Insight into the intrinsic flexibility of the SL1 stem-loop from genomic RNA of HIV-1 as probed by molecular dynamics simulation

The SL1 stem-loop is the dimerization initiation site for linking the two copies of the RNA forming the HIV-1 genome. The 26 nucleotides stem contains a defect consisting on a highly conserved G-rich 1-3 asymmetrical internal loop, which is a major site for nucleocapsid protein binding. Several NMR attempts were undertaken to determine the internal loop structure in the SL1 monomer. However, the RNA constructs used in the different studies were largely mutated, in particular with replacement of the nine nucleotides apical loop by a tetraloop, and divergent results were obtained ranging from a rigid structure to a particularly large flexibility. To investigate the reasons for such discrepancies, we used molecular dynamics simulation of the SL1 monomer to probe the effect of mutations on the conformational stability of the internal loop and of the whole stem. It is found that in the wild-type sequence, the internal loop displays conformational variability originating mainly from the nine nucleotides apical loop flexibility that causes large conformational fluctuations (without changing the average structure) in the 7 bp duplex linking the apical and internal loops. The large amplitude atomic motions in the duplex are transmitted to the internal loop in which they induce conformational changes characterized by a labile hydrogen bond network such as G5 successively H-bonded to A29 and G30. On the contrary, with a four nucleotides apical loop, conformational fluctuations in the duplex are reduced by a factor of 2 and are not sufficiently energizing for promoting changes in the internal loop structure at the time scale of the simulations.     

On the stability of different experimental dimeric structures of the SL1 sequence from the genomic RNA of HIV-1 in solution: a molecular dynamics simulation and electrophoresis study

SL1 is a stem-loop RNA sequence from the genome of HIV-1 thought to be the initiation site for the dimerization of the retroviral genomic RNA. The aim of this study is to check the stability in solution of different experimental dimeric structures available in the literature. Two kinds of dimer have been evidenced: an extended duplex looking like a double helix with two internal bulges and a kissing complex in which the monomers with a stem/loop conformation are linked by intermolecular loop-loop interactions. Two divergent experimental structures of the kissing complex from the Lai isolate are reported in the literature, one obtained from NMR (Mujeeb et al., Nature Structural Biology, 1998, Vol. 5, pp. 432-436) and the other one from x-ray crystallography (Ennifar et al., Nature Structural Biology, 2001, Vol. 8, pp. 1064-1068). A crystallographic structure of the Mal isolate was also reported (Ennifar et al., Nature Structure Biology, 2001, Vol. 8, pp. 1064-1068). Concerning the extended duplex, a NMR structure is available for Lai (Girard et al., Journal of Biomolecular Structure and Dynamics, 1999, Vol. 16, pp. 1145-1157) and a crystallographic structure for Mal (Ennifar et al., Structure, 1999, Vol. 7, pp. 1439-1449). Using a molecular dynamics technique, all these experimental structures have been simulated in solution with explicit water and counterions. We show that both extended duplex structures are stable. On the contrary, the crystallographic structures of the Lai and Mal kissing complexes are rapidly destabilized in aqueous environment. Finally, the NMR structure of the Lai loop-loop kissing complex remains globally stable over a 20 ns MD simulation, although large rearrangements occur at the level of the stem/loop junctions that are flexible, as shown from free energy calculations. These results are compared to electrophoresis experiments on dimer formation.     

Conformational transitions of flanking purines in HIV-1 RNA dimerization initiation site kissing complexes studied by CHARMM explicit solvent molecular dynamics

Dimerization of HIV-1 genomic RNA is initiated by kissing loop interactions at the Dimerization Initiation Site (DIS). Dynamics of purines that flank the 5' ends of the loop-loop helix in HIV-1 DIS kissing complex were explored using explicit solvent molecular dynamics (MD) simulations with the CHARMM force field. Multiple MD simulations (200 ns in total) of X-ray structures for HIV-1 DIS Subtypes A, B, and F revealed conformational variability of flanking purines. In particular, the flanking purines, which in the starting X-ray structures are bulged-out and stack in pairs, formed a consecutive stack of four bulged-out adenines at the beginning of several simulations. This conformation is seen in the crystal structure of DIS Subtype F with no interference from crystal packing, and was frequently reported in our preceding MD studies performed with the AMBER force field. However, as CHARMM simulations progressed, the four continuously stacked adenines showed conformational transitions from the bulged-out into the bulged-in geometries. Although such an arrangement has not been seen in any X-ray structure, it has been suggested by a recent NMR investigation. In CHARMM simulations, in the longer time scale, the flanking purines display the tendency to move to bulged-in conformations. This is in contrast with the AMBER simulations, which indicate a modest prevalence for bulged-out flanking base positions in line with the X-ray data. The simulations also suggest that the intermolecular stacking between purines from the opposite hairpins can additionally stabilize the kissing complex.     

