Application of antihelix antibodies in proteinstructure determinationJi Won Kima,1, Songwon Kimb,1, Haerim Leec, Geunyoung Choa, Sun Chang Kimd, Hayyoung Leee, Mi Sun Jinb,2,and Jie-Oh Leea,2

aDepartment of Life Sciences, Pohang University of Science and Technology, Nam-gu, Pohang 37673, Korea; bSchool of Life Sciences, Gwangju Institute ofScience and Technology, Buk-gu, Gwangju 61005, Korea; cDepartment of Chemistry, Korea Advanced Institute of Science and Technology, Yuseong-gu,Daejeon 34141, Korea; dDepartment of Biological Sciences, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon 34141, Korea;and eInstitute of Biotechnology, Chungnam National University, Yuseong-gu, Daejeon 34134, Korea

Edited by K. C. (Chris) Christopher Garcia, Stanford University, Stanford, CA, and approved July 10, 2019 (received for review June 16, 2019)

Antibodies are indispensable tools in protein engineering andstructural biology. Antibodies suitable for structural studies shouldrecognize the 3-dimensional (3D) conformations of target proteins.Generating such antibodies and characterizing their complexes withantigens take a significant amount of time and effort. Here, weshow that we can expand the application of well-characterizedantibodies by “transplanting” the epitopes that they recognize toproteins with completely different structures and sequences. Previ-ously, several antibodies have been shown to recognize the alpha-helical conformation of antigenic peptides. We demonstrate thatthese antibodies can be made to bind to a variety of unrelated“off-target” proteins by modifying amino acids in the preexistingalpha helices of such proteins. Using X-ray crystallography, we de-termined the structures of the engineered protein–antibody com-plexes. All of the antibodies bound to the epitope-transplantedproteins, forming accurately predictable structures. Furthermore,we showed that binding of these antihelix antibodies to the engi-neered target proteins can modulate their catalytic activities bytrapping them in selected functional states. Our method is simpleand efficient, and it will have applications in protein X-ray crystal-lography, electron microscopy, and nanotechnology.

X-ray crystallography | antibody | protein design

Antibody Fab or Fv fragments have been found to be usefulfor structural studies of challenging proteins. Currently,

high-resolution structural studies of proteins by single-particleelectron microscopy are limited to those with molecular massesabove ∼50 kDa (1). It has been proposed that, for smaller pro-teins, binding of 1- or 2-antibody Fab fragments can increase theeffective size of the target proteins. Antibodies can also be usefulfor X-ray crystallography of proteins (2, 3). They can freeze theconformation of a protein with multiple structural states into asingle uniform state, and they can hide hydrophobic or flexiblesurfaces that interfere with crystallization. Antibodies suitablefor structural studies need to recognize the 3-dimensional (3D)conformation of the target protein, because the protein–antibodycomplex formed must have a stable and rigid structure for effectiveaveraging of images and for crystallization. Antibodies that bind toflexible linear regions of proteins cannot form rigid complexes andare not useful for structural analysis. However, the generation of“conformation-specific” antibodies is not straightforward for manyproteins and requires special screening methods (4–6). Antibodiesare essential tools in protein nanotechnology as well (7–9). Manyantibodies are being used to construct complex nanostructures,because they can bind to specific components of the nano-assemblies, forming stable and predictable structures. They arealso useful in functionalizing nanostructures, because antibodiescan bind proteins or other molecules with useful functions. Map-ping epitopes of the antibodies and characterizing structures of theantibody–antigen complexes are often the most time-consumingsteps in this application, because they should be determined em-pirically for every antibody.

Previously, many antibodies have been shown to bind shortalpha-helical peptides present in antigenic proteins. Some ofthese antibodies bind exclusively to a single alpha helix and havelittle or no interaction with other parts of a protein. For example,the 3MNZ antibody is one of numerous antibodies raised againstthe gp41 peptide of HIV (10). In this article, we use Protein DataBank (PDB) ID codes to designate antibodies to avoid confu-sion. Gp41 is a subunit of the HIV envelope protein and forms ahomotrimeric complex containing 6 alpha helices. The 3LRHintrabody is another example of an antihelix antibody (11). Intra-bodies are antibodies or antibody fragments that work within thecell to bind to intracellular proteins. The 3LRH intrabody is anengineered variable domain fragment of an immunoglobulin lightchain that binds to an alpha-helical stretch of 14 amino acidsoriginating from human huntingtin protein. The binding site of theantigenic peptide is located in the concave surface formed by thebeta sheet that normally interacts with the heavy-chain variabledomain. When the peptide antigen binds to 3LRH intrabody, itadopts a near-ideal alpha-helical structure. The 1P4B antibodyalso recognizes a helical epitope with a different amino acid se-quence (12). It was initially generated and selected by ribosome

