- Email: [email protected]
Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation
Biochemical and Biophysical Research Communications 466 (2015) 388e392
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation Shoji J. Ohuchi a, Fumihiko Sagawa a, Taiichi Sakamoto b, Tan Inoue a, * a
Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba, 275-0016, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2015 Accepted 6 September 2015 Available online 9 September 2015
RNA-protein complexes (RNPs) are useful for constructing functional nano-objects because a variety of functional proteins can be displayed on a designed RNA scaffold. Here, we report circular permutations of an RNA-binding protein L7Ae based on the three-dimensional structure information to alter the orientation of the displayed proteins on the RNA scaffold. An electrophoretic mobility shift assay and atomic force microscopy (AFM) analysis revealed that most of the designed circular permutants formed an RNP nano-object. Moreover, the alteration of the enhanced green fluorescent protein (EGFP) orientation was confirmed with AFM by employing EGFP on the L7Ae permutant on the RNA. The results demonstrate that targeted fine-tuning of the stereo-specific fixation of a protein on a protein-binding RNA is feasible by using the circular permutation technique. © 2015 Elsevier Inc. All rights reserved.
Keywords: Circular permutation RNA-protein complex RNP nano-object
1. Introduction In nanobiotechnology, highly sophisticated nano-objects have been constructed by employing RNA because naturally occurring RNAs utilize a variety of three-dimensional (3D) structural motifs that are unavailable for DNA [1e3]. However, it is difficult to add a desired functionality to these nano-objects owing to the limited number of available functional RNAs. By contrast, proteins possess sophisticated functionality, such as a high affinity and specificity for antibodies and enzyme catalytic activity. However, the molecular design of proteins remains difficult at the refined 3D level due to complicated folding processes. Thus, functional nano-objects with a defined 3D structure and high functionality have been designed and constructed by the combined use of RNA and protein to overcome these problems. The signs of the RNA-protein world remain present in living organisms because many functional RNA-protein complexes (RNPs), represented by ribosomes and spliceosomes, exist in present cells. This indicates that artificial RNP engineering is feasible
Abbreviations: RNP, RNA-protein complex. * Corresponding author. E-mail address: [email protected] (T. Inoue). http://dx.doi.org/10.1016/j.bbrc.2015.09.035 0006-291X/© 2015 Elsevier Inc. All rights reserved.
and viable for exploring and modifying living systems. Recently, Sachdeva et al. reported the in vivo expression of an RNP nanoobject displaying two enzymes that catalyze a cascade reaction of a pentadecane production pathway [4]. They found that the reaction efficiency depended on the geometry of the enzyme placement on the scaffold RNA, which can be changed by rotation via altering the length of the flanking RNA stem. Thus, the distance between and relative orientation of the proteins can be controlled to optimize the functionality of RNP nano-objects by changing the RNA stem length. Such stereo-specific control of the displayed proteins would be valuable for the optimization of enzymatic cascade reactions, the recognition of a target by affinity proteins, and the induction of signal transduction by ligand proteins. However, the altered design of the RNA does not allow for the simultaneous regulation of the distance between the proteins and the relative orientation of those on the scaffold. Furthermore, the geometrical alteration of the protein placement is restricted by the circumferential outline of the stem axes. To solve these problems, we established an alternative strategy for the stereo-specific control of protein placement on RNA. Previously, we reported the design and construction of an RNP nano-object consisting of three proteins bound to an RNA scaffold by employing L7Ae, a ribosomal protein, and its binding partner,
