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The application of helix fusion methods in structural biology
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ScienceDirect The application of helix fusion methods in structural biology Na-Young Kwon, Youngjin Kim and Jie-Oh Lee Methods generating fusion proteins with rigid and predictable structures have been developed in recent years. Among them, helix fusion methods that link two proteins by connecting their terminal alpha helices into a single and extended alpha helix can be particularly useful because designing fusion helices is conceptually and technically simple. These methods have been shown crucial in obtaining crystals that diffract x-rays to high resolution or attaching large and symmetrical backbone proteins to small target proteins for cryo-EM analysis. The structural rigidity of the fusion helix is crucial for these applications, and the reduction of structural ambiguity and flexibility at the fusion sites will further enhance the usefulness of this method. Address Department of Life Sciences, POSTECH, Pohang, Republic of Korea Corresponding author: Lee, Jie-Oh ([email protected])
Current Opinion in Structural Biology 2020, 60:110–116 This review comes from a themed issue on Proteins Edited by Jan Steyaerts and Todd Yeates
https://doi.org/10.1016/j.sbi.2019.12.007 0959-440X/ã 2019 Elsevier Ltd. All rights reserved.
Background Linking two proteins into a fusion protein that has a stable and predictable structure is useful for generating protein complexes that have applications in various areas of protein biotechnology [1–4]. The helix fusion method is the simplest of these methods because it involves connecting the ends of two pre-existing alpha helices, one in each of the partner proteins [4,5]. If it is successful, the connected helices become a single extended helix. Because most of the alpha helices in proteins adopt a uniform backbone conformation, the fused proteins should have rigid and predictable structures. This method has several advantages over other rigid fusion methods. First, alpha helices are present in the majority of protein structures, and their structures can be predicted with reasonable accuracy from the amino acid sequences. Because of this, the fusion helix method can be used not only for target proteins of known structure but also for Current Opinion in Structural Biology 2020, 60:110–116
those with unknown structures. Second, helix fusion is conceptually simple and does not require complicated computation. Therefore, biochemistry labs without access to expensive computational facilities and expertise can use it in their research. Methods of helix fusion have been developed over the last ten years, and are starting to be used in applications including x-ray crystallography and cryo-EM. In this review, we will summarize recent advances in this methodology.
End-to-end fusion The simplest way to link two helices is by end-to-end fusion (Figure 1a). In order to adjust the length of the fusion helix, the helices are truncated, or a short alpha helical linker is inserted at the junction. This method of protein fusion for assembly of artificial protein complexes was proposed 19 years ago by Yeates et al. [4,6]. They connected N-terminal and C-terminal helices of two naturally dimeric proteins in order to generate a long fiber-like protein complex. Their attempt was only partially successful, as they obtained heterogeneous protein complexes. However, some of the protein complexes seen in negatively stained electron microscopy appeared to have the intended structure. They also designed cagelike protein assemblies to be obtained by connecting the terminal helices of naturally dimeric and trimeric proteins with a short alpha helical linker. After careful optimization of the linker sequences, they produced a protein cage with the desired structure [3]. In this case, the connecting linkers of some of the subunits adopted the expected helical structure. However, the linkers in other protein subunits had structures that diverged from the ideal alpha-helical geometry. This deviation was unexpected but apparently helped the formation of cages because they compensated for small errors in design. This example demonstrates that not only rigidity but also some flexibility is useful in protein design. Later they applied a similar strategy to generate a large protein cube by fusing helices from naturally trimeric and dimeric proteins with different linking angles, and the crystal structure demonstrated the success of their design [7]. Helix fusion has been proved very successful in crystallizing GPCRs. Transmembrane proteins are notoriously difficult to crystallize for high-resolution structural study. Fusion with easily crystallizable proteins such as lysozymes is among the most successful methods for crystallizing GPCRs [8–12]. The usefulness of this method was first demonstrated in the case of the beta2 adrenergic receptor [8,11]. Between their fifth and the sixth www.sciencedirect.com
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Figure 1
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The end-to-end helix fusion method. (a) The ends of the alpha helices of two proteins are fused (b) Crystal structure of the CXCR4 chemokine receptor-T4 lysozyme fusion protein (PDB ID, 3OE0). The T4 lysozyme sequence is inserted into the internal loop of the CXCR4 chemokine receptor. The N-terminal helix of the lysozyme is fused to the fifth helix of the CXCR4. (c) Cryo-EM structure of the T33-21 cage-DARPin fusion protein. The two proteins are linked by the helix fusion method (PDB ID, 6C9K). (d) Cryo-EM structure of GFP noncovalently bound to the T33-21DARPin fusion protein. Helix junctions are marked with vertical lines.