Significance

Antibodies have been found to be helpful for structural studies ofchallenging proteins by X-ray crystallography and electron mi-croscopy. They are being used to construct useful protein nano-structures as well. Antibodies suitable for structural study shouldrecognize the 3-dimensional conformations of target proteins.However, generating such antibodies and characterizing struc-tures of their complexes with antigens take months or even yearsof research. Here, we show that we can expand the application ofwell-characterized antibodies by “transplanting” the alpha-helicalepitopes that they recognize to proteins with completely differ-ent structures and sequences. Systematic screening of moreantihelix antibodies will greatly expand the scope of the method.

Author contributions: J.W.K., S.K., Haerim Lee, Hayyoung Lee, M.S.J., and J.-O.L. designedresearch; J.W.K., S.K., Haerim Lee, and G.C. performed research; J.W.K., S.K., Haerim Lee,G.C., and J.-O.L. analyzed data; and J.W.K., S.K., Haerim Lee, S.C.K., Hayyoung Lee, M.S.J.,and J.-O.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The atomic coordinates and X-ray diffraction data reported in this paperhave been deposited in the Protein Data Bank (PDB) under the following ID codes: 6K68for 8420-3MNZ, 6K65 for 9014-1P4B, 6K64 for 8188-3LRH, 6K3M for 8189-3LRH, 6K6B for8496-3LRH, 6K67 for 9011-3LRH, 6K69 for 9213-3LRH, and 6K6A for 8188cys-3LRHcys.

See Commentary on page 17611.1J.W.K. and S.K. contributed equally to this work.2To whom correspondence may be addressed. Email: misunjin@gist.ac.kr or jieoh@postech.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1910080116/-/DCSupplemental.

Published online August 1, 2019.

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display screening against a GCN4 peptide. The antibody binds to a12-residue GCN4 that adopts a near-ideal alpha-helical structurewhen bound to the antibody.Here, we show that helical epitopes for these antihelix anti-

bodies can be created in a variety of off-target proteins, becausemost of the alpha helices found in proteins adopt a uniformbackbone conformation. The resulting engineered proteins wereshown to bind to the antihelix antibodies with high affinity. The for-mation of these complexes with predicted structures was confirmed byX-ray crystallography and negative staining electron microscopy.

ResultsCreation of a 3MNZ Epitope in the C-Terminal Alpha Helix of Protein Aand Determination of the Crystal Structure of the Engineered ProteinA–3MNZ Single-Chain Fv Complex. The 3MNZ antibody recognizesan alpha-helical peptide with 14 amino acid residues and 2C-terminal amino acids of gp41 (Fig. 1A) (10). The core interactionin the peptide–antibody complex is mediated by one side of thehelix composed of hydrophobic amino acids L61, W66, and L69and hydrophilic amino acids E62, D64, K65, and S68. The otherside of the helix is fully exposed to solvent. The C-terminal N71is not part of the helix and interacts strongly via hydrogen bondswith the heavy chain of the antibody.The B1 domain of protein A was chosen as a test case for

creating a 3MNZ epitope, because it is composed of 3 alphahelices, is readily produced in Escherichia coli, and can be crys-tallized in a variety of conditions. To engineer the protein Adomain, the structure of gp41 peptide bound to the Fab fragmentof the 3MNZ antibody was superimposed on that of the

C-terminal helix of protein A while avoiding steric collisionof antibody and protein A (SI Appendix, Fig. S1). Because theN-terminal 3 amino acids, from N56 to Q58, of gp41 do not interactwith the antibody, the amino acids of protein A were used for thecorresponding positions (Fig. 1A). In the docked structure, aminoacids in the G44, E45, K47-L49, E51, S52, and A54 positions ofprotein A play key roles in antibody binding and were changed tothose in the gp41 sequence (Fig. 1B). Amino acids V42, L43, A46,and Q53 of protein A were not changed, because they point to thehydrophobic core of protein A. The N50 position of protein A isnot critical for binding; either Asn or Ala is compatible in thatposition, and alanine was chosen. The resulting #8420 protein Awas produced in E. coli and purified to homogeneity. The struc-tural integrity of the protein A does not seem to have been dis-turbed, because the protein can be overexpressed in E. coli, ishighly resistant to subtilisin digestion, and elutes as a monomer ingel filtration chromatography. The engineered protein A wasmixed with the purified single-chain Fv (scFv) fragment of the3MNZ antibody, and the structure of the complex was determinedby X-ray crystallography.The crystal structure of the engineered protein A–3MNZ scFv