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termed the box C/D kink-turn RNA motif [5]. The protein binding enables the box C/D motif to bend by ~60 at three tips to form a nearly equilateral triangle [6]. The triangular RNP can be functionalized if L7Ae fused with another functional protein is attached to the tips of the RNA [5,7e9]. For example, a fusion protein with an affinity protein enabled the construction of an RNP displaying three affinity moieties, and this triangular RNP showed a superior binding profile compared with a monomeric affinity protein due to the avidity effect on the human cell surface [7,8]. Another example is the display of a ligand protein, galectin 1, that initiates an apoptotic signal in human cells [8]. Precise regulation of the receptor assemblage was achieved for the quantitative ON/OFF signal transduction by controlling the sizes of the fusion RNP because the distance between the ligands is determined by the size of the triangular-shaped scaffold RNA. To further enhance this molecular design technique, we report here the stereo-specific azimuth adjustment of L7Ae protein at the tips of the scaffold RNA using the circular permutation technique. 2. Materials and methods RNA preparation was carried out as previously described, employing PCR products or partially double-stranded synthetic DNAs as a transcription template [9]. All synthetic DNAs were purchased from Eurofins Genomics Japan (Japan). L7Ae, cpL7Ae, and their EGFP-fusion proteins were expressed and purified as previously described [5,8]. Analyses of the RNP complex by SPR, EMSA, and AFM were carried out as described previously [5,8,9]. For the detection of the RNP with the EGFP fusion proteins by EMSA, the gels were stained with ethidium bromide because the fluorescence wavelength of SYBR green staining overlaps that of EGFP. Detailed experimental methods are described in Supplemental Information. 3. Results The circular permutation technique can reorganize the topology of an existing protein [10,11]. This technique has been utilized for the improvement of protein properties, the construction of artificial allosteric proteins and other applications by connecting the native terminals with a linker peptide and cleaving an existing peptide chain to generate new terminals. We applied this technique for altering the relative orientation of a protein fused to mutated L7Ae on triangular RNA (Fig. S1). The circular permutations were rationally designed based on the available 3D crystal structure of the L7Ae protein-box C/D RNA complex to investigate RNA binding capacity and protein orientation on RNA (Fig. S1). The resulting mutated L7Aes were intended to create new termini that are physically different from one another, which should in turn provide several independent stereochemical positions of the displayed L7Ae and its fused proteins (Fig. 1). Eight residues (Gly26, Gly42, Glu57, Asn70, Ser78, Gly87, Asn99, and Gly101) were selected to form a new N-terminus from the protein surface residues that are not involved in RNA recognition and the stabilization of the protein structure (Fig. 1 and Fig. S2) [6]. The binding activities of the designed circular permutants (cpL7Aes) to the box C/D motif were analyzed by surface plasmon resonance spectroscopy. Five variants (cpL7Ae-Gly26, cpL7Ae-Asn70, cpL7AeGly87, cpL7Ae-Asn99, and cpL7Ae-Gly101) revealed the activities comparable to that of the wild-type L7Ae (Table S1 and Fig. S3). To investigate the ability of forming the triangular RNP, an electrophoretic mobility shift assay (EMSA) was performed by employing a scaffold RNA with a 26-base pair (bp) stem, Tri26L/S (Fig. S4). The wild-type L7Ae produces three upshifted RNA bands that correspond to the RNP with one, two, and three L7Ae proteins. As
Fig. 1. Three dimensional structure of the L7Ae-box C/D RNA complex (PDB ID 1RLG). The structure in (B) was obtained by 90 rotation around the horizontal axis to that in (A). The C-terminal residue (Lys119) and the eight residues (Gly26, Gly42, Glu57, Asn70, Ser78, Gly87, Asn99, and Gly101) chosen as a new N-terminus are shown as colored space-filling amino acids. The box C/D RNA is shown as a stick model colored in light blue. Images were prepared using UCSF Chimera [12]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