transmembrane alpha helices all GPCRs have a highly flexible loop that interacts with heterotrimeric G proteins inside the cell. To reduce the flexibility of this loop, a T4 lysozyme sequence was inserted into this loop. The fusion protein could be crystallized and its structure could be determined by x-ray crystallography. After this success, several crystallization chaperones have been used in structural study of GPCRs not only by inserting chaperones into the internal loop but also by attaching them to the N-terminus or C-terminus of the target GPCR [9,13–19]. Some of these chaperones, such as T4 lysozyme, cytochrome b562, and glycogen synthase contain alpha helices at their N-termini and/or C-termini. www.sciencedirect.com
In some of the fusion proteins formed with GPCRs, these alpha helices have been fused to the transmembrane helices of the target GPCR by end-to-end fusion (Figure 1b). To reduce the structural flexibility of the fusion sites, many fusion proteins need to be constructed and tested. Among them, those that show cooperativity and improved thermal stability are chosen for crystallization. This method has been used for crystallizing and determining the structure of more than 90 GPCRs, which are deposited in the PDB database. Many of these fusion proteins had helical conformations significantly distorted from the ideal helix geometry at the fusion site, demonstrating the strength and weakness of this fusion method. Current Opinion in Structural Biology 2020, 60:110–116
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Liu et al. showed that the helix fusion method has potential applications in single-particle analysis in cryoelectron microscopy (cryo-EM) as well (Figure 1c and d) [20]. One of the major obstacles to cryo-EM study is the size limitation of target proteins [21]. Although there are several examples of proteins significantly smaller than 100 kDa being successfully studied by cryo-EM, this is still challenging in the majority of cases [21–24]. Attaching large backbone proteins to small target proteins by the helix fusion method increases the overall size and therefore increases the chance that high-resolution structural analysis of the target protein [20,25,26–28]. Liu et al. fused the N-terminal alpha helix of a DARPin protein to the C-terminal alpha helix of a large and symmetrical protein cage. DARPins are artificial ankyrin repeat proteins that are developed to replace antibodies for therapeutic purposes. A DARPin molecule that can bind to a given target can be selected by screening a randomized library [29,30]. T33-21 is a computationally designed protein that assembles into a large cage-like structure [2,20,25]. The cage is composed of
24 subunits, consisting of four trimers of two different subunit types. The trimeric subunits contain alpha helices at their C-termini. Liu et al. designed several end-to-end fusions of the C-terminal helix of T33-21 and the N-terminal helix of the DARPin using different fusion sequences. Of the five fusion proteins they produced, one had a rigid structure, and this structure could be determined by the single particle analysis method using cryo-EM [20]. In the structure, the DARPin was linked to the cage by the helix fusion but the DARPin part of the fusion protein was less well resolved than the cage part due to its greater flexibility. Nevertheless, this study demonstrated that the helix fusion method can be used for structural study of many small proteins. Later, the authors determined the structure of a DARPin-cage fusion protein together with GFP noncovalently bound to it [25]. They proposed that by selecting DARPins that can bind to target proteins of unknown structure, this method can be used for high-resolution structural study of small target proteins. A similar strategy is used by others [26,27]. In these studies, they fused a DARPin to aldolase
Figure 2
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Helix fusion stabilized by the EY-CBS cross-linker. (a) Two proteins are linked by the helix fusion and reacted with the EY-CBS cross-linker. The i and i+12 positions of the fusion alpha helices are mutated to cysteines for the cross-linking reaction. (b) Crystal structure of the DARPin-protein A fusion stabilized by EY-CBS (PDB ID, 5CBN). (c) Crystal structure of a T4 lysozyme-protein A fusion stabilized by EY-CBS (PDB ID, 5EWX). The protein A sequence is inserted into a flexible region in the lysozyme structure. Current Opinion in Structural Biology 2020, 60:110–116
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or maltose binding protein (MBP) to glutamine synthetase. A GFP molecule is noncovalently bound to the DARPin, mimicking a target protein. Structures were determined with 5–8 A˚ resolution in the MBP or the GFP region. It appears flexibility at the helix junctions of the fused proteins limited the overall resolution of the targets.