complex shows that modification of the C-terminal helix causespractically no change in the overall structure of protein A (Fig.1C), and the wild-type and engineered protein A could besuperimposed with a Cα root mean square deviation (r.m.s.d.)value of 0.77 Å (SI Appendix, Fig. S2). The artificially createdepitope adopts an alpha-helical structure as intended, and the aminoacids engineered to interact with the antibody are superimposablewith those of gp41 bound to 3MNZ scFv (Fig. 1D). These crystal-lographic observations prove that the epitope of 3MNZ scFv can beengineered in the C-terminal helix of protein A.

Engineering of 3LRH Epitopes in the Protein A Domain. To test ifother antibodies can be used as antialpha helix antibodies, wechose 3LRH intrabody for the next test, because the peptide–antibody interaction is mediated by one side of the helix and theother side is fully exposed to solvent. It is easy to produce in E.coli and has a high affinity for alpha-helical antigens. We createdan epitope of 3LRH at 3 different positions in protein A andsolved the structures of their complexes with 3LRH (Fig. 2).The 3LRH intrabody binds to a peptide originating from hu-

man huntingtin protein. To create the epitope, the first 10 aminoacids of the huntingtin peptide and the last 10 amino acids of theC-terminal helix of protein A were overlapped by a molecularmodeling program (Fig. 2A and SI Appendix, Fig. S3). Aminoacids K48, E51, and S52 in protein A point to the antibody andseem critical for binding (SI Appendix, Fig. S4). Therefore, theywere changed to those of the huntingtin peptide. K48 did notneed to be changed, because lysines are found in the corre-sponding positions of both the huntingtin peptide and the pro-tein A sequences. The resulting #8188 protein A bound 3LRHwith high affinity, and the antibody–antigen complex was puri-fied from unbound excess proteins by gel filtration chromatog-raphy for crystallization. In the crystal structure, the modified C-terminal alpha helix of protein A interacts with the antibody asintended (Fig. 2B and SI Appendix, Fig. S5). The conformationsof the amino acids interacting with the antibody were practicallyidentical with those in the native huntingtin peptide–3LRHcomplex, and the overall structures of the mutant and the nativeproteins A could be superimposed with a Cα r.m.s.d. of 0.41 Å asplanned (SI Appendix, Figs. S5 and S6). Introduction of a disulfidebridge enhances stability of the 3LRH complex (SI Appendix, Fig.S7). This may be useful for demanding nanotechnology applica-tions, such as those encountered in animal experiments.To prove that the epitope could be created at different posi-

tions of protein A, 2 additional epitope sites were generated inprotein A (SI Appendix, Figs. S8 and S9). The first, #8189, wascreated by shifting the docking area by 1 helical turn from that of#8188 protein A. For this, an artificial C-terminal epitope wascreated by superimposing the 7 amino acids of the huntingtinpeptide and the C-terminal helix of protein A (SI Appendix, Fig.

D

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Fig. 1. Crystal structure of the 3MNZ–protein A #8420 complex. (A) Aminoacid sequence of the engineered epitope in protein A #8420. The sequencesof native protein A and the gp41 peptide are aligned after structuraldocking of the alpha helices (SI Appendix, Fig. S1). The overlap region of thedocked helices is indicated by the green line. (B) The predicted structure ofthe engineered protein A with the 3MNZ epitope. The amino acid residuespointing to 3MNZ scFv and the host protein are shown in red and gray, re-spectively. (C) Crystal structure of the 3MNZ scFv and protein A #8420complex. The amino acid residues mutated to create the epitope are in red.(D) Close-up view of the engineered epitope in the protein A #8420 struc-ture. The crystal structures of protein A #8420 and the gp41 peptide boundto the 3MNZ scFv are aligned, and their amino acid residues that interactwith the antibody are shown in gray and red, respectively. The view isidentical to that of C.