anticipated, the variants with the binding affinity comparable to that of the wild-type protein formed a triangular RNP (Fig. 2). In addition to the variants with the binding affinity comparable to that of the wild-type protein, the moderately reduced affinity of the cpL7Ae-Gly42 variant suggested that this structure also forms a triangular RNP (Fig. 2C). Furthermore, the variant with reduced affinity (two orders of magnitude less), cpL7Ae-Glu57, formed a triangular RNP (Fig. 2D), whereas the other low-affinity variant, cpL7Ae-Ser78, hardly formed a triangular RNP even in the presence of the highly concentrated protein (Fig. 2F). This observation indicates a possibility that a certain structural difference exists between the box C/D motif on Tri26L/S RNA and the free box C/D motif. The triangular RNP formations of these variants were also confirmed by high-speed liquid-phase atomic force microscopy (AFM) (Fig. S5). Next, fusion proteins of cpL7Ae and enhanced green fluorescent protein (EGFP) were examined whether the designed circular permutants can form the triangular RNA (Fig. 3). The EGFP fusion proteins of cpL7Ae-Gly26, cpL7Ae-Asn70, cpL7Ae-Asn99 and cpL7Ae-Gly101 formed a triangular RNP as efficiently as the wildtype L7Ae-EGFP fusion. The EGFP fusion proteins of the three variants with reduced affinity (cpL7Ae-Gly42, cpL7Ae-Glu57, and cpL7Ae-Ser78) formed incomplete RNPs with one or two proteins
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Fig. 2. EMSA analysis of the triangular RNP formation with wild-type or circular permutant L7Ae. The triangular RNPs were prepared by mixing 25 nmol Tri26L/S RNA and the indicated concentrations of wild-type L7Ae (A), cpL7Ae-Gly26 (B), cpL7Ae-Gly42 (C), cpL7Ae-Glu57 (D), cpL7Ae-Asn70 (E), cpL7Ae-Ser78 (F), cpL7Ae-Gly87 (G), cpL7Ae-Asn99 (H), or cpL7Ae-Gly101 (I). In Figures (C), (D), (F), and (H), the triangular RNP prepared with wild-type protein (‘WT’) was also loaded as a control. The gel was stained with SYBR green to scan for the RNAs. The three upshifted bands correspond to the RNPs with one, two and three proteins.
Fig. 3. EMSA analysis of the triangular RNP formation with EGFP fusions of the wild-type or circular permutant L7Ae. The triangular RNPs were prepared by mixing 200 nmol Tri26L/S RNA and the indicated concentrations of wild-type L7Ae-EGFP (A), cpL7Ae-Gly26-EGFP (B), cpL7Ae-Gly42-EGFP (C), cpL7Ae-Glu57-EGFP (D), cpL7Ae-Asn70-EGFP (E), cpL7Ae-Ser78-EGFP (F), cpL7Ae-Gly87-EGFP (G), cpL7Ae-Asn99-EGFP (H), or cpL7Ae-Gly101-EGFP (I). In Figures (B) to (I), the triangular RNP prepared with wild-type L7Ae-EGFP (‘WT’) was also loaded as a control. The gel was stained with ethidium bromide to scan for the RNAs. The three upshifted bands correspond to the RNPs with one, two and three protein. An EMSA assay was performed to analyze the triangular RNP formed with 200 nM Tri26L/S RNA and varying concentrations of EGFP fusion protein.
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bound to the RNA. However, the EGFP fusion protein of cpL7AeGly87 hardly bound to the RNA even in the presence of high concentrations of the protein (Fig. 3G). The resulting difference from the data with the non-fused protein (Fig. 2G) was perhaps caused by the steric hindrance of EGFP because Gly87 is located very close to the box C/D RNA in the complex (Fig. 1 and Fig. S2). Finally, fusion proteins of cpL7Ae and EGFP were examined
