Polymorphism of bulged-out residues in HIV-1 RNA DIS kissing complex and structure comparison with solution studies

All retroviruses encapsidate their genome as a dimer of homologous single-stranded RNAs. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) is located in the 5'-untranslated region of the viral genome and consists of a hairpin with a 6 nt self-complementary loop sequence. Genomic RNA dimerization, a crucial step for virion infectivity, is promoted by the formation of a loop-loop complex (or kissing complex) between two DIS hairpins. Crystal structures for the subtypes A, B and F of the HIV-1 DIS kissing complex have now been solved at 2.3 A, 1.9 A and 1.6 A, respectively. They revealed a polymorphism of bulged-out residues showing clearly that their conformation is not a mere consequence of crystal packing. They also provide more insights into ion binding, hydration, and RNA conformation and flexibility. In particular, we observed the binding of spermine to the loop-loop helix, which displaced a magnesium cation important for subtype A DIS dimerization. The excellent agreement between X-ray structures and the results of chemical probing and interference data on larger viral RNA fragments shows that the crystal structures are relevant for the DIS kissing complex present in solution and in viral particles. Accordingly, these structures will be helpful for designing new drugs derived from aminoglycoside antibiotics and targeted against the RNA dimerization step of the viral life-cycle.     

Molecular modeling and dynamics studies of HIV-1 kissing loop structures

Recognition of an RNA loop by another RNA loop is involved in several biological functions. The dimerization of two copies of the HIV-1 genomic RNA is thought to be involved in several steps of the retroviral life cycle. It has been shown that the dimerization of the two HIV-1 RNA genomes is initiated by the so called kissing loop. The 9nt kissing loop consists of a palindromic 6nt sequence that forms Watson-Crick base-pairs at the kissing site in HIV-1. We report the results of our molecular modeling and dynamics studies on two major subtype isolates (MAL and LAI) of HIV-1 kissing loop structures. From our modeling studies, we conclude that the conformation of the loop in the monomer might be closer to the A-RNA-like conformation in order to form an initial kissing structure. This is achieved by the stacking interactions of the bases at the 3' end of the loop and by the intramolecular tertiary interactions of a single linker nucleotide. We discuss the effect of the loop size and the structural limitations on the formation of kissing loop structures. Also, we propose a possible mechanism to convert the kissing loop structure to a stable extended duplex structure without unwinding the stems.     

Determination of thermodynamic parameters for HIV DIS type loop-loop kissing complexes

The HIV-1 type dimerization initiation signal (DIS) loop was used as a starting point for the analysis of the stability of Watson–Crick (WC) base pairs in a tertiary structure context. We used ultraviolet melting to determine thermodynamic parameters for loop–loop tertiary interactions and compared them with regular secondary structure RNA helices of the same sequences. In 1 M Na⁺ the loop–loop interaction of a HIV-1 DIS type pairing is 4 kcal/mol more stable than its sequence in an equivalent regular and isolated RNA helix. This difference is constant and sequence independent, suggesting that the rules governing the stability of WC base pairs in the secondary structure context are also valid for WC base pairs in the tertiary structure context. Moreover, the effect of ion concentration on the stability of loop–loop tertiary interactions differs considerably from that of regular RNA helices. The stabilization by Na⁺ and Mg²⁺ is significantly greater if the base pairing occurs within the context of a loop–loop interaction. The dependence of the structural stability on salt concentration was defined via the slope of a T_m/log [ion] plot. The short base-paired helices are stabilized by 8°C/log [Mg²⁺] or 11°C/log [Na⁺], whereas base-paired helices forming tertiary loop–loop interactions are stabilized by 16°C/log [Mg²⁺] and 26°C/log [Na⁺]. The different dependence on ionic strength that is observed might reflect the contribution of specific divalent ion binding to the preformation of the hairpin loops poised for the tertiary kissing loop–loop contacts.     

Pseudopeptides and beta folding: x-ray structures compared with structures in solution

In order to restrain the flexibility of the peptide molecules and reduce their biodegradation, modifications of the main chain are now introduced in pseudopeptide analogues. Surprisingly, there is very little data on the conformational properties of these derivatives. We have examined pseudopeptide analogues of RCO-X-Y-NHR' model dipeptides in the depsi, N-methylated, reduced, retro, alpha, beta-dehydro, beta-amino acid, and hydrazino series, in the solid state by x-ray diffraction, and in solution by ir and 1H-nmr spectroscopy. This study provides us with accurate dimensions of the peptide surrogates, and gives some information on the conformational tendencies induced by these substitutions, with reference to those of the related dipeptide sequences.     

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5′-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a ‘kissing complex’ between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg²⁺, but not by K⁺ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg²⁺ ions.     

Preliminary x-ray diffraction studies of EcoRI restriction endonuclease-DNA complex

A complex between EcoRI restriction endonuclease and cognate DNA fragment, 5′-G-A-A-T-T-C C-T-T-A-A-G-5′, has been crystallized. The space group is P42₁2 with $\text{a = b = 183.2}\text{A}\text{?}\text{, c = 49.7}\text{A}\text{?}\text{, α = β = γ = 90 °}$. The unit cell contains four enzyme monomers plus two duplex DNA fragments in an asymmetric unit. High quality crystals of the enzyme alone have also been obtained.     

Crystal and solution structures of an HslUV protease-chaperone complex

HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.     

Conformations of flanking bases in HIV-1 RNA DIS kissing complexes studied by molecular dynamics

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.     

