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Structural basis for specific single-stranded RNA recognition by designer pentatricopeptide repeat proteins.

Shen C, Zhang D, Guan Z, Liu Y, Yang Z, Yang Y, Wang X, Wang Q, Zhang Q, Fan S, Zou T, Yin P - Nat Commun (2016)

Bottom Line: The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA.Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G.These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

No MeSH data available.


Structural plasticity of U8C2.(a) All dPPR repeats of dPPR-U8C2 exhibit a nearly identical conformation. Each repeat is organized into two helices (a and b) followed by a short loop. (b) The contact between adjacent dPPR repeats is primarily mediated by van der Waals interactions. Repeats 5 and 6 from dPPR-U8C2 are coloured cyan and green, respectively. Two perpendicular views are presented, with amino acids that participate in the interaction shown in violet and magenta at repeats 5 and 6, respectively. (c) RNA-bound dPPR-U8C2 exhibits a more compressed conformation than RNA-free cPPR and synthPPR3.5. The three structures are superimposed using the first PPR repeat of each protein, and dPPR-U8C2, cPPR and synthPPR3.5 are coloured green, light blue and orange. The blue dashed arrow indicates the conformational differences between dPPR-U8C2 and cPPR.
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f3: Structural plasticity of U8C2.(a) All dPPR repeats of dPPR-U8C2 exhibit a nearly identical conformation. Each repeat is organized into two helices (a and b) followed by a short loop. (b) The contact between adjacent dPPR repeats is primarily mediated by van der Waals interactions. Repeats 5 and 6 from dPPR-U8C2 are coloured cyan and green, respectively. Two perpendicular views are presented, with amino acids that participate in the interaction shown in violet and magenta at repeats 5 and 6, respectively. (c) RNA-bound dPPR-U8C2 exhibits a more compressed conformation than RNA-free cPPR and synthPPR3.5. The three structures are superimposed using the first PPR repeat of each protein, and dPPR-U8C2, cPPR and synthPPR3.5 are coloured green, light blue and orange. The blue dashed arrow indicates the conformational differences between dPPR-U8C2 and cPPR.

Mentions: Similarly to RNA-free PPR10, all dPPR-U8C2 repeats exhibit a nearly identical conformation except for the short turns connecting helix a and helix b (Fig. 3a). In addition, all dPPR repeats exhibit a high degree of structural homology with the repeats in RNA-bound PPR10 (PDB ID: 4M59)22, artificially engineered cPPR-polyC (PDB ID: 4WSL)29 and synthetic PPR protein synthPPR3.5 (PDB ID: 4OZS)35, indicating that our RNA-bound dPPR motifs fold similarly to the natural ones and to engineered proteins in their RNA-free form. In the dPPR-U8C2 structure, each helix b stacks against the helix a of the following repeat through extensive van der Waals interactions, forming an inter-repeat structure similar to a three-helix bundle (Fig. 3b). Furthermore, although the structures of cPPR-polyC, synthPPR3.5, RNA-bound PPR10 (repeat 6–15) and dPPR-U8C2 all exhibit superhelical spiral shapes, they are distinct from one another in their configurational details. Although it has a similar diameter to RNA-free cPPR-polyC and synthPPR3.5, RNA-bound dPPR-U8C2 exhibits a more compact conformation with a helical period length of ∼70 Å, which is shorter than that of cPPR proteins (∼90 Å; Fig. 3c). The fact that the three types of engineered PPR motifs (dPPR, cPPR and synthPPR) are almost identical indicates that RNA binding may change the interaction between PPR hairpin-shaped motifs and induce conformational changes. Compared with RNA-bound PPR10 (repeats 6–15), RNA-bound dPPR-U8C2 also exhibits in a tighter form, and there is a 20-Å difference between their diameters (Supplementary Fig. 5). According to previous studies, repeats 6–15 of PPR10 fail to bind RNA perfectly, thus suggesting that RNA binding might contribute to subtle conformational variations. We speculate that these differences may be gradually amplified over an increasing number of repeats, ultimately leading to the prominent compression of the superhelix. This conformational plasticity appears to be a result of extensive van der Waals interactions between adjacent repeats. A similar phenomenon has been observed in DNA-bound/unbound TALE crystal structures3637 but not in the PUF–RNA interaction838.


Structural basis for specific single-stranded RNA recognition by designer pentatricopeptide repeat proteins.