Chemical cross-linking Although applications of the end-to-end helix fusion method have been successful in protein engineering and structural biology, the lengthy procedure required to identify successful helix fusions among the many candidates can be a problem when designing more elaborate nano-assemblies or studying challenging target proteins. This is because the amino acids at the fusion sites are surrounded by non-native amino acids from the two connected partner proteins, and these amino acids may not support a stable alpha helical structure at the fusion site [31–34]. In order to solve this problem, Jeong et al. used a chemical cross-linker that stabilizes the alpha-helical structure (Figure 2a) [35]. EY-CBS is a thiol-reactive crosslinker [36]. Unlike other cross-linkers, this compound contains two functional groups that are connected in a rigid way: the distance between the two reactive halo-carbonyl groups is fixed at 17 A˚, which matches the distance between the i and i+12 residues in an ideal alpha helix. Because of this, EY-CBS has been shown to stabilize the alpha helical structure of short peptides. Jeong et al. fused alpha helices in several pairs of model proteins by the end-to-end helix fusion method. They then mutated amino acids at the i and i+12 positions in the fused alpha helix to cysteines. Some of these fusion proteins showed 100% reactivity to the EY-CBS cross-linker. Their crystal structures demonstrated that helix fusion had indeed been successful and the fusion helix had the near-ideal geometry of an alpha helix (Figure 2b and c). The fusion protein that had not been reacted with EY-CBS could also be crystallized but the fusion helix deviated substantially from the ideal alpha helical structure, demonstrating that the EY-CBS crosslinker was required to enforce the alpha helical conformation at the fusion site. This chemical cross-linking method has the advantage that it guarantees the existence of helical structure at the fusion site without the need to determine the structure of a fusion that has been generated. However, it requires a mutation of two cysteines at precise positions in the fusion helix for the cross-linking reaction. Therefore, proteins that have cysteines essential for function or structure cannot be used because these cysteines may react with the crosslinker.
Shared helix method In order to overcome problems associated with the endto-end and cross-linking methods, a shared helix method has been developed [37,38]. In this method, the alpha helices of the two proteins are partially overlapped using www.sciencedirect.com
molecular modeling software. In the overlapped region, amino acids were selected from one or other of the two natural sequences in such a way as to stabilize the shared helix. That is, protein 1 residues were chosen for amino acid positions pointing to the core of protein 1, and protein 2 residues for those pointing to the core of protein 2 (Figure 3a). This is because most of the alpha helices found in globular proteins are amphipathic. In other words, one side of the helix is hydrophilic and exposed to the solvent while the other side is hydrophobic and interacts with the hydrophobic core of the protein. The hydrophobic side of the helix is crucial for stabilizing the native alpha helical structure while the hydrophilic amino acids are usually dispensable for the structural stability of the protein. Batyuk et al. used this method and were able to connect the c-terminal alpha helix of a DARPin with the n-terminal alpha helix of beta-lactamase (Figure 3b) [37]. They generated five different fusion proteins and showed by x-ray crystallography that the fusion helices indeed formed one extended alpha helix. The same group used a similar strategy to connect two DARPin proteins by the shared helix fusion [39]. They proposed that this method could be used to determine the crystal structures of many target proteins because it could connect DARPins bound to target proteins and DARPins bound to crystallization chaperones in a rigid and crystallizable fashion [40]. Youn et al. independently showed that the shared helix method could be used to connect a variety of proteins other than DARPins [38]. They were able in this way to use fusion alpha helices to create cagelike protein complexes decorated with small proteins or to generate artificial repeat proteins. They also used this method to connect small target proteins to a large backbone protein and proposed that it could be applied to structure determination of proteins smaller than 100 kDa by cryo-EM (Figure 3c).
