolerance. AcrB is a homotrimer that acts as a tripartite complex with the outer membrane channel ToIC and the membrane fusion protein AcrA. Recently, the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin were reported. Here we performed 20 ns molecular dynamics (MD) simulations of the AcrB-rifampicin-minocycline complex in a lipid bilayer and explicit water. We found that the bound drugs, rifampicin and erythromycin, made a unidirectional peristaltic movement towards the extrusion funnel of ToIC, which was facilitated by the water efflux in the channel of AcrB. With a shift of the Phe-617 loop, rifampicin in the access monomer moved towards the entrance of the distal binding pocket. Minocycline in the binding monomer moved from the distal binding pocket towards the gate of the central funnel. The channel between the entrance and the gate made a concerted opening during the MD simulations, which was helpful for the peristaltic movement. Our results showed that the mutations of Gly616Pro and Gly619Pro prevented the movement of the Phe-617 loop, which indicated the critical role of the flexibility of the Phe-617 loop. In addition, three putative proton translocation channels were proposed based on our results. Our study provided dynamical information and important residues for the peristaltic movement in AcrB, which were critical for substrate uptake and extrusion function.     

Isolation of bioactive peptides from tryptone that modulate lipase production in Yarrowia lipolytica

In this work the effect of several organic nitrogen sources on lipase production in Yarrowia lipolytica LgX64.81 overproducing mutant was studied. Among them, tryptone and peptone showed the most prominent stimulatory effect. Interestingly, only tryptic and peptic casein digest were found to highly induce lipase biosynthesis while lipase production was very limited in the presence of casein digest from papain and pronase-catalysed hydrolysis and absent in case of chymotryptic digest. It was also demonstrated that the stimulatory peptides should be present in the culture medium at specific proportions and molecular size to match the physiological requirement of Yarrowia lipolytica strain for lipase biosynthesis.     

Quinol oxidation by c-type cytochromes: structural characterization of the menaquinol binding site of NrfHA

Membrane-bound cytochrome c quinol dehydrogenases play a crucial role in bacterial respiration by oxidizing menaquinol and transferring electrons to various periplasmic oxidoreductases. In this work, the menaquinol oxidation site of NrfH was characterized by the determination of the X-ray structure of Desulfovibrio vulgaris NrfHA nitrite reductase complex bound to 2-heptyl-4-hydroxyquinoline-N-oxide, which is shown to act as a competitive inhibitor of NrfH quinol oxidation activity. The structure, at 2.8-Å resolution, reveals that the inhibitor binds close to NrfH heme 1, where it establishes polar contacts with two essential residues: Asp89, the residue occupying the heme distal ligand position, and Lys82, a strictly conserved residue. The menaquinol binding cavity is largely polar and has a wide opening to the protein surface. Coarse-grained molecular dynamics simulations suggest that the quinol binding site of NrfH and several other respiratory enzymes lie in the head group region of the membrane, which probably facilitates proton transfer to the periplasm. Although NrfH is not a multi-span membrane protein, its quinol binding site has several characteristics similar to those of quinone binding sites previously described. The data presented here provide the first characterization of the quinol binding site of the cytochrome c quinol dehydrogenase family.     

The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones

The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such “alternative” NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C₁₂ (HDQ) and C₁₄ are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C₅ and C₆) displayed significantly higher IC₅₀ values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.     

Lipid a from lipopolysaccharide recognition: Structure,dynamics and cooperativity by molecular dynamics simulations

Molecular dynamics simulations of Lipid A and its natural precursor Lipid IVA from E.coli have been carried out free in solution, bound to the myeliod differentiation protein 2 (MD2) and in the complex of MD2 with the toll like receptor 4 (TLR4). In addition, simulations of the ligand free MD2 and MD2‐TLR4 complex were performed. A structural and energetic characterization of the bound and unbound states of Lipid A/IVA was generated. As the crystal structures depict, the main driving force for MD2‐Lipid A/IVA are the hydrophobic interactions between the aliphatic tails and the MD2 cavity. The charged phosphate groups do strongly interact with positively charged residues, located at the surface of MD2. However, they are not essential for keeping the lipids in the cavity, indicating a more prominent role in binding recognition and ionic interactions with TLR4 at the MD2/TLR4 interface. Interestingly, in the absence of any ligand MD2 rapidly closes, blocking the binding cavity. The presence of TLR4, though changing the dynamics, was not able to impede the aforementioned closing event. We hypothesize that fluctuations of the H1 region are essential for this phenomenon, and it is plausible that an equilibrium between the open and closed states exists, although the lengths of our simulations are not sufficient to encompass the reversible process. The MD2/Lipid A‐TLR4 complex simulations show that the presence of the ligand energetically stabilizes the complex relative to the ligand‐free structures, indicating cooperativity in the binding process. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Can molecular dynamics simulations improve the structural accuracy and virtual screening performance of GPCR models?

The determination of G protein-coupled receptor (GPCR) structures at atomic resolution has improved understanding of cellular signaling and will accelerate the development of new drug candidates. However, experimental structures still remain unavailable for a majority of the GPCR family. GPCR structures and their interactions with ligands can also be modelled computationally, but such predictions have limited accuracy. In this work, we explored if molecular dynamics (MD) simulations could be used to refine the accuracy of in silico models of receptor-ligand complexes that were submitted to a community-wide assessment of GPCR structure prediction (GPCR Dock). Two simulation protocols were used to refine 30 models of the D₃ dopamine receptor (D₃R) in complex with an antagonist. Close to 60 μs of simulation time was generated and the resulting MD refined models were compared to a D₃R crystal structure. In the MD simulations, the receptor models generally drifted further away from the crystal structure conformation. However, MD refinement was able to improve the accuracy of the ligand binding mode. The best refinement protocol improved agreement with the experimentally observed ligand binding mode for a majority of the models. Receptor structures with improved virtual screening performance, which was assessed by molecular docking of ligands and decoys, could also be identified among the MD refined models. Application of weak restraints to the transmembrane helixes in the MD simulations further improved predictions of the ligand binding mode and second extracellular loop. These results provide guidelines for application of MD refinement in prediction of GPCR-ligand complexes and directions for further method development.     

Molecular Basis for the Dissociation Dynamics of Protein A-Immunoglobulin G1 Complex

Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.     

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.     

Molecular dynamics simulations of sarcin-ricin rRNA motif

Explicit solvent molecular dynamics (MD) simulations were carried out for sarcin–ricin domain (SRD) motifs from 23S (Escherichia coli) and 28S (rat) rRNAs. The SRD motif consists of GAGA tetraloop, G-bulged cross-strand A-stack, flexible region and duplex part. Detailed analysis of the overall dynamics, base pairing, hydration, cation binding and other SRD features is presented. The SRD is surprisingly static in multiple 25 ns long simulations and lacks any non-local motions, with root mean square deviation (r.m.s.d.) values between averaged MD and high-resolution X-ray structures of 1–1.4 Å. Modest dynamics is observed in the tetraloop, namely, rotation of adenine in its apex and subtle reversible shift of the tetraloop with respect to the adjacent base pair. The deformed flexible region in low-resolution rat X-ray structure is repaired by simulations. The simulations reveal few backbone flips, which do not affect positions of bases and do not indicate a force field imbalance. Non-Watson–Crick base pairs are rigid and mediated by long-residency water molecules while there are several modest cation-binding sites around SRD. In summary, SRD is an unusually stiff rRNA building block. Its intrinsic structural and dynamical signatures seen in simulations are strikingly distinct from other rRNA motifs such as Loop E and Kink-turns.