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S8). The amino acids in the protein A structure pointing to the3LRH intrabody were changed to those of the huntingtin pep-tide, and the amino acids pointing to the core of protein A werenot changed. The second epitope was generated by mutating theN-terminal helix of protein A using a similar strategy (SI Appendix,Fig. S9). As intended, the 2 mutant proteins formed stable com-plexes with 3LRH intrabody, the complexes were not dissociatedby gel filtration chromatography, and their structures could bedetermined. The crystal structures showed that the amino acidsmutated to create the epitopes interacted with the antibody asplanned, and their structures were superimposable on the designedstructures (Fig. 2 C and D and SI Appendix, Fig. S10).

Engineering of the 3LRH Epitope in Calmodulin. To test if artificialhelical epitopes could be created in proteins other than proteinA, the 3LRH epitope was engineered in the N-terminal helix ofcalmodulin (Fig. 3A and SI Appendix, Fig. S11). Calmodulin is acalcium sensor and binds to calcium ions via EF hand motifs(13). It is composed of N- and C-terminal domains that arehomologous in sequence as well as in structure. Each domaincontains 2 calcium binding EF hand motifs. To generate the3LRH epitope, the N-terminal helix of the N-terminal domain ofcalmodulin was docked with the huntingtin peptide by over-lapping the first 8 amino acids of the calmodulin helix and thelast 8 amino acids of the huntingtin helix (Fig. 3A). As withprotein A, the amino acids pointing to the solvent in the over-lapped region were changed to those of the huntingtin peptide (SIAppendix, Fig. S11). The resulting mutant N-terminal fragment ofcalmodulin binds strongly to 3LRH intrabody, and the complexcan be crystallized. As designed, the structure of the calmodulinpart of the calmodulin–3LRH complex is not changed with theexception of the last 3 amino acids, the structure of which appearsunstable in the absence of the C-terminal calmodulin fragment(SI Appendix, Fig. S12A). The 2 calcium binding sites of theengineered calmodulin fragment are occupied by calcium ions, dem-onstrating that the functional and structural integrity of the protein

is not disturbed (Fig. 3B). The overall structure of the engineered3LRH epitope is superimposable with that of the huntingtinpeptide in the 3LRH complex, proving the success of the design(Fig. 3B and SI Appendix, Fig. S12B).

Engineering of the 3LRH Epitope in an Internal Alpha Helix. To showthat helical epitopes can be created in internal helices as well asin terminal helices, we engineered a 3LRH epitope in an internalhelix of T4 lysozyme. The lysozyme is composed of 11 alphahelices and 3 short beta strands, and we created the 3LRHepitope in the second alpha helix (Fig. 3C and SI Appendix, Fig.S13). To connect the third beta strand and the engineered sec-ond alpha helix, which is extended by 3 additional helical turns,we inserted a linker with 12 flexible amino acids (Fig. 3D and SIAppendix, Table S1). The mutated lysozyme protein was pro-duced in E. coli and crystallized as a complex with 3LRHintrabody. The crystal structure confirms that the 3LRH epitopehas been created and that the mutated lysozyme binds to theantibody (Fig. 3D). The side chains interacting with the antibodyin the 3LRH complex have practically identical conformations tothose in the huntingtin peptide (SI Appendix, Fig. S14).

Engineering of the 1P4B Epitope in a Protein A Domain and CrystalStructure of the Protein A–1P4B scFv Complex. As the last test case,we chose the 1P4B antibody because of its high affinity for thealpha-helical antigen of GCN4 and its efficient expression in E.coli. The peptide–antibody interaction of the 1P4B antibody isagain mediated by one side of the helix, and the other side is fullyexposed to solvent. As in the other cases, we docked the 1P4B–GCN4 structure to the C-terminal helix of the protein A domainwhile avoiding steric collision of protein A and antibody. Then,the amino acid residues interacting with the antibody were mu-tated to those of the GCN4 peptide (Fig. 4A and SI Appendix,Fig. S15). Protein A and the scFv fragment of 1P4B formed a

overlap

Aprotein A

huntingtin

engineeredepitope

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47

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protein A #8188N

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protein A #8189N

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3LRH intrabody

56

18

60

Fig. 2. Crystal structure of 3LRH–protein A complexes. (A) Amino acid se-quence of the engineered epitope in protein A #8188. The native protein Aand huntingtin sequences are aligned after structural docking of the alphahelices. The overlapped regions of the docked helices are marked with agreen line. (B) Crystal structure of the 3LRH intrabody and protein A #8188complex. The amino acid residues mutated to create the epitope are in red.(C) Crystal structure of the 3LRH intrabody and protein A #8189 complex.The amino acid residues mutated to create the epitope are in red. (D) Crystalstructure of the 3LRH intrabody and protein A #8496 complex.

