Structural Dynamics of Precursor and Product of the RNA Enzyme from the Hepatitis Delta Virus as Revealed by Molecular Dynamics Simulations

Krasovska, Maryna V.; Sefcikova, Jana; Špačková, Nad'a; Šponer, Jiřı́; Walter, Nils G.

doi:10.1016/j.jmb.2005.06.016

Cited by 61 publications

(157 citation statements)

References 78 publications

Supporting

Mentioning

143

Contrasting

Order By: Relevance

“…The two simplest explanations for this lack of electron density are that a significant fraction of molecules degraded during crystal growth or that the crystal contains a conformational ensemble with some flexibility and/or disparity around the active site, a possibility favored by the authors (Chen et al 2010). To test this notion and provide a window into the rapid (sub-microsecond), small-scale dynamics of the ribozyme, not easily accessible by experimental techniques (Ditzler et al 2010), we performed MD simulations on the crystallized trans-acting precursor using our established protocols (Krasovska et al 2005(Krasovska et al , 2006Rhodes et al 2006;McDowell et al 2007McDowell et al , 2010Sefcikova et al 2007a,b;Ditzler et al 2010). We used the latest parmbsc0χ OL3 (Perez et al 2007;Zgarbova et al 2011) variant of the Cornell et al AMBER force field (Cornell et al 1995), which is essential for stable RNA simulations (Banas et al 2010).…”

Section: Resultsmentioning

confidence: 99%

“…Previous MD studies have identified two protonated cytosines as structurally important: C41, that upon protonation forms a rare stabilizing quadruple interaction just below the active site (Krasovska et al 2005;Veeraraghavan et al 2010), and C75, that upon protonation promotes a stable active site in the context of the trans-acting precursor (Veeraraghavan et al 2011) as well as cis-acting product structure (Krasovska et al 2005). Protonation in the context of the new, trans-acting ribozyme is justified since the crystal structure was obtained at a pH of 5.0 (Chen et al 2010); however, since the pK a of C75 of the HDV ribozyme was measured as ∼6.4 (Nakano et al 2000;Gong et al 2007), at the more physiological pH of 7.5 used in our solution, probing a protonated C75H…”

Section: (B)mentioning

confidence: 99%

“…The emergent crystal structure of the inactivated cis-acting C75U mutant precursor at up to 2.2 Å-resolution (Ke et al 2004) was probed by independent molecular dynamics (MD) studies in which residue 75 was changed back to C (Krasovska et al 2005). In combination, these studies led to the discovery of a structural change upon release of the 5 ′ product and catalytic metal ion, as C75 moves deeper into the active site cleft to form hydrogen bonds with C22's nonbridging oxygen and the 5 ′ OH leaving group of G1, while the adjacent G25 rotates in conformation from anti to syn (Ke et al 2004;Krasovska et al 2005). These changes result in a collapse of the P1 and P3 helices toward the active site and a more compact catalytic core and concomitant overall P2-P4 axial lengthening of the product (Fig.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

Sripathi

Tay

Banáš

et al. 2014

RNA

Self Cite

View full text Add to dashboard Cite

The hepatitis delta virus (HDV) ribozyme is a member of the class of small, self-cleaving catalytic RNAs found in a wide range of genomes from HDV to human. Both pre-and post-catalysis (precursor and product) crystal structures of the cis-acting genomic HDV ribozyme have been determined. These structures, together with extensive solution probing, have suggested that a significant conformational change accompanies catalysis. A recent crystal structure of a trans-acting precursor, obtained at low pH and by molecular replacement from the previous product conformation, conforms to the product, raising the possibility that it represents an activated conformer past the conformational change. Here, using fluorescence resonance energy transfer (FRET), we discovered that cleavage of this ribozyme at physiological pH is accompanied by a structural lengthening in magnitude comparable to previous trans-acting HDV ribozymes. Conformational heterogeneity observed by FRET in solution appears to have been removed upon crystallization. Analysis of a total of 1.8 µsec of molecular dynamics (MD) simulations showed that the crystallographically unresolved cleavage site conformation is likely correctly modeled after the hammerhead ribozyme, but that crystal contacts and the removal of several 2 ′ -oxygens near the scissile phosphate compromise catalytic in-line fitness. A cisacting version of the ribozyme exhibits a more dynamic active site, while a G-1 residue upstream of the scissile phosphate favors poor fitness, allowing us to rationalize corresponding changes in catalytic activity. Based on these data, we propose that the available crystal structures of the HDV ribozyme represent intermediates on an overall rugged RNA folding free-energy landscape.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: (B)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

