Structural dynamics of individual Holliday junctions

The four-way DNA (Holliday) junction is the central intermediate of genetic recombination, but the dynamic aspects of this important structure are presently unclear. Although transitions between alternative stacking conformers have been predicted, conventional kinetic studies are precluded by the inability to synchronize the junction in a single conformer in bulk solution. Using single-molecule fluorescence methodology we have been able to detect these transitions. The sequence dependence, the influence of counterions and measured energetic barriers indicate that the conformer transition and branch migration processes share the unstacked, open structure as the common intermediate but have different rate-limiting steps. Relative rates indicate that multiple conformer transitions occur at each intermediate step of branch migration, allowing the junction to reach conformational equilibrium. This provides a mechanism whereby the sequence-dependent conformational bias could determine the extent of genetic exchange upon junction resolution.     

Chrysophanol anthrone: a theoretical study on the potential energy surface

The potential energy surface (PES) of chrysophanol anthrone, the active component of Goa Powder, was systematically explored and thoroughly scrutinised via density functional theory, in order to gain an understanding of its physicochemical properties. In particular, we focused on the rotations of the two hydroxyl-phenyl dihedral angles. A picture with a four stable rotamers emerged where only the most stable conformer has a planar structure and the less stable conformer has the maximum deviation from planarity. The computed PES shows that the energy barriers for the conformer interconversion are less than 15 kcal/mol. From the analysis of the calculated intramolecular hydrogen bond enthalpy, we conclude that the number of the intramolecular hydrogen bonds governs the conformer stability. Additionally, the conformational equilibrium was pursued by means of an analysis of the energy of OH internal rotation barriers. The total energy changes were decomposed in an electrostatic decomposition scheme in order to gain an insight into the effects governing the torsional barrier and preferred conformations. This analysis shows that the interplay between the repulsive and attractive potentials causes the conformer stability, where the attractive term dominates the conformer stabilisation.     

Helical junctions as determinants for RNA folding: origin of tertiary structure stability of the hairpin ribozyme

Helical junctions are ubiquitous structural elements that govern the folding and tertiary structure of RNAs. The tobacco ringspot virus hairpin ribozyme consists of two helix-loop-helix elements that lie on adjacent arms of a four-way junction. In the active form of the hairpin ribozyme, the loops are in proximity. The nature of the helical junction determines the stability of the hairpin ribozyme tertiary structure [Walter, N. G., Burke, J. M., and Millar, D. P. (1999) Nat. Struct. Biol. 6, 544-549] and thus its catalytic activity. We used two-, three-, and four-way junction hairpin ribozymes as model systems to investigate the thermodynamic basis for the different tertiary structure stabilities. The equilibrium between docked and extended conformers was analyzed as a function of temperature using time-resolved fluorescence resonance energy transfer (trFRET). As the secondary and tertiary structure transitions overlap, information from UV melting curves and trFRET had to be combined to gain insight into the thermodynamics of both structural transitions. It turned out that the higher tertiary structure stability observed in the context of a four-way junction is the result of a lower entropic cost for the docking process. In the two- and three-way junction ribozymes, a high entropic cost counteracts the favorable enthalpic term, rendering the docked conformer only marginally stable. Thus, two- and three-way junction tertiary structures are more sensitive toward regulation by ligands, whereas four-way junctions provide a stable scaffold. Altogether, RNA folding and stability appear to be governed by principles similar to those for the folding of proteins.     

The dynamic nature of the four-way junction of the hepatitis C virus IRES

Translation is initiated within the RNA of the hepatitis C virus at the internal ribosome entry site (IRES). The IRES is a 341-nucleotide element that contains a four-way helical junction (IIIabc) as a functionally important element of the secondary structure. The junction has three additional, nonpaired nucleotides at the point of strand exchange on one diagonal. We have studied the global conformation and folding of this junction in solution, using comparative gel electrophoresis and steady-state and time-resolved fluorescence resonance energy transfer. In the absence of divalent metal ions, the junction adopts an extended-square structure, in contrast to perfect four-way RNA junctions, which retain coaxial helical stacking under all conditions. The IIIabc junction is induced to fold on addition of Mg(2+), by pairwise coaxial stacking of arms, into the conformer in which the unpaired bases are located on the exchanging strands. Fluorescence lifetime measurements indicate that in the presence of Mg(2+) ions, the IIIabc junction exists in a dynamic equilibrium comprising approximately equal populations of antiparallel and parallel species. These dynamic properties may be important in mediating interactions between the IRES and the ribosome and initiation factors.     