Transplantation of helix epitopes Antibodies have been found to be helpful for structural studies of challenging proteins by X-ray crystallography and electron microscopy. Antibodies that are suitable for this application should recognize the three-dimensional conformations of target proteins because flexibility in the protein complexes is harmful to crystallization or image processing. However, generating such antibodies and characterizing structures of their complexes with antigens take months or even years of research. Recently Kim et al. showed that a short alpha helical epitope can be added to various host proteins in a rigid and predictable structure by fusing it to host alpha helices (Figure 4a) [41]. There are many antibody structures with short alpha helical peptides bound to them in the PDB database. They proposed that these previously characterized anti-helix antibodies could be used as Current Opinion in Structural Biology 2020, 60:110–116
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Figure 3
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The shared helix method. (a) Schematic representation of the shared helix method. (b) Crystal structure of a DARPin-lactamase fusion protein. The two proteins are linked by the shared helix method (PDB ID, 5AQ9). (c) Crystal structure of a calmodulin-ZpA963 fusion protein noncovalently attached to the YgiG-protein A fusion proteins (PDB ID, 5H7D). YgiG forms a stable tetramer. The c-terminal helix of the YgiG subunit and the Nterminal helix of protein A are fused by the shared helix method. The N-terminal domain of calmodulin is also linked to ZpA963 by the shared helix method. Protein A and ZpA963 form a stable heterodimer.
crystallization chaperones because they can bind to offtarget proteins after fusion of the epitopes to alpha helices of the host proteins. These engineered epitopes can be added to the terminal alpha helices or inserted into internal alpha helices (Figure 4b and c). Using x-ray crystallography, they demonstrated that all the epitopetransplanted proteins bound to the anti-helix antibodies form accurately predictable structures. Furthermore, they showed that binding of these anti-helix antibodies to the engineered target proteins can modulate their catalytic activities by trapping the host protein in a selected functional state. This epitope transplantation method can significantly reduce the amount of time required to generate antibodies not only for structural study but also for building protein nano-assemblies because the positions and projection angles of the antibody can be Current Opinion in Structural Biology 2020, 60:110–116
accurately predicted. Systematic screening of more anti-helix antibodies or antibody mimicking proteins should greatly expand the scope of the suggested method [41,42].
Summary Various helix fusion methods have been developed in recent years. These methods have been successful in determining the structures of many GPCRs and may have applications in the cryo-EM analysis of proteins significantly smaller than those that can be studied currently. Because of the technical and conceptual simplicity of these methods they should have wide applications in protein engineering and structural biology. The reduction of structural ambiguity and flexibility at the fusion sites will further enhance the usefulness of these methods. www.sciencedirect.com
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Figure 4
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Transplantation of alpha helical epitopes to off-target proteins. (a) Schematic representation of the epitope transplantation method. (b) Crystal structure of engineered protein A bound to the Fv domain of an anti-helix antibody. The alpha helical epitope of the antibody is fused to the Nterminal alpha helix of protein A by the shared helix method (PDB ID, 6K68). (c) Crystal structure of engineered T4 lysozyme bound to an anti-helix antibody. The alpha helical epitope of the antibody is fused to an internal alpha helix of the lysozyme (PDB ID, 6K69).
Conflict of interest statement
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Nothing declared.
Acknowledgements This research was supported by grants from the National Research Foundation (NRF- 2017R1A2A1A17069497, NRF-2017M3A9F6029753 and NRF-2019M3E5D6066058) funded by the Ministry of Science, ICT of Korea.
