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A posteriori design of crystal contacts to improve the X-ray diffraction properties of a small RNA enzyme.

MacElrevey C, Spitale RC, Krucinska J, Wedekind JE - Acta Crystallogr. D Biol. Crystallogr. (2007)

Bottom Line: This investigation describes the use of a dangling 5'-U to form an intermolecular U.U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L).In contrast, C3L variants diffracted to 3.35 A and exhibited a 15 A expansion of the c axis.The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

ABSTRACT
The hairpin ribozyme is a small catalytic RNA comprising two helix-loop-helix domains linked by a four-way helical junction (4WJ). In its most basic form, each domain can be formed independently and reconstituted without a 4WJ to yield an active enzyme. The production of such minimal junctionless hairpin ribozymes is achievable by chemical synthesis, which has allowed structures to be determined for numerous nucleotide variants. However, abasic and other destabilizing core modifications hinder crystallization. This investigation describes the use of a dangling 5'-U to form an intermolecular U.U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L). Both linker constructs demonstrated similar enzymatic activity, but S9L constructs yielded crystals that diffracted to 2.65 A resolution or better. In contrast, C3L variants diffracted to 3.35 A and exhibited a 15 A expansion of the c axis. Crystal packing of the C3L construct showed a paucity of 6(1) contacts, which comprise numerous backbone to 2'-OH hydrogen bonds in junctionless and S9L complexes. Significantly, the crystal packing in minimal structures mimics stabilizing features observed in the 4WJ hairpin ribozyme structure. The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects.

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A stereoview stick diagram of a superposition of the 4WJ, C3L and S9L hairpin-ribozyme structures. The orientation of the stereoview is rotated 180° around the vertical axis compared with Fig. 6 ▶ and was chosen to emphasize the agreement between residues of the intramolecular four-way junction with those of the intermolecular packing environment observed in the C3L and S9L structures. The 4WJ structure is shown in salmon, C3L in blue and S9L in magenta. Residues are numbered according to hairpin-ribozyme conventions and are labeled only where the sequence is conserved in all three structures except position 56 of the natural 4WJ..
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fig7: A stereoview stick diagram of a superposition of the 4WJ, C3L and S9L hairpin-ribozyme structures. The orientation of the stereoview is rotated 180° around the vertical axis compared with Fig. 6 ▶ and was chosen to emphasize the agreement between residues of the intramolecular four-way junction with those of the intermolecular packing environment observed in the C3L and S9L structures. The 4WJ structure is shown in salmon, C3L in blue and S9L in magenta. Residues are numbered according to hairpin-ribozyme conventions and are labeled only where the sequence is conserved in all three structures except position 56 of the natural 4WJ..

Mentions: A novel feature of the minimal ribozyme lattice was observed during the comparison of minimally hinged and 4WJ structures. The prevalence of pseudo-continuous helical packing within the lattice of minimal constructs (Fig. 6 ▶ a) enabled the formation of a rudimentary 4WJ motif. H4′ of one symmetry mate stacked bluntly onto H3, while H2′ of another symmetry mate stacked onto H2 in a staggered fashion through the U·U interaction (e.g. Fig. 6 ▶ b); additionally, the 2′-OH of a symmetry-related U−5 molecule was positioned appropriately to engage in an ∼3 Å hydrogen bond with a nonbridging phosphoryl O atom of either linker (Figs. 6 ▶ b and 6 ▶ c). Overall, the stacking interactions at this helical intersection bury 490 and 540 Å2 of surface area within the C3L and S9L structures, respectively, and exhibit an uncanny resemblance to the 4WJ motif (Fig. 6 ▶ d). A similar series of interactions were described above as stabilizing forces for the interdomain linker residue, A14, which is present in the 4WJ structure. In the latter molecule, the base pair between U−5 and A14 at the top of H2 was flanked by a coaxial interaction to form an energetically favorable flush stack; the end of H3 was supported similarly (Fig. 6 ▶ d; Rupert et al., 2002 ▶). The strand of RNA in the 4WJ structure that comprised these two coaxial stacks was single-stranded as it crossed over A14, further contributing to the ∼710 Å2 of surface area buried in the interface of this biologically relevant motif. Although the natural 4WJ is an intramolecular interaction, in contrast to the intermolecular contacts of the minimal constructs, a superposition of these three structures further emphasizes the excellent agreement between the modes of stacking at this interface (Fig. 7 ▶).


A posteriori design of crystal contacts to improve the X-ray diffraction properties of a small RNA enzyme.