Sripathi

Tay

Banáš

et al. 2014

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, the active site environment could optimize the microscopic pK a in order to increase catalytic efficiency, similar to the well-studied HDV. In HDV, the pK a of N3 of C76 is perturbed toward neutrality, and the perturbed C76 has been shown to act as the general acid in the self-cleavage, based on experimental and computational studies (Nakano et al 2000;Shih and Been 2001;Krasovska et al 2005). Also, using Raman crystallography, Guo et al (2009) showed that the pK a of N1 of A38 in the hairpin ribozyme (HPRZ) FIGURE 1.…”

Section: Introductionmentioning

confidence: 99%

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Yao¹,

Hamelberg²

2010

RNA

View full text Add to dashboard Cite

The GlmS ribozyme is believed to exploit a general acid-base catalytic mechanism in the presence of glucosamine-6-phosphate (GlcN6P) to accelerate self-cleavage by approximately six orders of magnitude. The general acid and general base are not known, and the role of the GlcN6P cofactor is even less well understood. The amine group of GlcN6P has the ability to either accept or donate a proton and could therefore potentially act as an acid or a base. In order to decipher the role of GlcN6P in the self-cleavage of glmS, we have determined the preferred protonation state of the amine group in the wild-type and an inactive G40A mutant using molecular dynamics simulations and free energy calculations. Here we show that, upon binding of GlcN6P to wild-type glmS, the pK a of the amine moiety is altered by the active site environment, decreasing by about 2.2 from a solution pK a of about 8.2. On the other hand, we show that the pK a of the amine group slightly increases to about 8.4 upon binding to the G40A inactive mutant of glmS. These results suggest that GlcN6P acts as a general acid in the self-cleavage of glmS. Upon binding to glmS, GlcN6P can easily release a proton to the 59-oxygen of G1 during self-cleavage of the backbone phosphodiester bond. However, in the G40A inactive mutant of glmS, the results suggest that the ability of GlcN6P to easily release its proton is diminished, in addition to the possible lack of G40 as an effective base.

show abstract

“…Several computational approaches have also been used to probe the mechanism of the HDV ribozyme; however, some results support GAB model 1 (Krasovska et al 2005(Krasovska et al , 2006, while others support GAB model 2 Wei et al 2007). In general, biochemical data are more consistent with GAB model 2, in which C75 acts as a general acid and a hydrated Mg 2+ hydroxide ion acts as general base (Nakano et al , 2001Das and Piccirilli 2005).…”

Section: Introductionmentioning

confidence: 99%

Mechanistic characterization of the HDV genomic ribozyme: The cleavage site base pair plays a structural role in facilitating catalysis

Cerrone-Szakal¹,

Chadalavada²,

Golden³

et al. 2008

RNA

View full text Add to dashboard Cite

The hepatitis delta virus (HDV) ribozyme occurs in the genomic and antigenomic strands of the HDV RNA and within mammalian transcriptomes. Previous kinetic studies suggested that a wobble pair (is preferred at the cleavage site; however, the reasons for this are unclear. We conducted sequence comparisons, which indicated that while G d U is the most prevalent combination at the cleavage site, G-C occurs to a significant extent in genomic HDV isolates, and G d U, G-C, and A-U pairs are present in mammalian ribozymes. We analyzed the folding of genomic HDV ribozymes by free energy minimization and found that variants with purine-pyrimidine combinations at the cleavage site are predicted to form native structures while pyrimidine-purine combinations misfold, consistent with earlier kinetic data and sequence comparisons. To test whether the cleavage site base pair contributes to catalysis, we characterized the pH and Mg 2+ -dependence of reaction kinetics of fastfolding genomic HDV ribozymes with cleavage site base pair purine-pyrimidine combinations: G d U, A-U, G-C, and A C ribozyme as being more active with a wobble pair at the cleavage site than with no base pair at all. Overall, the data support a model in which the cleavage site base pair provides a structural role in catalysis and does not need to be a wobble pair.

show abstract

Structural Dynamics of Precursor and Product of the RNA Enzyme from the Hepatitis Delta Virus as Revealed by Molecular Dynamics Simulations

Cited by 61 publications

References 78 publications

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

Disparate HDV ribozyme crystal structures represent intermediates on a rugged free-energy landscape

Deciphering the role of glucosamine-6-phosphate in the riboswitch action of glmS ribozyme

Mechanistic characterization of the HDV genomic ribozyme: The cleavage site base pair plays a structural role in facilitating catalysis

Contact Info

Product

Resources

About