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ScienceDirect The application of helix fusion methods in structural biology Na-Young Kwon, Youngjin Kim and Jie-Oh Lee Methods generating fusion proteins with rigid and predictable structures have been developed in recent years. Among them, helix fusion methods that link two proteins by connecting their terminal alpha helices into a single and extended alpha helix can be particularly useful because designing fusion helices is conceptually and technically simple. These methods have been shown crucial in obtaining crystals that diffract x-rays to high resolution or attaching large and symmetrical backbone proteins to small target proteins for cryo-EM analysis. The structural rigidity of the fusion helix is crucial for these applications, and the reduction of structural ambiguity and flexibility at the fusion sites will further enhance the usefulness of this method. Address Department of Life Sciences, POSTECH, Pohang, Republic of Korea Corresponding author: Lee, Jie-Oh ([email protected])
Current Opinion in Structural Biology 2020, 60:110–116 This review comes from a themed issue on Proteins Edited by Jan Steyaerts and Todd Yeates
https://doi.org/10.1016/j.sbi.2019.12.007 0959-440X/ã 2019 Elsevier Ltd. All rights reserved.
Background Linking two proteins into a fusion protein that has a stable and predictable structure is useful for generating protein complexes that have applications in various areas of protein biotechnology [1–4]. The helix fusion method is the simplest of these methods because it involves connecting the ends of two pre-existing alpha helices, one in each of the partner proteins [4,5]. If it is successful, the connected helices become a single extended helix. Because most of the alpha helices in proteins adopt a uniform backbone conformation, the fused proteins should have rigid and predictable structures. This method has several advantages over other rigid fusion methods. First, alpha helices are present in the majority of protein structures, and their structures can be predicted with reasonable accuracy from the amino acid sequences. Because of this, the fusion helix method can be used not only for target proteins of known structure but also for Current Opinion in Structural Biology 2020, 60:110–116
those with unknown structures. Second, helix fusion is conceptually simple and does not require complicated computation. Therefore, biochemistry labs without access to expensive computational facilities and expertise can use it in their research. Methods of helix fusion have been developed over the last ten years, and are starting to be used in applications including x-ray crystallography and cryo-EM. In this review, we will summarize recent advances in this methodology.
End-to-end fusion The simplest way to link two helices is by end-to-end fusion (Figure 1a). In order to adjust the length of the fusion helix, the helices are truncated, or a short alpha helical linker is inserted at the junction. This method of protein fusion for assembly of artificial protein complexes was proposed 19 years ago by Yeates et al. [4,6]. They connected N-terminal and C-terminal helices of two naturally dimeric proteins in order to generate a long fiber-like protein complex. Their attempt was only partially successful, as they obtained heterogeneous protein complexes. However, some of the protein complexes seen in negatively stained electron microscopy appeared to have the intended structure. They also designed cagelike protein assemblies to be obtained by connecting the terminal helices of naturally dimeric and trimeric proteins with a short alpha helical linker. After careful optimization of the linker sequences, they produced a protein cage with the desired structure [3]. In this case, the connecting linkers of some of the subunits adopted the expected helical structure. However, the linkers in other protein subunits had structures that diverged from the ideal alpha-helical geometry. This deviation was unexpected but apparently helped the formation of cages because they compensated for small errors in design. This example demonstrates that not only rigidity but also some flexibility is useful in protein design. Later they applied a similar strategy to generate a large protein cube by fusing helices from naturally trimeric and dimeric proteins with different linking angles, and the crystal structure demonstrated the success of their design [7]. Helix fusion has been proved very successful in crystallizing GPCRs. Transmembrane proteins are notoriously difficult to crystallize for high-resolution structural study. Fusion with easily crystallizable proteins such as lysozymes is among the most successful methods for crystallizing GPCRs [8–12]. The usefulness of this method was first demonstrated in the case of the beta2 adrenergic receptor [8,11]. Between their fifth and the sixth www.sciencedirect.com
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The end-to-end helix fusion method. (a) The ends of the alpha helices of two proteins are fused (b) Crystal structure of the CXCR4 chemokine receptor-T4 lysozyme fusion protein (PDB ID, 3OE0). The T4 lysozyme sequence is inserted into the internal loop of the CXCR4 chemokine receptor. The N-terminal helix of the lysozyme is fused to the fifth helix of the CXCR4. (c) Cryo-EM structure of the T33-21 cage-DARPin fusion protein. The two proteins are linked by the helix fusion method (PDB ID, 6C9K). (d) Cryo-EM structure of GFP noncovalently bound to the T33-21DARPin fusion protein. Helix junctions are marked with vertical lines.