MacElrevey C, Spitale RC, Krucinska J, Wedekind JE - Acta Crystallogr. D Biol. Crystallogr. (2007)

A stereoview stick diagram of a superposition of the 4WJ, C3L and S9L hairpin-ribozyme structures. The orientation of the stereoview is rotated 180° around the vertical axis compared with Fig. 6 ▶ and was chosen to emphasize the agreement between residues of the intramolecular four-way junction with those of the intermolecular packing environment observed in the C3L and S9L structures. The 4WJ structure is shown in salmon, C3L in blue and S9L in magenta. Residues are numbered according to hairpin-ribozyme conventions and are labeled only where the sequence is conserved in all three structures except position 56 of the natural 4WJ..
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483500&req=5

fig7: A stereoview stick diagram of a superposition of the 4WJ, C3L and S9L hairpin-ribozyme structures. The orientation of the stereoview is rotated 180° around the vertical axis compared with Fig. 6 ▶ and was chosen to emphasize the agreement between residues of the intramolecular four-way junction with those of the intermolecular packing environment observed in the C3L and S9L structures. The 4WJ structure is shown in salmon, C3L in blue and S9L in magenta. Residues are numbered according to hairpin-ribozyme conventions and are labeled only where the sequence is conserved in all three structures except position 56 of the natural 4WJ..
Mentions: A novel feature of the minimal ribozyme lattice was observed during the comparison of minimally hinged and 4WJ structures. The prevalence of pseudo-continuous helical packing within the lattice of minimal constructs (Fig. 6 ▶ a) enabled the formation of a rudimentary 4WJ motif. H4′ of one symmetry mate stacked bluntly onto H3, while H2′ of another symmetry mate stacked onto H2 in a staggered fashion through the U·U interaction (e.g. Fig. 6 ▶ b); additionally, the 2′-OH of a symmetry-related U−5 molecule was positioned appropriately to engage in an ∼3 Å hydrogen bond with a nonbridging phosphoryl O atom of either linker (Figs. 6 ▶ b and 6 ▶ c). Overall, the stacking interactions at this helical intersection bury 490 and 540 Å2 of surface area within the C3L and S9L structures, respectively, and exhibit an uncanny resemblance to the 4WJ motif (Fig. 6 ▶ d). A similar series of interactions were described above as stabilizing forces for the interdomain linker residue, A14, which is present in the 4WJ structure. In the latter molecule, the base pair between U−5 and A14 at the top of H2 was flanked by a coaxial interaction to form an energetically favorable flush stack; the end of H3 was supported similarly (Fig. 6 ▶ d; Rupert et al., 2002 ▶). The strand of RNA in the 4WJ structure that comprised these two coaxial stacks was single-stranded as it crossed over A14, further contributing to the ∼710 Å2 of surface area buried in the interface of this biologically relevant motif. Although the natural 4WJ is an intramolecular interaction, in contrast to the intermolecular contacts of the minimal constructs, a superposition of these three structures further emphasizes the excellent agreement between the modes of stacking at this interface (Fig. 7 ▶).

Bottom Line: This investigation describes the use of a dangling 5'-U to form an intermolecular U.U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L).In contrast, C3L variants diffracted to 3.35 A and exhibited a 15 A expansion of the c axis.The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

ABSTRACT
The hairpin ribozyme is a small catalytic RNA comprising two helix-loop-helix domains linked by a four-way helical junction (4WJ). In its most basic form, each domain can be formed independently and reconstituted without a 4WJ to yield an active enzyme. The production of such minimal junctionless hairpin ribozymes is achievable by chemical synthesis, which has allowed structures to be determined for numerous nucleotide variants. However, abasic and other destabilizing core modifications hinder crystallization. This investigation describes the use of a dangling 5'-U to form an intermolecular U.U mismatch, as well as the use of synthetic linkers to tether the loop A and B domains, including (i) a three-carbon propyl linker (C3L) and (ii) a nine-atom triethylene glycol linker (S9L). Both linker constructs demonstrated similar enzymatic activity, but S9L constructs yielded crystals that diffracted to 2.65 A resolution or better. In contrast, C3L variants diffracted to 3.35 A and exhibited a 15 A expansion of the c axis. Crystal packing of the C3L construct showed a paucity of 6(1) contacts, which comprise numerous backbone to 2'-OH hydrogen bonds in junctionless and S9L complexes. Significantly, the crystal packing in minimal structures mimics stabilizing features observed in the 4WJ hairpin ribozyme structure. The results demonstrate how knowledge-based design can be used to improve diffraction and overcome otherwise destabilizing defects.

Show MeSH
Related in: MedlinePlus