Modeling the dynamics of a mutated stem-loop in the SL1 domain of HIV-1Lai genomic RNA by 1H-NOESY spectra

The cross-peaks of ¹H-NOESY spectra at different time delays are compared to a mode-coupling diffusion (MCD) calculation, including the evaluation of the full ¹H relaxation matrix, in the case of a 23 nucleotide fragment of the stem-loop SL1 domain of HIV-1_Lai genomic RNA mutated in a single position. The MCD theory gives significant agreement with ¹H relaxation experiments enabling a thorough understanding of the differential local dynamics along the sequence and particularly of the dynamics of nucleotides in the stem and in the loop. The differential dynamics of this hairpin structure is important in directing the dimerization of the retroviral genome, a fundamental step in the infectious process. The demonstration of a reliable use of time dependent NOE cross-peaks, largely available from NMR solution structure determination, coupled to MCD analysis, to probe the local dynamics of biological macromolecules, is a result of general interest of this paper.     

Modeling the dynamics of the solvated SL1 domain of HIV-1 genomic RNA

A mode-coupling solution of the Smoluchowski diffusion equation (MCD theory), designed to describe the dynamics of wobbling macromolecules in water, is applied to a macromolecular bead model including water beads in the nearest layers. The necessary statistical averages are evaluated by time averaging along a molecular dynamics (MD) trajectory where both solute and water are introduced as atomistic models. The cross peaks in (1)H nuclear Overhauser effect spectroscopy (NOESY) NMR spectra that are routinely measured to determine biological structures are here calculated for the mutated 23 nucleotides stem-loop fragment of the SL1 domain in the HIV-1(Lai) genomic RNA. The calculations are in acceptable agreement with experiments without requiring any screening of the hydrodynamic interactions. The screening of hydrodynamics was necessary in previous MCD calculations obtained by using the same full atomistic MD trajectory, but a nonsolvated frictional model.     

Extending the capabilities of targeted molecular dynamics: simulation of a large conformational transition in plasminogen activator inhibitor 1

Plasminogen activator inhibitor type 1 (PAI-1) is an inhibitor of plasminogen activators such as tissue-type plasminogen activator or urokinase-type plasminogen activator. For this molecule, different conformations are known. The inhibiting form that interacts with the proteinases is called the active form. The noninhibitory, noncleavable form is called the latent form. X-ray and modeling studies have revealed a large change in position of the reactive center loop (RCL), responsible for the interaction with the proteinases, that is inserted into a beta-sheet (s4A) in the latent form. The mechanism underlying this spontaneous conformational change (half-life = 2 h at 37 degrees C) is not known in detail. This investigation attempts to predict a transition path from the active to the latent structure at the atomic level, by using simulation techniques. Together with targeted molecular dynamics (TMD), a plausible assumption on a rigid body movement of the RCL was applied to define an initial guess for an intermediate. Different pathways were simulated, from the active to the intermediate, from the intermediate to the latent structure and vice versa under different conditions. Equilibrium simulations at different steps of the path also were performed. The results show that a continuous pathway from the active to the latent structure can be modeled. This study also shows that this approach may be applied in general to model large conformational changes in any kind of protein for which the initial and final three-dimensional structure is known.     