transmembrane alpha helices all GPCRs have a highly flexible loop that interacts with heterotrimeric G proteins inside the cell. To reduce the flexibility of this loop, a T4 lysozyme sequence was inserted into this loop. The fusion protein could be crystallized and its structure could be determined by x-ray crystallography. After this success, several crystallization chaperones have been used in structural study of GPCRs not only by inserting chaperones into the internal loop but also by attaching them to the N-terminus or C-terminus of the target GPCR [9,13–19]. Some of these chaperones, such as T4 lysozyme, cytochrome b562, and glycogen synthase contain alpha helices at their N-termini and/or C-termini. www.sciencedirect.com
In some of the fusion proteins formed with GPCRs, these alpha helices have been fused to the transmembrane helices of the target GPCR by end-to-end fusion (Figure 1b). To reduce the structural flexibility of the fusion sites, many fusion proteins need to be constructed and tested. Among them, those that show cooperativity and improved thermal stability are chosen for crystallization. This method has been used for crystallizing and determining the structure of more than 90 GPCRs, which are deposited in the PDB database. Many of these fusion proteins had helical conformations significantly distorted from the ideal helix geometry at the fusion site, demonstrating the strength and weakness of this fusion method. Current Opinion in Structural Biology 2020, 60:110–116
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Liu et al. showed that the helix fusion method has potential applications in single-particle analysis in cryoelectron microscopy (cryo-EM) as well (Figure 1c and d) [20]. One of the major obstacles to cryo-EM study is the size limitation of target proteins [21]. Although there are several examples of proteins significantly smaller than 100 kDa being successfully studied by cryo-EM, this is still challenging in the majority of cases [21–24]. Attaching large backbone proteins to small target proteins by the helix fusion method increases the overall size and therefore increases the chance that high-resolution structural analysis of the target protein [20,25,26–28]. Liu et al. fused the N-terminal alpha helix of a DARPin protein to the C-terminal alpha helix of a large and symmetrical protein cage. DARPins are artificial ankyrin repeat proteins that are developed to replace antibodies for therapeutic purposes. A DARPin molecule that can bind to a given target can be selected by screening a randomized library [29,30]. T33-21 is a computationally designed protein that assembles into a large cage-like structure [2,20,25]. The cage is composed of
24 subunits, consisting of four trimers of two different subunit types. The trimeric subunits contain alpha helices at their C-termini. Liu et al. designed several end-to-end fusions of the C-terminal helix of T33-21 and the N-terminal helix of the DARPin using different fusion sequences. Of the five fusion proteins they produced, one had a rigid structure, and this structure could be determined by the single particle analysis method using cryo-EM [20]. In the structure, the DARPin was linked to the cage by the helix fusion but the DARPin part of the fusion protein was less well resolved than the cage part due to its greater flexibility. Nevertheless, this study demonstrated that the helix fusion method can be used for structural study of many small proteins. Later, the authors determined the structure of a DARPin-cage fusion protein together with GFP noncovalently bound to it [25]. They proposed that by selecting DARPins that can bind to target proteins of unknown structure, this method can be used for high-resolution structural study of small target proteins. A similar strategy is used by others [26,27]. In these studies, they fused a DARPin to aldolase
Figure 2
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Helix fusion stabilized by the EY-CBS cross-linker. (a) Two proteins are linked by the helix fusion and reacted with the EY-CBS cross-linker. The i and i+12 positions of the fusion alpha helices are mutated to cysteines for the cross-linking reaction. (b) Crystal structure of the DARPin-protein A fusion stabilized by EY-CBS (PDB ID, 5CBN). (c) Crystal structure of a T4 lysozyme-protein A fusion stabilized by EY-CBS (PDB ID, 5EWX). The protein A sequence is inserted into a flexible region in the lysozyme structure. Current Opinion in Structural Biology 2020, 60:110–116
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or maltose binding protein (MBP) to glutamine synthetase. A GFP molecule is noncovalently bound to the DARPin, mimicking a target protein. Structures were determined with 5–8 A˚ resolution in the MBP or the GFP region. It appears flexibility at the helix junctions of the fused proteins limited the overall resolution of the targets.