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huntingtin 567E

50E

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Fig. 3. Crystal structure of the 3LRH–calmodulin and the 3LRH–T4 lysozymecomplexes. (A) Amino acid sequence of the engineered epitope in calmod-ulin #9011. The native calmodulin and huntingtin sequences are alignedafter structural docking of the alpha helices. The overlapped region of thedocked helices is marked with a green line. (B) Crystal structure of the 3LRHintrabody and calmodulin #9011 complex. The amino acid residues mutatedto create the epitope are in red. The calcium ions bound to the calmodulinare drawn as green balls. (C) Amino acid sequence of the engineered epitopein lysozyme #9213. The native lysozyme and huntingtin sequences are alignedafter structural docking of the alpha helices. The overlapped regions of thedocked helices are marked with a green line. (D) Crystal structure of the 3LRHintrabody and lysozyme #9213 complex. The amino acid residues mutated tocreate the epitope are colored in red. The flexible “VEGGGGSGGGGS” linker isnot visible in the electron density map and is marked with a broken gray line.

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stable complex, and the purified complex was crystallized forstructural analysis. The mutated protein A, #9014, binds to theantibody fragment, forming the intended structure, and the aminoacids mutated to bind to the antibody fragment adopt practicallyidentical conformations to those of the GCN4 peptide in the 1P4Bcomplex (Fig. 4B and SI Appendix, Figs. S16 and S17).

Engineering of the 1P4B Epitope in the ABCB6 Transporter.We testedif our antihelix antibodies are useful for study of membraneproteins with unknown structures. Human ABCB6 was chosen asthe target protein. ABCB6 is a mitochondrial Adenosine tri-phosphate (ATP) binding cassette (ABC) transporter that regulatesheme and porphyrin synthesis by translocating coproporphyrinogenIII from the cytoplasm into mitochondria (14). The monomericABCB6 is a half transporter, containing 1 transmembrane domain(TMD) and 1 nucleotide binding domain (NBD) that becomesactive by forming a homodimer. Crystal structure of the isolatedNBD has been determined (15). However, the structure of ABCB6containing TMD has not been reported yet. Therefore, we pre-dicted its structure using a homology modeling technique. Structureof Atm1-type ABC exporter (NaAtm1; PDB ID code 4MRS) thathas 46% homology in sequence with human ABCB6 was used asthe template, and the program MODELER was used for calcula-tion (16, 17). We observed that the homology model of the NBDclosely matches the crystal structure, demonstrating accuracy of themodeling (SI Appendix, Fig. S18) (15). The NBD of ABCB6 con-tains a short helix at its C terminus (SI Appendix, Fig. S18B). Totransplant the 1P4B epitope onto it, the C-terminal helix of theNBD domain of ABCB6 was docked with the GCN4 peptide byoverlapping the last 3 amino acids of ABCB6 and the first 3 aminoacids of the GCN4 helix (SI Appendix, Fig. S19). The amino acidsexposed to the solvent in the overlap region were again changed tothose of GCN4 peptide, while the amino acids pointing to ABCB6were not changed. Q823 of ABCB6 is not in the overlap region butappeared to collide with the W109 and S111 residues of 1P4B scFvin the docking model (SI Appendix, Fig. S20). Therefore, the glu-tamine was changed to alanine (Q823A) to avoid this collision.To facilitate visualization of ABCB6 in the micrographs, we

introduced 2 mutations. First, we truncated the lysosomal tar-geting segment of human ABCB6, residues 1 to 205, to improvethe homogeneity of the protein as shown previously (18). Sec-ond, we also introduced an E659Q mutation into the NBD do-main of ABCB6 to disrupt ATP hydrolysis activity and stabilizethe outward-facing structure (19–23). The engineered ABCB6was produced in Pichia pastoris, and purified ABCB6 was in-cubated with 1P4B Fab at a molar ratio of 1:3 to form stablecomplexes. The complexes and excess antibody were separatedby size exclusion chromatography (Fig. 5 A and B). The pooledfractions containing the ABCB6–Fab complex were then mixedwith ATP/Mg2+ to trap ABCB6 in the ATP-bound, outward-facingcatalytic state. The resulting protein complex was subjected to

