Limits...
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.

Show MeSH

Related in: MedlinePlus

Schematic surface and ball-and-stick diagrams illustrating the 61 packing interactions of hinged hairpin-ribozyme constructs. (a) Comparison of C3L (blue) and S9L (magenta) unit cells. The perspective represents the interaction of molecules about the 61 screw axis. (For twofold and 21 operations, refer to Fig. 6 ▶                  a.) Superposition of C3L onto S9L initiates at the third molecule from the bottom (i.e. the ‘reference’ molecule), represented in bold magenta and blue overlay. Symmetry operations used to generate the remaining molecules demonstrate the degeneration of the superposition as a result of the 15 Å elongated unit cell of the C3L structure. C3L molecules are shown in blue and green, with S9L structures shown in pink. The dashed box denotes the inset for (b) and (c). (b) Expanded view of the 61 packing scheme for the S9L structure. Atoms engaged in potential hydrogen bonds are depicted as white spheres. Helices are labeled as in Fig. 1 ▶(c) and primes (′) denote symmetry-related molecules. An additional H3′′ helix that is packed in a blunt-ended base stack with H4 has been omitted for clarity. The asymmetric unit (H4) is colored magenta, with symmetry molecules in red (H3′) or light pink (H4′). (c) The C3L 61 packing scheme as described in (b), but the asymmetric unit is colored blue and symmetry mates are coloured teal (H3′) or green (H4′). White spheres identify equivalent atoms in the C3L structure that are engaged in hydrogen bonding in the S9L structure. (d)–(g) Detailed view of hydrogen-bond interactions with each of four H4 residues. The A31 and U31 residues are base-paired; A31 ends the linker strand, while U31 begins the S-turn strand. (h)–(k) Equivalent distances in the C3L structure demonstrate the loss of 61-fold packing interactions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2483500&req=5

fig5: Schematic surface and ball-and-stick diagrams illustrating the 61 packing interactions of hinged hairpin-ribozyme constructs. (a) Comparison of C3L (blue) and S9L (magenta) unit cells. The perspective represents the interaction of molecules about the 61 screw axis. (For twofold and 21 operations, refer to Fig. 6 ▶ a.) Superposition of C3L onto S9L initiates at the third molecule from the bottom (i.e. the ‘reference’ molecule), represented in bold magenta and blue overlay. Symmetry operations used to generate the remaining molecules demonstrate the degeneration of the superposition as a result of the 15 Å elongated unit cell of the C3L structure. C3L molecules are shown in blue and green, with S9L structures shown in pink. The dashed box denotes the inset for (b) and (c). (b) Expanded view of the 61 packing scheme for the S9L structure. Atoms engaged in potential hydrogen bonds are depicted as white spheres. Helices are labeled as in Fig. 1 ▶(c) and primes (′) denote symmetry-related molecules. An additional H3′′ helix that is packed in a blunt-ended base stack with H4 has been omitted for clarity. The asymmetric unit (H4) is colored magenta, with symmetry molecules in red (H3′) or light pink (H4′). (c) The C3L 61 packing scheme as described in (b), but the asymmetric unit is colored blue and symmetry mates are coloured teal (H3′) or green (H4′). White spheres identify equivalent atoms in the C3L structure that are engaged in hydrogen bonding in the S9L structure. (d)–(g) Detailed view of hydrogen-bond interactions with each of four H4 residues. The A31 and U31 residues are base-paired; A31 ends the linker strand, while U31 begins the S-turn strand. (h)–(k) Equivalent distances in the C3L structure demonstrate the loss of 61-fold packing interactions.

Mentions: Contraction of the C3L H3 helical end affects H4′ of the symmetry-related molecule owing to pseudo-continuous helical crystal-packing interactions (Fig. 4 ▶ d). The terminal base pair of H4′ (A31′–U31′) experiences a modest lateral movement similar to, but less than, that observed for H3. This is illustrated in a comparison of the distances between the C3′ of C49 and the O4′ of U31′ (4 Å in S9L versus 2.9 Å in C3L; Figs. 4 ▶ d and 4 ▶ e). Because the lateral shift exhibited by the A31′–U31′ base pair in the C3L structure is not fully commensurate with that of H3, it is necessarily accompanied by a 1.5 Å upward shift of U31′ (Fig. 4 ▶ f). This upward movement circumvents an otherwise inescapable steric clash between the ribose moieties of U31′ and C49. The net result is a more flush base stack between H3 and H4′ in the C3L structure compared with the more staggered interaction observed in the S9L (Figs. 4 ▶ d and 4 ▶ e) and JL structures (not shown). From an engineering and design perspective, there are two opposing factors that appear to influence this end-to-end stacking interaction. The first is the local influence of the shortened C3 linker, which draws the G15–C49 base pair closer to the center of mass of the RNA, effectively improving the crystallographic base-stacking interactions. The second factor originates from crystal contacts observed in both the S9L and JL hairpin-ribozyme structures. In these two structures the terminal residues of both H3′ and H4 are engaged in backbone and minor-groove hydrogen bonds that confer stability along the 61-fold screw axis (for perspective, see Figs. 5 ▶ a and 5 ▶ b). These interactions support the more staggered helical packing, but are notably absent from the C3L structure (Fig. 5 ▶ c, cleft), which is relevant to understanding the principles of RNA interaction that influence high-resolution X-ray diffraction.