Chemical cross-linking Although applications of the end-to-end helix fusion method have been successful in protein engineering and structural biology, the lengthy procedure required to identify successful helix fusions among the many candidates can be a problem when designing more elaborate nano-assemblies or studying challenging target proteins. This is because the amino acids at the fusion sites are surrounded by non-native amino acids from the two connected partner proteins, and these amino acids may not support a stable alpha helical structure at the fusion site [31–34]. In order to solve this problem, Jeong et al. used a chemical cross-linker that stabilizes the alpha-helical structure (Figure 2a) [35]. EY-CBS is a thiol-reactive crosslinker [36]. Unlike other cross-linkers, this compound contains two functional groups that are connected in a rigid way: the distance between the two reactive halo-carbonyl groups is fixed at 17 A˚, which matches the distance between the i and i+12 residues in an ideal alpha helix. Because of this, EY-CBS has been shown to stabilize the alpha helical structure of short peptides. Jeong et al. fused alpha helices in several pairs of model proteins by the end-to-end helix fusion method. They then mutated amino acids at the i and i+12 positions in the fused alpha helix to cysteines. Some of these fusion proteins showed 100% reactivity to the EY-CBS cross-linker. Their crystal structures demonstrated that helix fusion had indeed been successful and the fusion helix had the near-ideal geometry of an alpha helix (Figure 2b and c). The fusion protein that had not been reacted with EY-CBS could also be crystallized but the fusion helix deviated substantially from the ideal alpha helical structure, demonstrating that the EY-CBS crosslinker was required to enforce the alpha helical conformation at the fusion site. This chemical cross-linking method has the advantage that it guarantees the existence of helical structure at the fusion site without the need to determine the structure of a fusion that has been generated. However, it requires a mutation of two cysteines at precise positions in the fusion helix for the cross-linking reaction. Therefore, proteins that have cysteines essential for function or structure cannot be used because these cysteines may react with the crosslinker.
Shared helix method In order to overcome problems associated with the endto-end and cross-linking methods, a shared helix method has been developed [37,38]. In this method, the alpha helices of the two proteins are partially overlapped using www.sciencedirect.com
molecular modeling software. In the overlapped region, amino acids were selected from one or other of the two natural sequences in such a way as to stabilize the shared helix. That is, protein 1 residues were chosen for amino acid positions pointing to the core of protein 1, and protein 2 residues for those pointing to the core of protein 2 (Figure 3a). This is because most of the alpha helices found in globular proteins are amphipathic. In other words, one side of the helix is hydrophilic and exposed to the solvent while the other side is hydrophobic and interacts with the hydrophobic core of the protein. The hydrophobic side of the helix is crucial for stabilizing the native alpha helical structure while the hydrophilic amino acids are usually dispensable for the structural stability of the protein. Batyuk et al. used this method and were able to connect the c-terminal alpha helix of a DARPin with the n-terminal alpha helix of beta-lactamase (Figure 3b) [37]. They generated five different fusion proteins and showed by x-ray crystallography that the fusion helices indeed formed one extended alpha helix. The same group used a similar strategy to connect two DARPin proteins by the shared helix fusion [39]. They proposed that this method could be used to determine the crystal structures of many target proteins because it could connect DARPins bound to target proteins and DARPins bound to crystallization chaperones in a rigid and crystallizable fashion [40]. Youn et al. independently showed that the shared helix method could be used to connect a variety of proteins other than DARPins [38]. They were able in this way to use fusion alpha helices to create cagelike protein complexes decorated with small proteins or to generate artificial repeat proteins. They also used this method to connect small target proteins to a large backbone protein and proposed that it could be applied to structure determination of proteins smaller than 100 kDa by cryo-EM (Figure 3c).