negative staining electron microscopy (EM) and single-particleanalysis. In total, 153,945 and 138,419 particles were collectedand used to obtain 2-dimensional (2D) class averages of ABCB6with and without bound 1P4B Fab, respectively. The major 2Dclass averages of ABCB6 without the bound antibody exhibitedthe characteristic structural features of ABC transporters: theTMDs embedded in the detergent micelles and the NBDs forminga closed dimer. By contrast, the EM images of the ABCB6–1P4BFab complex clearly included extra densities corresponding todumbbell-shaped Fab bound to the presumed NBD binding site(Fig. 5 C and D). The 3D reconstructions confirmed that the lo-cation of the Fab density was in agreement with the designedstructure, thus showing that the protein with artificially designedepitope adopted the intended structure (Fig. 5 E and F). Becauseof the flexibility between the variable and constant regions of Fab,only the Fv region is seen in the final density map.In our structure, only 1 molecule of Fab is bound to 1 NBD

domain, because steric hindrance prevents binding to the otherdomain as predicted. During purification, cholesteryl hemi-succinate was added to the detergent to stabilize the structure ofABCB6 and maintain its activity, and this may be responsible forthe slightly larger dimensions of the detergent micelles (Fig. 5 Eand F) (24). Because the resolution of our EM maps was notsufficient to build a model for ABCB6 directly, we fitted theknown structure of a bacterial homolog of P-glycoprotein (P-gp),

overlap

A

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protein A #9014

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12

Fig. 4. Crystal structure of the 1P4B–protein A #9014 complex. (A) Aminoacid sequence of the engineered epitope in the protein A #9014. The nativeprotein A and the GCN4 sequences are aligned after structural docking of thealpha helices. The overlapped regions of the docked helices are marked with agreen line. (B) Crystal structure of the 1P4B scFv and protein A #9014 complex.The amino acid residues mutated to create the epitope are colored in red.

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Fig. 5. The interaction between human ABCB6 and the 1P4B Fab. (A) Sizeexclusion chromatography profiles and (B) SDS-PAGE analysis of the ABCB6–1P4B Fab complex. The ABCB6 and 1P4B Fab bands are indicated by blackarrows. (C and D) EM micrographs of ABCB6 with and without the 1P4B Fab.A few representative particles are boxed, and major 2D average classes areshown underneath. The red, yellow, and white arrows indicate detergentmicelles, the cytosolic NBD domain, and 1P4B Fab bound to ABCB6, re-spectively. (E and F) EM maps with the docked crystal structure of a bacterialmultidrug ABC transporter, Sav1866, with PDB ID code 2ONJ in the outward-facing conformation.

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Sav1866 from Staphylococcus aureus, to the final density map(14). Our EM maps closely match the Sav1866 structure, in-dicating that ABCB6 has an outward-facing TMD (i.e., open tothe mitochondrial intermembrane space) and a closed NBDconformation. High-resolution cryo-EM study of the ABCB6–1P4B complex is underway.

Engineering of the 1P4B and 3LRH Epitopes in P-gp and Inhibition ofIts ATP Hydrolysis Activity. To test if the antihelix antibodies can beused to stabilize specific conformational states of proteins, wechose Caenorhabditis elegans P-gp as a model protein (25). P-gp,a member of the ABC transporter superfamily, is a major mul-tidrug transporter that can confer drug resistance by pumpinganticancer drugs out of cancer cells. The functional unit of P-gpis composed of homologous N-terminal and C-terminal halves,each containing a TMD (TMD1 and TMD2) and a cytoplasmicNBD (NBD1 and NBD2) in a single polypeptide (Fig. 6 A andB). The TMDs recognize various substrates and facilitate theirtranslocation across the membrane, while the NBDs bind andhydrolyze ATP, providing the energy for substrate translocation.To introduce a 1P4B epitope into P-gp, a short helix at the C

terminus of NBD1 was docked with the GCN4 peptide byoverlapping the last 12 amino acids of the NBD1 helix and thefirst 12 amino acids of the GCN4 peptide (Fig. 6A and SI Ap-pendix, Fig. S21 A and B). The amino acids of the NBD1 helixexposed to solvent were substituted for those of the GCN4peptide, while the amino acids pointing toward the center ofNBD1 were not. Using a similar strategy, the 3LRH epitope wasalso created on the P-gp NBD1, but its docking area was shiftedby 1 helical turn from that of 1P4B to avoid steric collision withthe host protein (Fig. 6B and SI Appendix, Fig. S21 C and D).To verify the interactions of the engineered P-gps with anti-