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whether the designed circular permutants alter the orientation of the protein on the triangular RNA by using AFM (Fig. 4). A newly designed scaffold RNA containing parallel double stems, Tri48stem RNA, was employed to distinguish the front and back face of the triangular RNP (Fig. S4, Fig. S6, and Fig. S7). Consistent with the previous observations [5], EGFP orientations of the triangular RNP with wild-type L7Ae-EGFP were in the distal direction (Fig. 4A and C). Interestingly, EGFP orientations of the one variant, cpL7AeAsn70-EGFP, were apparently different from those of wild-type L7Ae-EGFP (Fig. 4B and C). As observed, the EGFP orientations were consistent with the model structure (Fig. 4B), indicating that the triangular RNP adopted the designed structure. Triangular RNP composed of cpL7Ae-Asn70-EGFP and Tri26L/S RNA also exhibited similar EGFP orientations, indicating that the observed orientation does not depend on the scaffold RNA (Fig. S8). The EGFP orientations of three other variants were not observed as clearly as that of cpL7Ae-Asn70-EGFP under the same conditions (Fig. S9). This is likely due to the intrinsic flexibility of the linker between cpL7Ae and EGFP, the high flexibility of the residue chosen for a C-terminus, and/or the non-uniform adsorption property of the protein moieties onto the mica surface. 4. Discussion RNPs are useful for constructing functional nano-objects because a variety of functional proteins can be attached to a designed RNA scaffold. To further improve the molecular design technique, an alternative stereo-specific fixation of functional protein displayed on the RNP is desired. In this study, we designed cpL7Aes to alter the orientation of the displayed protein on RNA. Among eight cpL7Aes designed, one variant, cpL7Ae-Asn70-EGFP, showed the altered orientation of EGFP on the triangular RNP as designed. Our results demonstrate that fine-tuning of the stereospecific fixation of proteins on RNA is feasible using the circular permutation technique. These results constitute a step further in RNP design and indicate that the stereochemical control of functional proteins on RNA enables a high level of precision nanobiotechnology. In the application of this technique, the display of an affinity protein at the desired position with fixed orientation highly contributes to efficient target recognition due to the lower degree of entropy change upon the allosteric binding. Accordingly, the stereo-specific control of the displayed ligand to a receptor protein has potential to improve the efficiency of signal induction [8]. The stereo-specific control of the displayed enzymes will improve nanobiotechnology for optimizing enzymatic cascade reactions [4]. Acknowledgments
Fig. 4. Structures of the triangular RNP constructed with EGFP fusion proteins. (A and B) Model structure (upper images) and AFM images (lower images) of the triangular RNP composed of Tri48stem RNA and wild-type L7Ae-EGFP (A) and cpL7Ae-Asn70EGFP (B). The upper left and upper right images show the front and back faces of the model structure of the triangular RNP, respectively. The branched stem-forming RNA, long RNA, and short RNA strands are shown in light blue, green, and dark blue, respectively. L7Ae variants, linker residues, and EGFP moieties are shown in yellow, orange, and red, respectively. The model structure images were prepared using the ICM-Pro software package. For the AFM analyses, the triangular RNPs were prepared by mixing 5 nmol Tri48stem RNA and 40 nmol fusion protein. The branched stems (bumps) are emphasized by the white arrowheads. The white bars on the lower left two images indicate 50 nm, and the right six images were acquired on smaller scales (30 nm 30 nm). (C) Orientations of EGFP displayed on the triangular RNP. The three tips of the triangle with black lines indicate the highest positions of the wild-type L7Ae or cpL7Ae-Asn70 moieties on the front face of the RNP of the AFM image. The highest positions of the EGFP moieties and the L7Ae or cpL7Ae-Asn70 moieties are linked by bold blue and red lines, respectively. The average orientations of the EGFP moieties were obtained from 28 images of the RNP (N ¼ 54) and are represented as the degree of the angles between a side of the triangle and bold blue or red lines. Thin blue and red lines indicate standard deviation values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
This work was supported by grants from the Japan Society for the Promotion of Science (23221011 and 23119005). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.09.035. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.035. References [1] W.W. Grabow, L. Jaeger, RNA self-assembly and RNA nanotechnology, Acc. Chem. Res. 47 (2014) 1871e1880.