Transplantation of helix epitopes Antibodies have been found to be helpful for structural studies of challenging proteins by X-ray crystallography and electron microscopy. Antibodies that are suitable for this application should recognize the three-dimensional conformations of target proteins because flexibility in the protein complexes is harmful to crystallization or image processing. However, generating such antibodies and characterizing structures of their complexes with antigens take months or even years of research. Recently Kim et al. showed that a short alpha helical epitope can be added to various host proteins in a rigid and predictable structure by fusing it to host alpha helices (Figure 4a) [41]. There are many antibody structures with short alpha helical peptides bound to them in the PDB database. They proposed that these previously characterized anti-helix antibodies could be used as Current Opinion in Structural Biology 2020, 60:110–116
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Figure 3
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The shared helix method. (a) Schematic representation of the shared helix method. (b) Crystal structure of a DARPin-lactamase fusion protein. The two proteins are linked by the shared helix method (PDB ID, 5AQ9). (c) Crystal structure of a calmodulin-ZpA963 fusion protein noncovalently attached to the YgiG-protein A fusion proteins (PDB ID, 5H7D). YgiG forms a stable tetramer. The c-terminal helix of the YgiG subunit and the Nterminal helix of protein A are fused by the shared helix method. The N-terminal domain of calmodulin is also linked to ZpA963 by the shared helix method. Protein A and ZpA963 form a stable heterodimer.
crystallization chaperones because they can bind to offtarget proteins after fusion of the epitopes to alpha helices of the host proteins. These engineered epitopes can be added to the terminal alpha helices or inserted into internal alpha helices (Figure 4b and c). Using x-ray crystallography, they demonstrated that all the epitopetransplanted proteins bound to the anti-helix antibodies form accurately predictable structures. Furthermore, they showed that binding of these anti-helix antibodies to the engineered target proteins can modulate their catalytic activities by trapping the host protein in a selected functional state. This epitope transplantation method can significantly reduce the amount of time required to generate antibodies not only for structural study but also for building protein nano-assemblies because the positions and projection angles of the antibody can be Current Opinion in Structural Biology 2020, 60:110–116
accurately predicted. Systematic screening of more anti-helix antibodies or antibody mimicking proteins should greatly expand the scope of the suggested method [41,42].
Summary Various helix fusion methods have been developed in recent years. These methods have been successful in determining the structures of many GPCRs and may have applications in the cryo-EM analysis of proteins significantly smaller than those that can be studied currently. Because of the technical and conceptual simplicity of these methods they should have wide applications in protein engineering and structural biology. The reduction of structural ambiguity and flexibility at the fusion sites will further enhance the usefulness of these methods. www.sciencedirect.com
Helix fusion methods in structural biology Kwon, Kim and Lee 115
Figure 4
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Transplantation of alpha helical epitopes to off-target proteins. (a) Schematic representation of the epitope transplantation method. (b) Crystal structure of engineered protein A bound to the Fv domain of an anti-helix antibody. The alpha helical epitope of the antibody is fused to the Nterminal alpha helix of protein A by the shared helix method (PDB ID, 6K68). (c) Crystal structure of engineered T4 lysozyme bound to an anti-helix antibody. The alpha helical epitope of the antibody is fused to an internal alpha helix of the lysozyme (PDB ID, 6K69).
Conflict of interest statement
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Nothing declared.
Acknowledgements This research was supported by grants from the National Research Foundation (NRF- 2017R1A2A1A17069497, NRF-2017M3A9F6029753 and NRF-2019M3E5D6066058) funded by the Ministry of Science, ICT of Korea.
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