Shen C, Zhang D, Guan Z, Liu Y, Yang Z, Yang Y, Wang X, Wang Q, Zhang Q, Fan S, Zou T, Yin P - Nat Commun (2016)

Structural plasticity of U8C2.(a) All dPPR repeats of dPPR-U8C2 exhibit a nearly identical conformation. Each repeat is organized into two helices (a and b) followed by a short loop. (b) The contact between adjacent dPPR repeats is primarily mediated by van der Waals interactions. Repeats 5 and 6 from dPPR-U8C2 are coloured cyan and green, respectively. Two perpendicular views are presented, with amino acids that participate in the interaction shown in violet and magenta at repeats 5 and 6, respectively. (c) RNA-bound dPPR-U8C2 exhibits a more compressed conformation than RNA-free cPPR and synthPPR3.5. The three structures are superimposed using the first PPR repeat of each protein, and dPPR-U8C2, cPPR and synthPPR3.5 are coloured green, light blue and orange. The blue dashed arrow indicates the conformational differences between dPPR-U8C2 and cPPR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837458&req=5

f3: Structural plasticity of U8C2.(a) All dPPR repeats of dPPR-U8C2 exhibit a nearly identical conformation. Each repeat is organized into two helices (a and b) followed by a short loop. (b) The contact between adjacent dPPR repeats is primarily mediated by van der Waals interactions. Repeats 5 and 6 from dPPR-U8C2 are coloured cyan and green, respectively. Two perpendicular views are presented, with amino acids that participate in the interaction shown in violet and magenta at repeats 5 and 6, respectively. (c) RNA-bound dPPR-U8C2 exhibits a more compressed conformation than RNA-free cPPR and synthPPR3.5. The three structures are superimposed using the first PPR repeat of each protein, and dPPR-U8C2, cPPR and synthPPR3.5 are coloured green, light blue and orange. The blue dashed arrow indicates the conformational differences between dPPR-U8C2 and cPPR.
Mentions: Similarly to RNA-free PPR10, all dPPR-U8C2 repeats exhibit a nearly identical conformation except for the short turns connecting helix a and helix b (Fig. 3a). In addition, all dPPR repeats exhibit a high degree of structural homology with the repeats in RNA-bound PPR10 (PDB ID: 4M59)22, artificially engineered cPPR-polyC (PDB ID: 4WSL)29 and synthetic PPR protein synthPPR3.5 (PDB ID: 4OZS)35, indicating that our RNA-bound dPPR motifs fold similarly to the natural ones and to engineered proteins in their RNA-free form. In the dPPR-U8C2 structure, each helix b stacks against the helix a of the following repeat through extensive van der Waals interactions, forming an inter-repeat structure similar to a three-helix bundle (Fig. 3b). Furthermore, although the structures of cPPR-polyC, synthPPR3.5, RNA-bound PPR10 (repeat 6–15) and dPPR-U8C2 all exhibit superhelical spiral shapes, they are distinct from one another in their configurational details. Although it has a similar diameter to RNA-free cPPR-polyC and synthPPR3.5, RNA-bound dPPR-U8C2 exhibits a more compact conformation with a helical period length of ∼70 Å, which is shorter than that of cPPR proteins (∼90 Å; Fig. 3c). The fact that the three types of engineered PPR motifs (dPPR, cPPR and synthPPR) are almost identical indicates that RNA binding may change the interaction between PPR hairpin-shaped motifs and induce conformational changes. Compared with RNA-bound PPR10 (repeats 6–15), RNA-bound dPPR-U8C2 also exhibits in a tighter form, and there is a 20-Å difference between their diameters (Supplementary Fig. 5). According to previous studies, repeats 6–15 of PPR10 fail to bind RNA perfectly, thus suggesting that RNA binding might contribute to subtle conformational variations. We speculate that these differences may be gradually amplified over an increasing number of repeats, ultimately leading to the prominent compression of the superhelix. This conformational plasticity appears to be a result of extensive van der Waals interactions between adjacent repeats. A similar phenomenon has been observed in DNA-bound/unbound TALE crystal structures3637 but not in the PUF–RNA interaction838.

Bottom Line: The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA.Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G.These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China.

ABSTRACT
As a large family of RNA-binding proteins, pentatricopeptide repeat (PPR) proteins mediate multiple aspects of RNA metabolism in eukaryotes. Binding to their target single-stranded RNAs (ssRNAs) in a modular and base-specific fashion, PPR proteins can serve as designable modules for gene manipulation. However, the structural basis for nucleotide-specific recognition by designer PPR (dPPR) proteins remains to be elucidated. Here, we report four crystal structures of dPPR proteins in complex with their respective ssRNA targets. The dPPR repeats are assembled into a right-handed superhelical spiral shell that embraces the ssRNA. Interactions between different PPR codes and RNA bases are observed at the atomic level, revealing the molecular basis for the modular and specific recognition patterns of the RNA bases U, C, A and G. These structures not only provide insights into the functional study of PPR proteins but also open a path towards the potential design of synthetic sequence-specific RNA-binding proteins.

No MeSH data available.