bodies in vitro, pull-down assays were performed. For this, theengineered P-gps with eGFP and decahistidine tags at their C

termini were bound to nickel-nitrilotriacetic acid (Ni-NTA) resinand incubated with 1P4B scFv or 3LRH intrabody in molar ratiosof 1:10. After thorough washing, the protein mixtures wereseparated on sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE) gels. Wild-type P-gp was used as thefirst negative control, and empty resin without bound P-gp wasused as the second negative control to exclude the possibility ofbackground binding. Our results revealed that the engineeredP-gps interacted strongly with both antibodies (SI Appendix, Fig.S22). The formation of stable complexes was confirmed by sizeexclusion chromatography as well: both antibodies eluted in thehigh-molecular mass fraction typical of complexes stabilized bystrong interactions (SI Appendix, Fig. S23).It has been demonstrated that the formation of a closed NBD

dimer is a prerequisite for the ATPase activity of ABC trans-porters (23, 26). Our structure-based docking models suggest thatbinding of antihelix antibodies to the engineered P-gp inhibits itsATP hydrolysis activity by sterically preventing the closing motionof the NBD domains. To confirm this idea, we measured theATPase activity of the purified P-gp in the presence and absenceof antibodies using the potent anticancer drugs, Paclitaxel andActinomycin D, as drug substrates. As expected for an ABCtransporter, all of the P-gp structures without antibodies displayeddrug-stimulated ATPase activities, and engineering of the P-gphad almost no effect on its enzymatic characteristics, such asdrug concentration dependence, maximum level of stimulation bydrugs, and biphasic responses to substrates, with low concentra-tions stimulatory and high concentrations inhibitory (Fig. 6 C andD). This strongly suggests that transplanting helix epitopes to thehost proteins did not affect their intrinsic function or damage theiroverall structures. However, both the 1P4B scFv and the 3LRHintrabody inhibited the basal and drug-stimulated ATPase activi-ties of the engineered P-gps (Fig. 6 C and D). This demonstratesthat binding of antihelix antibodies to the engineered P-gpsinhibited the structural switching of the protein to the closedstate that is essential for ATPase activity. This is highly reminis-cent of the effect of binding of nanobody to mouse P-gp NBD1 onits ATPase activity (27).

DiscussionWe have shown that several antibodies against alpha helices canbe made to bind diverse unrelated proteins by mutating solvent-exposed amino acids in the alpha helix to those that can interactwith the antibodies. The residues important for structural stabilityof the alpha helix are not changed in order to not disrupt thenative structures of the host proteins. We have shown that ourmethod is valid by determining the structures of 8 engineeredprotein–antibody complexes by X-ray crystallography. In all cases,the overall structure of each mutated protein is not seriouslydisturbed, and the mutated helix interacts with the antihelix an-tibodies as intended. Moreover, the amino acids directly inter-acting with the antibody adopt conformations that are identical tothose found in the native peptide–antibody complexes.Our antihelix method can be used in the following situations.

First, if there is structural information on paralog or orthologproteins, the method can be used to design epitopes in the targetproteins by homology modeling. Second, it can be used when thestructure of part of a protein complex is known. For example, ifthe structure of a ligand protein is known, it can be used todesign epitopes in the ligand protein part of a ligand–receptorcomplex with structure that is unknown. Third, our method couldbe particularly useful in structural studies of transmembraneproteins, because most of them are composed entirely of longalpha helices. Antibody Fab fragments have been successfullyused in structural analysis of transmembrane proteins (4, 5).However, the production of high-affinity antibodies againsttransmembrane proteins with small hydrophilic areas takes signifi-cant amount of time and effort. Fourth, in drug design experiments,one needs to determine the structures of hundreds of protein–drugcomplexes in a high-throughput fashion, and the bound antibodymay be helpful for identifying crystallization or cryo-EM conditions

P-gp #3273

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Fig. 6. Engineering of the 1P4B or 3LRH epitopes into the C. elegans P-gp.(A) The predicted structure of the 1P4B scFv and P-gp #3273 complexes.Closing motion of the NBD induced by ATP hydrolysis is prohibited by thebound antibody. The amino acid residues changed to create the 1P4B epi-tope are in red. (B) The predicted structure of the 3LRH intrabody and P-gp#3274 complex. The amino acid residues changed to create the 3LRH epitopeare in red. (C and D) ATPase activity in the presence and absence of antibody.Drug-stimulated ATPase activities were measured with (C) paclitaxel and(D) actinomycin D. Data points indicate the average activities from 3 separateexperiments. Error bars indicate ±SDs. WT, wild type.