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)

Schematic surface and ball-and-stick diagrams illustrating the 61 packing interactions of hinged hairpin-ribozyme constructs. (a) Comparison of C3L (blue) and S9L (magenta) unit cells. The perspective represents the interaction of molecules about the 61 screw axis. (For twofold and 21 operations, refer to Fig. 6 ▶                  a.) Superposition of C3L onto S9L initiates at the third molecule from the bottom (i.e. the ‘reference’ molecule), represented in bold magenta and blue overlay. Symmetry operations used to generate the remaining molecules demonstrate the degeneration of the superposition as a result of the 15 Å elongated unit cell of the C3L structure. C3L molecules are shown in blue and green, with S9L structures shown in pink. The dashed box denotes the inset for (b) and (c). (b) Expanded view of the 61 packing scheme for the S9L structure. Atoms engaged in potential hydrogen bonds are depicted as white spheres. Helices are labeled as in Fig. 1 ▶(c) and primes (′) denote symmetry-related molecules. An additional H3′′ helix that is packed in a blunt-ended base stack with H4 has been omitted for clarity. The asymmetric unit (H4) is colored magenta, with symmetry molecules in red (H3′) or light pink (H4′). (c) The C3L 61 packing scheme as described in (b), but the asymmetric unit is colored blue and symmetry mates are coloured teal (H3′) or green (H4′). White spheres identify equivalent atoms in the C3L structure that are engaged in hydrogen bonding in the S9L structure. (d)–(g) Detailed view of hydrogen-bond interactions with each of four H4 residues. The A31 and U31 residues are base-paired; A31 ends the linker strand, while U31 begins the S-turn strand. (h)–(k) Equivalent distances in the C3L structure demonstrate the loss of 61-fold packing interactions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Schematic surface and ball-and-stick diagrams illustrating the 61 packing interactions of hinged hairpin-ribozyme constructs. (a) Comparison of C3L (blue) and S9L (magenta) unit cells. The perspective represents the interaction of molecules about the 61 screw axis. (For twofold and 21 operations, refer to Fig. 6 ▶ a.) Superposition of C3L onto S9L initiates at the third molecule from the bottom (i.e. the ‘reference’ molecule), represented in bold magenta and blue overlay. Symmetry operations used to generate the remaining molecules demonstrate the degeneration of the superposition as a result of the 15 Å elongated unit cell of the C3L structure. C3L molecules are shown in blue and green, with S9L structures shown in pink. The dashed box denotes the inset for (b) and (c). (b) Expanded view of the 61 packing scheme for the S9L structure. Atoms engaged in potential hydrogen bonds are depicted as white spheres. Helices are labeled as in Fig. 1 ▶(c) and primes (′) denote symmetry-related molecules. An additional H3′′ helix that is packed in a blunt-ended base stack with H4 has been omitted for clarity. The asymmetric unit (H4) is colored magenta, with symmetry molecules in red (H3′) or light pink (H4′). (c) The C3L 61 packing scheme as described in (b), but the asymmetric unit is colored blue and symmetry mates are coloured teal (H3′) or green (H4′). White spheres identify equivalent atoms in the C3L structure that are engaged in hydrogen bonding in the S9L structure. (d)–(g) Detailed view of hydrogen-bond interactions with each of four H4 residues. The A31 and U31 residues are base-paired; A31 ends the linker strand, while U31 begins the S-turn strand. (h)–(k) Equivalent distances in the C3L structure demonstrate the loss of 61-fold packing interactions.
Mentions: Contraction of the C3L H3 helical end affects H4′ of the symmetry-related molecule owing to pseudo-continuous helical crystal-packing interactions (Fig. 4 ▶ d). The terminal base pair of H4′ (A31′–U31′) experiences a modest lateral movement similar to, but less than, that observed for H3. This is illustrated in a comparison of the distances between the C3′ of C49 and the O4′ of U31′ (4 Å in S9L versus 2.9 Å in C3L; Figs. 4 ▶ d and 4 ▶ e). Because the lateral shift exhibited by the A31′–U31′ base pair in the C3L structure is not fully commensurate with that of H3, it is necessarily accompanied by a 1.5 Å upward shift of U31′ (Fig. 4 ▶ f). This upward movement circumvents an otherwise inescapable steric clash between the ribose moieties of U31′ and C49. The net result is a more flush base stack between H3 and H4′ in the C3L structure compared with the more staggered interaction observed in the S9L (Figs. 4 ▶ d and 4 ▶ e) and JL structures (not shown). From an engineering and design perspective, there are two opposing factors that appear to influence this end-to-end stacking interaction. The first is the local influence of the shortened C3 linker, which draws the G15–C49 base pair closer to the center of mass of the RNA, effectively improving the crystallographic base-stacking interactions. The second factor originates from crystal contacts observed in both the S9L and JL hairpin-ribozyme structures. In these two structures the terminal residues of both H3′ and H4 are engaged in backbone and minor-groove hydrogen bonds that confer stability along the 61-fold screw axis (for perspective, see Figs. 5 ▶ a and 5 ▶ b). These interactions support the more staggered helical packing, but are notably absent from the C3L structure (Fig. 5 ▶ c, cleft), which is relevant to understanding the principles of RNA interaction that influence high-resolution X-ray diffraction.

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