Investigating the disorder–order transition of calmodulin binding domain upon binding calmodulin using molecular dynamics simulation

We have studied the conformational transition of the calmodulin binding domains (CBD) in several calmodulin‐binding kinases, in which CBD changes from the disordered state to the ordered state when binding with calmodulin (CaM). Targeted molecular dynamics simulation was used to investigate the binding process of CaM and CBD of CaM‐dependent kinase I (CaMKI–CBD). The results show that CaMKI–CBD began to form an α‐helix and the interaction free energy between CaM and CaMKI–CBD increased once CaM fully encompassed CaMKI–CBD. Two series of CaM/CBD complex systems, including the complexes of CaM with the initially disordered and the final ordered CBD, were constructed to study the interaction using molecular dynamics simulations. Our analyses suggest that the VDW interaction plays a dominant role in CaM/CBD binding and is a key factor in the disorder–order transition of CBD. Additionally, the entropy effect is not in favor of the formation of the CaM/CBD complex, which is consistent with the experimental evidence. Based on the results, it appears that the CBD conformational change from a non‐compact extended structure to compact α‐helix is critical in gaining a favorable VDW interaction and interaction free energy. Copyright © 2009 John Wiley & Sons, Ltd.     

Base pairing at the stem-loop junction in the SL1 kissing complex of HIV-1 RNA: a thermodynamic study probed by molecular dynamics simulation

The thermodynamics of the opening/closure process of a GC base pair located at the stem-loop junction of the SL1 sequence from HIV-1(Lai) genomic RNA was investigated in the context of a loop-loop homodimer (or kissing complex) using molecular dynamics simulation. The free energy, enthalpy and entropy changes for the closing reaction are 0 kcal x mol(-1), -11 kcal x mol(-1) and -0.037 kcal x mol(-1) x K(-1) at 300 degrees K respectively. Furthermore it is found that the free energy change is the same for the formation of a 11 nucleotide loop closed with UG and for the formation of a 9 nucleotide loop closed with GC. Our study evidences the high flexibility of the nucleotides at the stem-loop junction explaining the weak stability of this structure.     

Identification of the PcrA DNA helicase reaction pathway by applying advanced targeted molecular dynamic simulations

PcrA DNA helicase uses the free energy of hydrolysis and binding of ATP to unwind double-stranded DNA (ds-DNA). There are two states of PcrA, termed the substrate and product complexes and, through the conformational changes between these two states, PcrA moves along ds-DNA and separates the two strands. In this study, two different methods, namely chain minimisation (CM, less reliable method) and auto targeted molecular dynamic (TMD) simulation (more reliable), were performed to generate two different initial reaction pathways between these two states, and then fixed root mean square distance (RMSD) TMD simulation was performed to optimise these two initial pathways. In general, the two optimised pathways share very similar major conformational changes, but are different in the minor motions. The potential energy profiles of the two improved pathways are generally similar, but the one generated by the improved TMD path is slightly lower. Considering the poor reliability of the initial path generated by CM and insignificant improvements of the auto-TMD path, our study suggests that fixed RMSD TMD simulation can generate reliable reaction pathways, but the different initial paths still have some influence on the detailed conformational analysis.     

Molecular dynamics simulation for probing the flexibility of the 35 nucleotide SL1 sequence kissing complex from HIV-1Lai genomic RNA

The SL1 stem-loop located in the encapsidation domain is responsible for initiating the dimerisation of HIV-1 genomic RNA by means of a loop-loop interaction known as Kissing Complex (KC). The SL1 secondary structure has been predicted as a 35 nucleotides [K. G. Murti, M. Bondurant, and A. Tereba. J Virol 37, 411-419 (1981)] stem-loop composed of a 4 base pairs (bp) terminal duplex, a 4 nt asymmetrical internal loop, a 7 bp internal duplex, and a 9 nt apical loop. Several high resolution structures of the monomer and of KC of a 23 nt sequence containing only the internal duplex and the apical loop of SL1 are available in the literature. No experimental high resolution structure of the complete native SL1 sequence has been reported so far, either for the monomer or for KC. The asymmetrical internal loop has been described from NMR studies of different monomeric hairpin sequences, leading to divergent results, which suggests its high flexibility. In this work, we built a SL1(35) KC model which was submitted to a 31 ns molecular dynamics simulation (MD). Our results allows to describe the internal dynamics of SL1(35) KC and the differences of behavior of the different parts of the dimer. Thus, we could show the stability of the interactions between the two apical loops and of the terminal duplexes, the destabilization of the internal duplexes and the high flexibility of the asymmetrical internal loops.     

Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag–gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5′-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein–protein interactions.     

Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six-transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd-numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge-pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt-bridge networks with a significant shift from inter- to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even-numbered helices can undergo a face rotation, whereas odd-numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor.     

HIV-1 viral RNA is selected in the form of monomers that dimerize in a three-step protease-dependent process; the DIS of stem-loop 1 initiates viral RNA dimerization

We have characterized the viral RNA conformation in wild-type, protease-inactive (PR-) and SL1-defective (DeltaDIS) human immunodeficiency virus type 1 (HIV-1), as a function of the age of the viruses, from newly released to grown-up (>or=24 h old). We report evidence for packaging HIV-1 genomic RNA (gRNA) in the form of monomers in PR- virions, viral RNA rearrangement (not maturation) within PR- HIV-1, protease-dependent formation of thermolabile dimeric viral RNAs, a new form of immature gRNA dimer at about 5 h post virion release, and slow-acting dimerization signals in SL1-defective viruses. The rates of gRNA dimer formation were >or=3-fold and >or=10-fold slower in DeltaDIS and PR- viruses than in wild-type, respectively. Thus, the DIS, i.e. the palindrome in the apical loop of SL1, is a dimerization initiation signal, but its role can be masked by one or several slow-acting dimerization site(s) when grown-up SL1-inactive virions are investigated. Grown-up PR- virions are not flawless models for immature virions because gRNA dimerization increases with the age of PR- virions, indicating that the PR- mutation does not "freeze" gRNA conformation in a nascent primordial state. Our study is the first on gRNA conformation in newly released mutant or primate retroviruses. It shows for the first time that the packaged retroviral gRNA matures in more than one step, and that formation of immature dimeric viral RNA requires viral protein maturation. The monomeric viral RNAs isolated from budding HIV-1, as modeled by newly released PR- virions, may be seen as dimers that are much more fragile than thermolabile dimers.     

Conformational changes induced by ATP-hydrolysis in an ABC transporter: a molecular dynamics study of the Sav1866 exporter

ATP-Binding Cassette (ABC) transporters are ubiquitous membrane proteins that use energy from ATP binding or/and hydrolysis to actively transport allocrites across membranes. In this study, we identify ATP-hydrolysis induced conformational changes in a complete ABC exporter (Sav1866) from Staphylococcus aureaus, using molecular dynamics (MD) simulations. By performing MD simulations on the ATP and ADP+IP bound states, we identify the conformational consequences of hydrolysis, showing that the major rearrangements are not restricted to the NBDs, but extend to the transmembrane domains (TMDs) external regions. For the first time, to our knowledge, we see, within the context of a complete transporter, NBD dimer opening in the ADP+IP state in contrast with all ATP-bound states. This opening results from the dissociation of the ABC signature motif from the nucleotide. In addition, in both states, we observe the opening of a gate entrance in the intracellular loop region leading to the exposure of the TMDs internal cavity to the cytoplasm. To see if this opening was large enough to allow allocrite transport, the adiabatic energy profile for doxorubicin passage was determined. For both states, this profile, although an approximation, is overall downhill from the cytoplasmatic to the extracellular side, and the local energy barriers along the TMDs are relatively small, evidencing the exporter nature of Sav1866. The major difference between states is an energy barrier located in the cytoplasmic gate region, which becomes reduced upon hydrolysis, suggesting that allocrite passage is facilitated, and evidencing a possible molecular mechanism for the active transport in these proteins.     

Evidence of salicylic acid pathway with EDS1 and PAD4 proteins by molecular dynamics simulation for grape improvement

Biotic stress is a major cause of heavy loss in grape productivity. In order to develop biotic stress-resistant grape varieties, the key defense genes along with its pathway have to be deciphered. In angiosperm plants, lipase-like protein phytoalexin deficient 4 (PAD4) is well known to be essential for systemic resistance against biotic stress. PAD4 functions together with its interacting partner protein enhanced disease susceptibility 1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defense pathway. Existence and structure of key protein of systemic resistance EDS1 and PAD4 are not known in grapes. Before SA pathway studies are taken in grape, molecular evidence of EDS1: PAD4 complex is to be established. To establish this, EDS1 protein sequence was retrieved from NCBI and homologous PAD4 protein was generated using Arabidopsis thaliana as template and conserved domains were confirmed. In this study, computational methods were used to model EDS1 and PAD4 and simulated the interactions of EDS1 and PAD4. Since no structural details of the proteins were available, homology modeling was employed to construct three-dimensional structures. Further, molecular dynamic simulations were performed to study the dynamic behavior of the EDS1 and PAD4. The modeled proteins were validated and subjected to molecular docking analysis. Molecular evidence of stable complex of EDS1:PAD4 in grape supporting SA defense pathway in response to biotic stress is reported in this study. If SA defense pathway genes are explored, then markers of genes involved can play pivotal role in grape variety development especially against biotic stress leading to higher productivity.     