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more convenient for high-throughput use. As with many otherprotein engineering methods being used in structural biology, theapplication of our antihelix antibody approach needs careful con-trol experiments to show that the functional and structural integrityof the host protein has not been compromised by mutations in-troduced into the helix.To test the validity of our method, we used 3 antibody frag-

ments (3MNZ, 3LRH, and 1P4B) that have been previouslyshown to bind alpha-helical peptides. All of the antibodies boundto the engineered proteins, forming the expected rigid structures.This success suggests that many other helix binding antibodiescould be applied in our antihelix antibody method. Thousands ofantibody–antigen structures have already been deposited in thePDB. A quick survey of these structures showed that more than10 antibodies could be used as antihelix antibodies. Moreover,since these antibodies were generated for other purposes, theantihelix antibody pool could be drastically increased if a moresystematic method of generating and screening antibodies for usein our antihelix antibody method was attempted. To apply ourantihelix method, the target alpha helices should be well exposedto the solvent, because steric collision of the antibodies and targetproteins would interfere with binding. Having a panel of anti-bodies with different sequences and structures would be useful,because different antibodies recognize different structures, and atleast 1 antibody that did not produce steric collision might well bepresent among a panel of candidate antihelix antibodies.In conclusion, we have shown that several antibodies reported

to bind to alpha helices can be used as semiuniversal antihelixantibodies. These antibodies will be convenient tools in thestructural study of proteins by X-ray crystallography and cryo-EM. They can also be used to assemble protein subunits withpredictable structures for protein nanotechnology. Systematicscreening of more antihelix antibodies should greatly expand thescope of the method.

Materials and MethodsPreparation of the Antibody-Engineered Protein Complexes. To make the3MNZ scFv–protein A #8420 complex, the purified antibody fragment andengineered protein were mixed in a 1:1 molar ratio for 1 h at 4 °C. Complexformation was monitored by native PAGE. Other antibody–protein com-plexes were purified after mixing by Superdex 200 gel filtration chroma-tography to remove unbound protein. The antibody–protein complexeswere concentrated to 5 mg/mL before crystallization.

Crystallization and Data Collection. The crystallization conditions for theantibody-engineered protein complexes and freezing conditions for theircrystals are summarized in SI Appendix, Table S2. All crystals were flashfrozen in liquid nitrogen at −170 °C. Diffraction data were collected at the7A and 5C beam lines of the Pohang Accelerator Laboratory. The packageHKL2000 was used to index, integrate, and scale the diffraction data (HKLResearch).

Structure Determination, Refinement, and Homology Modeling. Initial phaseswere calculated by the molecular replacement technique using the programPHASER (28). Atomic models were built by iterative modeling and re-finement using the programs COOT and PHENIX (29, 30). Crystallographicdata are summarized in SI Appendix, Tables S3 and S4. The homology modelof human ABCB6 without the lysosomal targeting segment (residues 1 to205) was generated by the program MODELER (https://salilab.org/modeller/)based on the structure of Atm1-type ABC exporter from Novosphingobiumaromaticivorans DSM 12444, NaAtm1 (16, 17). Protocols for preparation ofantibodies, preparation of engineered proteins, ATP hydrolysis activity,preparation of samples for negative EM and image acquisition, and EM dataprocessing and image analysis are described in SI Appendix. Atomic coor-dinates and diffraction data have been deposited in the PDB (SI Appendix,Tables S2 and S3).

ACKNOWLEDGMENTS. We thank the staff of the beam lines 5C and 7A,Pohang Accelerator Laboratory for help with data collection and Dr. JulianGross for critical reading of the manuscript. This research was supported byNational Research Foundation Grants NRF 2017R1A2A1A17069497 and NRF-2017M3A9F6029753 funded by the Ministry of Science and ICT of Korea andSamsung Science and Technology Foundation Grant SSTF-BA1702-14.

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