Role of a Novel PH-Kinase Domain Interface in PKB/Akt Regulation: Structural Mechanism for Allosteric Inhibition

Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB''s inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1). By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM), a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor) and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.     

The R163K mutant of human thymidylate synthase is stabilized in an active conformation: structural asymmetry and reactivity of cysteine 195

Loop 181-197 of human thymidylate synthase (hTS) populates two conformational states. In the first state, Cys195, a residue crucial for catalytic activity, is in the active site (active conformer); in the other conformation, it is about 10 A away, outside the active site (inactive conformer). We have designed and expressed an hTS variant, R163K, in which the inactive conformation is destabilized. The activity of this mutant is 33% higher than that of wt hTS, suggesting that at least one-third of hTS populates the inactive conformer. Crystal structures of R163K in two different crystal forms, with six and two subunits per asymmetric part of the unit cells, have been determined. All subunits of this mutant are in the active conformation while wt hTS crystallizes as the inactive conformer in similar mother liquors. The structures show differences in the environment of catalytic Cys195, which correlate with Cys195 thiol reactivity, as judged by its oxidation state. Calculations show that the molecular electrostatic potential at Cys195 differs between the subunits of the dimer. One of the dimers is asymmetric with a phosphate ion bound in only one of the subunits. In the absence of the phosphate ion, that is in the inhibitor-free enzyme, the tip of loop 47-53 is about 11 A away from the active site.     

Conformational model of the Holliday junction transition deduced from molecular dynamics simulations

Homologous recombination plays a key role in the restart of stalled replication forks and in the generation of genetic diversity. During this process, two homologous DNA molecules undergo strand exchange to form a four-way DNA (Holliday) junction. In the presence of metal ions, the Holliday junction folds into the stacked-X structure that has two alternative conformers. Experiments have revealed the spontaneous transitions between these conformers, but their detailed pathways are not known. Here, we report a series of molecular dynamics simulations of the Holliday junction at physiological and elevated (400 K) temperatures. The simulations reveal new tetrahedral intermediates and suggest a schematic framework for conformer transitions. The tetrahedral intermediates bear resemblance to the junction conformation in complex with a junction-resolving enzyme, T7 endonuclease I, and indeed, one intermediate forms a stable complex with the enzyme as demonstrated in one simulation. We also describe free energy minima for various states of the Holliday junction system, which arise during conformer transitions. The results show that magnesium ions stabilize the stacked-X form and destabilize the open and tetrahedral intermediates. Overall, our study provides a detailed dynamic model of the Holliday junction undergoing a conformer transition.     

Variants of human thymidylate synthase with loop 181–197 stabilized in the inactive conformation

Loop 181–197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180°. In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k_cat/K_m reduced by factors in a 2–12 range. One of the mutants, M190K, is however unique in having the value of k_cat/K_m smaller by a factor of ～7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181–197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188–194 of this loop, assumes a different conformation with the positions of C_α shifted up to 7.2 Å. This affects region 116–128, which became ordered in M190K while it is disordered in wt. The conformation of 116–128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.     

Global structure of four-way RNA junctions studied using fluorescence resonance energy transfer.

Four-way helical junctions are found widely in natural RNA species. In this study, we have studied the conformation of two junctions by fluorescence resonance energy transfer. We show that the junctions are folded by pairwise coaxial helical stacking, forming one predominant stacking conformer in both examples studied. At low magnesium ion concentrations, the helical axes of both junctions are approximately perpendicular. One junction undergoes a rotation into a distorted antiparallel structure induced by the binding of a single magnesium ion. By contrast, the axes of the four-way junction of the U1 snRNA remain approximately perpendicular under all conditions examined, and we have determined the stacking conformer adopted.     

Oxidative modification of Escherichia coli glutamine synthetase. Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation.

Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.     