A molecular dynamics simulation of the flavin mononucleotide–RNA aptamer complex

We report on an unrestrained molecular dynamics simulation of the flavin mononucleotide (FMN)–RNA aptamer. The simulated average structure maintains both cross‐strand and intermolecular FMN–RNA nuclear Overhauser effects from the nmr experiments and has all qualitative features of the nmr structure including the G10–U12–A25 base triple and the A13–G24, A8–G28, and G9–G27 mismatches. However, the relative orientation of the hairpin loop to the remaining part of the molecule differs from the nmr structure. The simulation predicts that the flexible phosphoglycerol part of FMN moves toward G27 and forms hydrogen bonds. There are structurally long‐lived water molecules in the FMN binding pocket forming hydrogen bonds within FMN and between FMN and RNA. In addition, long‐lived water is found bridging primarily RNA backbone atoms. A general feature of the environment of long‐lived “structural” water is at least two and in most cases three or four potential acceptor atoms. The 2′‐OH group of RNA usually acts as an acceptor in interactions with the solvent. There are almost no intrastrand O2′H(n)⋮O4′(n + 1) hydrogen bonds within the RNA backbone. In the standard case the preferred orientation of the 2′‐OH hydrogen atoms is approximately toward O3′ of the same nucleotide. However, a relatively large number of conformations with the backbone torsional angle γ in the trans orientation is found. A survey of all experimental RNA x‐ray structures shows that this backbone conformation occurs but is less frequent than found in the simulation. Experimental nmr RNA aptamer structures have a higher fraction of this conformation as compared to the x‐ray structures. The backbone conformation of nucleotide n + 1 with the torsional angle γ in the trans orientation leads to a relatively short distance between 2′‐OH(n) and O5′(n + 1), enabling hydrogen‐bond formation. In this case the preferred orientation of the 2′‐OH hydrogen atom is approximately toward O5′(n + 1). We find two relatively short and dynamically stable types of backbone–backbone next‐neighbor contacts, namely C2′(H)(n)⋮O4′(n + 1) and C5′(H)(n + 1)⋮O2′(n). These interactions may affect both backbone rigidity and thermodynamic stability of RNA helical structures. © 1999 John Wiley & Sons, Inc. Biopoly 50: 287–302, 1999     

The role of conformational selection in the molecular recognition of the wild type and mutants XPA67‐80 peptides by ERCC1

Molecular recognition is a fundamental step in the coordination of biomolecular pathways. Understanding how recognition and binding occur between highly flexible protein domains is a complex task. The conformational selection theory provides an elegant rationalization of the recognition mechanism, especially valid in cases when unstructured protein regions are involved. The recognition of a poorly structured peptide, namely XPA_67‐80, by its target receptor ERCC1, falls in this challenging study category. The microsecond molecular dynamics (MD) simulations, discussed in this work, show that the conformational propensity of the wild type XPA_67‐80 peptide in solution supports conformational selection as the key mechanism driving its molecular recognition by ERCC1. Moreover, all the mutations of the XPA_67‐80 peptide studied here cause a significant increase of its conformational disorder, relative to the wild type. Comparison to experimental data suggests that the loss of the recognized structural motifs at the microscopic time scale can contribute to the critical decrease in binding observed for one of the mutants, further substantiating the key role of conformational selection in recognition. Ultimately, because of the high sequence identity and analogy in binding, it is conceivable that the conclusions of this study on the XPA_67‐80 peptide also apply to the ERCC1‐binding domain of the XPA protein. Proteins 2015; 83:1341–1351. © 2015 Wiley Periodicals, Inc.