Structure of an integrin-ligand complex deduced from solution x-ray scattering and site-directed mutagenesis

The structural basis of the interaction of integrin heterodimers with their physiological ligands is poorly understood. We have used solution x-ray scattering to visualize the head region of integrin alpha 5 beta 1 in an inactive (Ca2+-occupied) state, and in complex with a fragment of fibronectin containing the RGD and synergy recognition sequences. Shape reconstructions of the data have been interpreted in terms of appropriate molecular models. The scattering data suggest that the head region undergoes no gross conformational changes upon ligand binding but do lend support to a proposed outward movement of the hybrid domain in the beta subunit. Fibronectin is observed to bind across the top of the head region, which contains an alpha subunit beta-propeller and a beta subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the beta-propeller domain. In support of this suggestion, mutagenesis of a prominent loop region on the side of the propeller identifies two residues (Tyr208 and Ile210) involved in recognition of the synergy region. Our data provide the first view of a complex between an integrin and a macromolecular ligand in solution, at a nominal resolution of approximately 10 A.     

Surface plasmon resonance kinetic studies of the HIV TAR RNA kissing hairpin complex and its stabilization by 2-thiouridine modification

Surface plasmon resonance (BIACORE) was used to determine the kinetic values for formation of the HIV TAR–TAR* (‘kissing hairpin’) RNA complex. The TAR component was also synthesized with the modified nucleoside 2-thiouridine at position 7 in the loop and the kinetics and equilibrium dissociation constants compared with the unmodified TAR hairpin. The BIACORE data show an equilibrium dissociation constant of 1.58 nM for the complex containing the s²U modified TAR hairpin, which is 8-fold lower than for the parent hairpin (12.5 nM). This is a result of a 2-fold faster k_a (4.14 × 10⁵ M^–1 s^–1 versus 2.1 × 10⁵ M^–1 s^–1) and a 4-fold slower k_d (6.55 × 10^–4 s^–1 versus 2.63 × 10^–3 s^–1). ¹H NMR imino spectra show that the secondary structure interactions involved in complex formation are retained in the s²U-modified complex. Magnesium has been reported to significantly stabilize the TAR–TAR* complex and we found that Mn²⁺ and Ca²⁺ are also strongly stabilizing, while Mg²⁺ exhibited the greatest effect on the complex kinetics. The stabilizing effects of 2-thiouridine indicate that this base modification may be generally useful as an antisense RNA modification for oligonucleotide therapeutics which target RNA loops.     

Structure of valinomycin-K+ complex in solution by extended x-ray absorption fine structure.

We used synchrotron radiation to measure the K-edge absorption spectra of the potassium ion in valinomycin-K+ complexes dissolved in ethanol and methanol. Our motivation is to study the structure of valinomycin around the potassium ion and the effect of solvents. From the extended x-ray absorption fine structure, we found that the mean distance from potassium to its coordination atoms, oxygen, is the same for both solvents, 2.79 +/- 0.02 A, compared with 2.76 A in crystal. The K-edge threshold spectra of the two solutions are almost identical but have a small difference in their relative peak intensities. The coincidence of their corresponding peak positions indicates that the strength of ligand field is about the same in these two samples. This agrees with the known binding energies of potassium ion to valinomycin in solutions. The difference in the relative peak intensities suggests a perturbation of ligand symmetry by solvents.     

Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal

We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ∼3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex.     

Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site

We describe the crystal structures of the RNA dimerization initiation sites (DIS) of HIV-1 subtypes A and B. Both molecules adopt a hairpin conformation, with loop sequences consisting of 272-AGGUGCACA-280 and 272-AAGCGCGCA-280, respectively. This includes a six-base self-complementary stretch (underlined) that allows homodimerization through the formation of a loop-loop, or 'kissing-loop', complex. The DISs for the two sequences have identical conformations, and the two interacting hairpins show a perfect coaxial alignment. The conserved purines, A272 and R273, are stacked in a bulged-out conformation and symmetrically join the upward and downward strands of each hairpin by crossing the helix major groove. There is good agreement between these structures and previous results from chemical probing in solution, as well as with differences in magnesium dependence for dimerization. The overall shape of the kissing-loop complex is very similar to that of the previously determined subtype A DIS duplex form. Unexpectedly, the purine R273 is the only base seen at a different position and is responsible for the difference in topology between the two forms. We propose that the transition from kissing-loop duplex could occur by a recombination mechanism based on symmetrical chain cleavage at R273 of each hairpin and subsequent cross-religation, rather than by base-pair melting and subsequent reannealing.     

MD and NMR studies of alpha-bungarotoxin surface accessibility

Protein surface accessibility represents a dimension of structural biology which has not been discussed in details so far, in spite of its fundamental role in controlling the molecular recognition process. In the present report the surface accessibility of alpha-bungarotoxin, a small and well characterized protein, has been investigated by analyzing its interaction with solvent and paramagnetic molecules in an integrated way. The presence of strong hydration sites, identified by a combined analysis of MD simulation and NMR results, seems to prevent the access of Gd(III)DTPA-BMA to the protein surface. On the contrary, the limited hydration of the alpha-bungarotoxin active site favors frequent encounters between the paramagnetic probe and the protein in the latter region. All the data obtained here for alpha-bungarotoxin suggest that shape and stability of the solvation shell control its surface accessibility and, hence, intermolecular interactions in a way which could be common to many other proteins.