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[2] J. Ishikawa, H. Furuta, Y. Ikawa, RNA tectonics (tectoRNA) for RNA nanostructure design and its application in synthetic biology, Wiley Interdiscip. Rev. RNA 4 (2013) 651e664. [3] W. Grabow, L. Jaeger, RNA modularity for synthetic biology, F1000Prime Rep. 5 (2013) 46. [4] G. Sachdeva, A. Garg, D. Godding, J.C. Way, P.A. Silver, In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner, Nucleic Acids Res. 42 (2014) 9493e9503. [5] H. Ohno, T. Kobayashi, R. Kabata, K. Endo, T. Iwasa, S.H. Yoshimura, K. Takeyasu, T. Inoue, H. Saito, Synthetic RNAeprotein complex shaped like an equilateral triangle, Nat. Nanotechnol. 6 (2011) 116e120. [6] T. Moore, Y. Zhang, M.O. Fenley, H. Li, Molecular basis of box C/D RNA-protein interactions; cocrystal structure of archaeal L7Ae and a box C/D RNA, Structure 12 (2004) 807e818. [7] E. Osada, Y. Suzuki, K. Hidaka, H. Ohno, H. Sugiyama, M. Endo, H. Saito, Engineering RNA-protein complexes with different shapes for imaging and
therapeutic applications, ACS Nano 8 (2014) 8130e8140. [8] Y. Fujita, R. Furushima, H. Ohno, F. Sagawa, T. Inoue, Cell-Surface receptor control that depends on the size of a synthetic equilateral-triangular RNAProtein complex, Sci. Rep. 4 (2014) 6422. [9] S.J. Ohuchi, F. Sagawa, H. Ohno, T. Inoue, A purification method for a molecular complex in which a scaffold molecule is fully loaded with heterogeneous molecules, PLoS One 10 (2015) e0120576. [10] Y. Yu, S. Lutz, Circular permutation: a different way to engineer enzyme structure and function, Trends Biotechnol. 29 (2011) 18e25. [11] U. Heinemann, M. Hahn, Circular permutation of polypeptide chains: implications for protein folding and stability, Prog. Biophys. Mol. Biol. 64 (1995) 121e143. [12] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimeraea visualization system for exploratory research and analysis, J. Comput. Chem. 25 (2004) 1605e1612.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Altering the orientation of a fused protein to the RNA-binding ribosomal protein L7Ae and its derivatives through circular permutation Shoji J. Ohuchi a, Fumihiko Sagawa a, Taiichi Sakamoto b, Tan Inoue a, * a
Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba, 275-0016, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2015 Accepted 6 September 2015 Available online 9 September 2015
RNA-protein complexes (RNPs) are useful for constructing functional nano-objects because a variety of functional proteins can be displayed on a designed RNA scaffold. Here, we report circular permutations of an RNA-binding protein L7Ae based on the three-dimensional structure information to alter the orientation of the displayed proteins on the RNA scaffold. An electrophoretic mobility shift assay and atomic force microscopy (AFM) analysis revealed that most of the designed circular permutants formed an RNP nano-object. Moreover, the alteration of the enhanced green fluorescent protein (EGFP) orientation was confirmed with AFM by employing EGFP on the L7Ae permutant on the RNA. The results demonstrate that targeted fine-tuning of the stereo-specific fixation of a protein on a protein-binding RNA is feasible by using the circular permutation technique. © 2015 Elsevier Inc. All rights reserved.
Keywords: Circular permutation RNA-protein complex RNP nano-object
1. Introduction In nanobiotechnology, highly sophisticated nano-objects have been constructed by employing RNA because naturally occurring RNAs utilize a variety of three-dimensional (3D) structural motifs that are unavailable for DNA [1e3]. However, it is difficult to add a desired functionality to these nano-objects owing to the limited number of available functional RNAs. By contrast, proteins possess sophisticated functionality, such as a high affinity and specificity for antibodies and enzyme catalytic activity. However, the molecular design of proteins remains difficult at the refined 3D level due to complicated folding processes. Thus, functional nano-objects with a defined 3D structure and high functionality have been designed and constructed by the combined use of RNA and protein to overcome these problems. The signs of the RNA-protein world remain present in living organisms because many functional RNA-protein complexes (RNPs), represented by ribosomes and spliceosomes, exist in present cells. This indicates that artificial RNP engineering is feasible
Abbreviations: RNP, RNA-protein complex. * Corresponding author. E-mail address: [email protected] (T. Inoue). http://dx.doi.org/10.1016/j.bbrc.2015.09.035 0006-291X/© 2015 Elsevier Inc. All rights reserved.