Quaternary structure and spin-state transition in azide methemoglobin A

The temperature-dependent ultraviolet and visible absorption changes of human azide methemoglobin with and without inositol hexaphosphate (IHP) were examined in a 4'-35 degrees C range. The 537-nm absorption change of IHP-free hemoglobin was about 1.2-fold larger than that of IHP-bound hemoglobin. The data were analyzed by considering the thermal spin equilibrium within the R and T conformers and the quaternary equilibrium between the two conformers. The spin equilibrium analysis suggested that the T conformer has a larger high-spin content than the R conformer. The quaternary equilibrium analysis, on the other hand, showed that the T conformer is more populated at lower temperature. The thermodynamic values for the quaternary equilibrium were determined to be delta H = -13.3 kcal/mol and delta S = -47.6 eu. The large negative delta H and delta S values were compensated for each other to give a small energy difference between the two quaternary states, e.g., delta G4 = 670 cal/mol of tetramer at 20 degrees C. The coincidence of the temperature-dependent IHP-induced changes in the visible and ultraviolet absorptions of heme and aromatic chromophores at the subunit boundaries suggested that the quaternary transition energy is not localized at heme moiety. The reverse temperature dependence of the T conformer fraction as compared with the high-spin fraction of heme iron was interpreted as indicating that the appearance of the T state is not directly coupled with an increase in the strain of Fe-N(F8 His) linkage in azide methemoglobin A.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

An alternative helix in the 26S rRNA promotes excision and integration of the Tetrahymena intervening sequence.

A highly conserved ribosomal stem-loop immediately upstream of the Tetrahymena splice junction can inhibit both forward and reverse self-splicing by competing with base pairing between the 5' exon and the guide sequence of the intervening sequence. Formation of this unproductive hairpin is preferred in precursor RNAs with short exons and results in a lower rate of splicing. Inhibition of self-splicing is not observed in longer precursors, suggesting that additional interactions in the extended exons can influence the equilibrium between the productive and unproductive hairpins at the 5' splice site. An alternative pairing upstream of the 5' splice site has been identified and is proposed to stabilize the active conformer of the pre-rRNA. Nucleotide changes that alter the ability to form this additional helix were made, and the self-splicing rates were compared. Precursors in which the proposed stem is stabilized splice more rapidly than the wild type, whereas RNAs that contain a base mismatch splice more slowly. The ability of DNA oligomers to bind the RNA, as detected by RNase H digestion, correlates with the predicted secondary structure of the RNA. We also show that a 236-nucleotide RNA containing the natural splice junction is a substrate for intervening sequence integration. As in the forward reaction, reverse splicing is enhanced in ligated exon substrates in which the alternative rRNA pairing is more stable.     

Atypical transient state kinetics of recombinant human dihydrofolate reductase produced by hysteretic behavior. Comparison with dihydrofolate reductases from other sources

The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faecium exhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t1/2 = 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Km for dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.     

General method for exact evaluation of parameters of the elementary steps of coupled reactions. Invariant analysis.

A general method has been devised for the exact evaluation of the rate constants of the elementary steps of a system consisting of any number of coupled reactions. The method is independent of the structure of the network of coupled steps. The precision of the data evaluation is solely dependent on the quality of the detection unit. The application of the method is illustrated with data collected for the binding of the competitive inhibitor proflavine to chymotrypsin under such conditions that five states of the enzyme are required to interpret the results. In the absence of a substance possessing the binding specificity, the enzyme is present as an equilibrium between an active and an inactive conformer. The latter prevails. The binding of the specific inhibitor releases a slow proton transfer from the medium to the alpha-amino group of Ile-16. Subsequently, the enzyme-inhibitor (or enzyme-substrate) complex re-arranges to the catalytically active form, which is retained until the supply of specific substrate is exhausted. The control features described are general, but are particularly conspicuous under the special environmental conditions used here. A comparison between data for alpha- and delta-forms of chymotrypsin showed that the chain ends of the former impeded the substrate binding and that the activity controlling conformational change occurred in the interior of the enzyme molecule.     

Tryptophan fluorescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis.

Single tryptophan residues were incorporated into each of three peptide segments that play key roles in the structural transition of ligand-free, inactive glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase to the active enzyme-substrate complex. Intrinsic tryptophan fluorescence and fluorescence quenching were used to monitor changes in a phosphoribosyltransferase (PRTase) "flexible loop", a "glutamine loop", and a C-terminal helix. Steady state fluorescence changes resulting from substrate binding were used to calculate binding constants and to detect the structural rearrangements that coordinate reactions at active sites for glutamine hydrolysis and PRTase catalysis. Pre-steady state kinetics of enzyme.PRPP and enzyme.PRPP.glutamine complex formation were determined from stopped-flow fluorescence measurements. The kinetics of the formation of the enzyme.PRPP complex were consistent with a model with two or more steps in which rapid equilibrium binding of PRPP is followed by a slow enzyme isomerization. This isomerization is ascribed to the closing of the PRTase flexible loop and is likely the rate-limiting step in the reaction of PRPP with NH(3). The pre-steady state kinetics for binding glutamine to the binary enzyme. PRPP complex could also be fit to a model involving rapid equilibrium binding of glutamine followed by an enzyme isomerization step. The changes monitored by fluorescence account for the interconversions between "end state" structures determined previously by X-ray crystallography and define an intermediate enzyme.PRPP conformer.     

Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.     

Thermal effects on an enzymatically latent conformation of coagulation factor VIIa.

Activation of the zymogen factor VII yields an enzyme form, factor VIIa, with only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Factor VIIa activity measured with a chromogenic peptide substrate is characterized by an unusual temperature dependency which indicates that the activated protease exists in an equilibrium between a latent (enzymatically inactive) and an active conformation. As shown by calorimetry and activity measurements the thermal effects on factor VIIa are fully reversible below the denaturation temperature of 58.1 degrees C. A model for factor VIIa has been proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Biol. Chem. 269, 18891-18898] in which the protease is supposed to exist primarily as a latent enzyme form because of the poor incorporation into the protease structure of the N-terminal Ile153 released by proteolytic cleavage during activation of factor VII. Binding of tissue factor to factor VIIa is assumed to shift the equilibrium towards an active conformation in which the N-terminal Ile153 forms a salt bridge with Asp343. We corroborate the validity of this model by: (a) chemical modification of factor VIIa; this suggests that the thermal effect on the equilibrium between the active and inactive conformation is reflected in the relative accessibility of the active site and the N-terminal Ile153; (b) measurements of factor VIIa binding to tissue factor indicating that complex formation is favoured by stabilization of the active conformation; and (c) activity measurements of a cross-linked factor VIIa-tissue factor complex; this showed that cross-linking stabilized the active conformation of factor VIIa and essentially prevented its thermally-induced transformation into the inactive state.     

3D-QSAR of N-myristoyltransferase inhibiting antifungal agents by CoMFA and CoMSIA methods

A series of benzofuran antifungals was examined to determine the structural requirements of N-myristoyltransferase (Nmt) enzyme inhibition by three-dimensional quantitative structure-activity relationship (3D-QSAR) using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. Evaluation of 20 compounds (training set) served to establish the model, which was validated by evaluation of a set of 6 compounds (test set). The lowest energy conformer of the most active molecule obtained from systematic search was used as the template structure for the alignment. The best predictions were obtained with the CoMFA model from RMS fit, with r(2)(cv)=0.828, r(2)(conv)=0.989, r(2)(pred)=0.754 and with the CoMSIA model combining hydrophobic, hydrogen bond donor and hydrogen bond acceptor fields with r(2)(cv)=0.821, r(2)(conv)=0.978 and r(2)(pred)=0.747. The models obtained from the present study can be useful for the development of new Nmt inhibitors as potential antifungals. The docking studies were also carried out wherein the active and inactive molecules were docked into the active site of the recently reported Candida albicans Nmt (CaNmt) crystal structure to analyze enzyme-inhibitor interactions. The results obtained from the present 3D-QSAR and docking studies were found complimentary.     

Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: different energetic contributions from a ribose zipper motif

The hairpin catalytic motif in tobacco ringspot virus satellite RNA consists of two helix-loop-helix elements on two adjacent arms of a four-way helical junction. The bases essential for catalytic activity are located in the loops that are brought into proximity by a conformational change as a prerequisite for catalysis. The two loops interact via a ribose zipper motif involving the 2'-hydroxyls of A10, G11, A24, and C25 [Rupert, P. B., and Ferre d'Amare, A. R. (2001) Nature 401, 780-786]. To quantify the energetic importance of the ribose zipper hydrogen bonds, we have incorporated deoxy modifications at these four positions and determined the resulting destabilization of the docked conformer by means of time-resolved fluorescence resonance energy transfer. In a minimal form of the ribozyme, in which the loops are placed on the arms of a two-way helical junction, all modifications lead to a significant loss in tertiary structure stability and altered Mg2+ binding. Surprisingly, no significant destabilization was seen with the natural four-way junction ribozyme, suggesting that hydrogen bonding interactions involving the 2'-hydroxyls do not contribute to the stability of the docked conformer. These results suggest that the energetic contributions of ribose zipper hydrogen bonds are highly context dependent and differ significantly for the minimal and natural forms of the ribozyme.