and viable for exploring and modifying living systems. Recently, Sachdeva et al. reported the in vivo expression of an RNP nanoobject displaying two enzymes that catalyze a cascade reaction of a pentadecane production pathway [4]. They found that the reaction efficiency depended on the geometry of the enzyme placement on the scaffold RNA, which can be changed by rotation via altering the length of the flanking RNA stem. Thus, the distance between and relative orientation of the proteins can be controlled to optimize the functionality of RNP nano-objects by changing the RNA stem length. Such stereo-specific control of the displayed proteins would be valuable for the optimization of enzymatic cascade reactions, the recognition of a target by affinity proteins, and the induction of signal transduction by ligand proteins. However, the altered design of the RNA does not allow for the simultaneous regulation of the distance between the proteins and the relative orientation of those on the scaffold. Furthermore, the geometrical alteration of the protein placement is restricted by the circumferential outline of the stem axes. To solve these problems, we established an alternative strategy for the stereo-specific control of protein placement on RNA. Previously, we reported the design and construction of an RNP nano-object consisting of three proteins bound to an RNA scaffold by employing L7Ae, a ribosomal protein, and its binding partner,
S.J. Ohuchi et al. / Biochemical and Biophysical Research Communications 466 (2015) 388e392
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termed the box C/D kink-turn RNA motif [5]. The protein binding enables the box C/D motif to bend by ~60 at three tips to form a nearly equilateral triangle [6]. The triangular RNP can be functionalized if L7Ae fused with another functional protein is attached to the tips of the RNA [5,7e9]. For example, a fusion protein with an affinity protein enabled the construction of an RNP displaying three affinity moieties, and this triangular RNP showed a superior binding profile compared with a monomeric affinity protein due to the avidity effect on the human cell surface [7,8]. Another example is the display of a ligand protein, galectin 1, that initiates an apoptotic signal in human cells [8]. Precise regulation of the receptor assemblage was achieved for the quantitative ON/OFF signal transduction by controlling the sizes of the fusion RNP because the distance between the ligands is determined by the size of the triangular-shaped scaffold RNA. To further enhance this molecular design technique, we report here the stereo-specific azimuth adjustment of L7Ae protein at the tips of the scaffold RNA using the circular permutation technique. 2. Materials and methods RNA preparation was carried out as previously described, employing PCR products or partially double-stranded synthetic DNAs as a transcription template [9]. All synthetic DNAs were purchased from Eurofins Genomics Japan (Japan). L7Ae, cpL7Ae, and their EGFP-fusion proteins were expressed and purified as previously described [5,8]. Analyses of the RNP complex by SPR, EMSA, and AFM were carried out as described previously [5,8,9]. For the detection of the RNP with the EGFP fusion proteins by EMSA, the gels were stained with ethidium bromide because the fluorescence wavelength of SYBR green staining overlaps that of EGFP. Detailed experimental methods are described in Supplemental Information. 3. Results The circular permutation technique can reorganize the topology of an existing protein [10,11]. This technique has been utilized for the improvement of protein properties, the construction of artificial allosteric proteins and other applications by connecting the native terminals with a linker peptide and cleaving an existing peptide chain to generate new terminals. We applied this technique for altering the relative orientation of a protein fused to mutated L7Ae on triangular RNA (Fig. S1). The circular permutations were rationally designed based on the available 3D crystal structure of the L7Ae protein-box C/D RNA complex to investigate RNA binding capacity and protein orientation on RNA (Fig. S1). The resulting mutated L7Aes were intended to create new termini that are physically different from one another, which should in turn provide several independent stereochemical positions of the displayed L7Ae and its fused proteins (Fig. 1). Eight residues (Gly26, Gly42, Glu57, Asn70, Ser78, Gly87, Asn99, and Gly101) were selected to form a new N-terminus from the protein surface residues that are not involved in RNA recognition and the stabilization of the protein structure (Fig. 1 and Fig. S2) [6]. The binding activities of the designed circular permutants (cpL7Aes) to the box C/D motif were analyzed by surface plasmon resonance spectroscopy. Five variants (cpL7Ae-Gly26, cpL7Ae-Asn70, cpL7AeGly87, cpL7Ae-Asn99, and cpL7Ae-Gly101) revealed the activities comparable to that of the wild-type L7Ae (Table S1 and Fig. S3). To investigate the ability of forming the triangular RNP, an electrophoretic mobility shift assay (EMSA) was performed by employing a scaffold RNA with a 26-base pair (bp) stem, Tri26L/S (Fig. S4). The wild-type L7Ae produces three upshifted RNA bands that correspond to the RNP with one, two, and three L7Ae proteins. As
Fig. 1. Three dimensional structure of the L7Ae-box C/D RNA complex (PDB ID 1RLG). The structure in (B) was obtained by 90 rotation around the horizontal axis to that in (A). The C-terminal residue (Lys119) and the eight residues (Gly26, Gly42, Glu57, Asn70, Ser78, Gly87, Asn99, and Gly101) chosen as a new N-terminus are shown as colored space-filling amino acids. The box C/D RNA is shown as a stick model colored in light blue. Images were prepared using UCSF Chimera [12]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
anticipated, the variants with the binding affinity comparable to that of the wild-type protein formed a triangular RNP (Fig. 2). In addition to the variants with the binding affinity comparable to that of the wild-type protein, the moderately reduced affinity of the cpL7Ae-Gly42 variant suggested that this structure also forms a triangular RNP (Fig. 2C). Furthermore, the variant with reduced affinity (two orders of magnitude less), cpL7Ae-Glu57, formed a triangular RNP (Fig. 2D), whereas the other low-affinity variant, cpL7Ae-Ser78, hardly formed a triangular RNP even in the presence of the highly concentrated protein (Fig. 2F). This observation indicates a possibility that a certain structural difference exists between the box C/D motif on Tri26L/S RNA and the free box C/D motif. The triangular RNP formations of these variants were also confirmed by high-speed liquid-phase atomic force microscopy (AFM) (Fig. S5). Next, fusion proteins of cpL7Ae and enhanced green fluorescent protein (EGFP) were examined whether the designed circular permutants can form the triangular RNA (Fig. 3). The EGFP fusion proteins of cpL7Ae-Gly26, cpL7Ae-Asn70, cpL7Ae-Asn99 and cpL7Ae-Gly101 formed a triangular RNP as efficiently as the wildtype L7Ae-EGFP fusion. The EGFP fusion proteins of the three variants with reduced affinity (cpL7Ae-Gly42, cpL7Ae-Glu57, and cpL7Ae-Ser78) formed incomplete RNPs with one or two proteins
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Fig. 2. EMSA analysis of the triangular RNP formation with wild-type or circular permutant L7Ae. The triangular RNPs were prepared by mixing 25 nmol Tri26L/S RNA and the indicated concentrations of wild-type L7Ae (A), cpL7Ae-Gly26 (B), cpL7Ae-Gly42 (C), cpL7Ae-Glu57 (D), cpL7Ae-Asn70 (E), cpL7Ae-Ser78 (F), cpL7Ae-Gly87 (G), cpL7Ae-Asn99 (H), or cpL7Ae-Gly101 (I). In Figures (C), (D), (F), and (H), the triangular RNP prepared with wild-type protein (‘WT’) was also loaded as a control. The gel was stained with SYBR green to scan for the RNAs. The three upshifted bands correspond to the RNPs with one, two and three proteins.
Fig. 3. EMSA analysis of the triangular RNP formation with EGFP fusions of the wild-type or circular permutant L7Ae. The triangular RNPs were prepared by mixing 200 nmol Tri26L/S RNA and the indicated concentrations of wild-type L7Ae-EGFP (A), cpL7Ae-Gly26-EGFP (B), cpL7Ae-Gly42-EGFP (C), cpL7Ae-Glu57-EGFP (D), cpL7Ae-Asn70-EGFP (E), cpL7Ae-Ser78-EGFP (F), cpL7Ae-Gly87-EGFP (G), cpL7Ae-Asn99-EGFP (H), or cpL7Ae-Gly101-EGFP (I). In Figures (B) to (I), the triangular RNP prepared with wild-type L7Ae-EGFP (‘WT’) was also loaded as a control. The gel was stained with ethidium bromide to scan for the RNAs. The three upshifted bands correspond to the RNPs with one, two and three protein. An EMSA assay was performed to analyze the triangular RNP formed with 200 nM Tri26L/S RNA and varying concentrations of EGFP fusion protein.
S.J. Ohuchi et al. / Biochemical and Biophysical Research Communications 466 (2015) 388e392
bound to the RNA. However, the EGFP fusion protein of cpL7AeGly87 hardly bound to the RNA even in the presence of high concentrations of the protein (Fig. 3G). The resulting difference from the data with the non-fused protein (Fig. 2G) was perhaps caused by the steric hindrance of EGFP because Gly87 is located very close to the box C/D RNA in the complex (Fig. 1 and Fig. S2). Finally, fusion proteins of cpL7Ae and EGFP were examined
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whether the designed circular permutants alter the orientation of the protein on the triangular RNA by using AFM (Fig. 4). A newly designed scaffold RNA containing parallel double stems, Tri48stem RNA, was employed to distinguish the front and back face of the triangular RNP (Fig. S4, Fig. S6, and Fig. S7). Consistent with the previous observations [5], EGFP orientations of the triangular RNP with wild-type L7Ae-EGFP were in the distal direction (Fig. 4A and C). Interestingly, EGFP orientations of the one variant, cpL7AeAsn70-EGFP, were apparently different from those of wild-type L7Ae-EGFP (Fig. 4B and C). As observed, the EGFP orientations were consistent with the model structure (Fig. 4B), indicating that the triangular RNP adopted the designed structure. Triangular RNP composed of cpL7Ae-Asn70-EGFP and Tri26L/S RNA also exhibited similar EGFP orientations, indicating that the observed orientation does not depend on the scaffold RNA (Fig. S8). The EGFP orientations of three other variants were not observed as clearly as that of cpL7Ae-Asn70-EGFP under the same conditions (Fig. S9). This is likely due to the intrinsic flexibility of the linker between cpL7Ae and EGFP, the high flexibility of the residue chosen for a C-terminus, and/or the non-uniform adsorption property of the protein moieties onto the mica surface. 4. Discussion RNPs are useful for constructing functional nano-objects because a variety of functional proteins can be attached to a designed RNA scaffold. To further improve the molecular design technique, an alternative stereo-specific fixation of functional protein displayed on the RNP is desired. In this study, we designed cpL7Aes to alter the orientation of the displayed protein on RNA. Among eight cpL7Aes designed, one variant, cpL7Ae-Asn70-EGFP, showed the altered orientation of EGFP on the triangular RNP as designed. Our results demonstrate that fine-tuning of the stereospecific fixation of proteins on RNA is feasible using the circular permutation technique. These results constitute a step further in RNP design and indicate that the stereochemical control of functional proteins on RNA enables a high level of precision nanobiotechnology. In the application of this technique, the display of an affinity protein at the desired position with fixed orientation highly contributes to efficient target recognition due to the lower degree of entropy change upon the allosteric binding. Accordingly, the stereo-specific control of the displayed ligand to a receptor protein has potential to improve the efficiency of signal induction [8]. The stereo-specific control of the displayed enzymes will improve nanobiotechnology for optimizing enzymatic cascade reactions [4]. Acknowledgments
Fig. 4. Structures of the triangular RNP constructed with EGFP fusion proteins. (A and B) Model structure (upper images) and AFM images (lower images) of the triangular RNP composed of Tri48stem RNA and wild-type L7Ae-EGFP (A) and cpL7Ae-Asn70EGFP (B). The upper left and upper right images show the front and back faces of the model structure of the triangular RNP, respectively. The branched stem-forming RNA, long RNA, and short RNA strands are shown in light blue, green, and dark blue, respectively. L7Ae variants, linker residues, and EGFP moieties are shown in yellow, orange, and red, respectively. The model structure images were prepared using the ICM-Pro software package. For the AFM analyses, the triangular RNPs were prepared by mixing 5 nmol Tri48stem RNA and 40 nmol fusion protein. The branched stems (bumps) are emphasized by the white arrowheads. The white bars on the lower left two images indicate 50 nm, and the right six images were acquired on smaller scales (30 nm 30 nm). (C) Orientations of EGFP displayed on the triangular RNP. The three tips of the triangle with black lines indicate the highest positions of the wild-type L7Ae or cpL7Ae-Asn70 moieties on the front face of the RNP of the AFM image. The highest positions of the EGFP moieties and the L7Ae or cpL7Ae-Asn70 moieties are linked by bold blue and red lines, respectively. The average orientations of the EGFP moieties were obtained from 28 images of the RNP (N ¼ 54) and are represented as the degree of the angles between a side of the triangle and bold blue or red lines. Thin blue and red lines indicate standard deviation values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
This work was supported by grants from the Japan Society for the Promotion of Science (23221011 and 23119005). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.09.035. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.035. References [1] W.W. Grabow, L. Jaeger, RNA self-assembly and RNA nanotechnology, Acc. Chem. Res. 47 (2014) 1871e1880.
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