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A de Novo Designed Template for Generating Conformation-specific Antibodies That Recognize α-Helices in Proteins*

  • Stephen M. Lu
    Affiliations
    From the Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262
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  • Robert S. Hodges
    Correspondence
    To whom correspondence should be addressed. Tel.: 303-315-8837; Fax: 303-315-1153;
    Affiliations
    From the Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262
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  • Author Footnotes
    * This work was supported by the University of Colorado Health Sciences Center and the Protein Engineering Network of Centres of Excellence at the University of Alberta.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:April 23, 2002DOI:https://doi.org/10.1074/jbc.M201981200
      The generation of antibodies directed toward the surface-exposed regions of a protein using synthetic peptides as immunogens representing surface loops and turns has been widely successful. However, peptides representing α-helical regions are typically unstructured in solution and unable to produce antibodies that recognize α-helices in native proteins. We describe a de novo designed parallel two-stranded α-helical coiled-coil template for immunization to prepare antibodies that recognize α-helical protein sequences in the native protein. This template was designed for maximum stability through an Ile/Leu hydrophobic core and an interchain disulfide bridge. Surface-exposed helical residues are inserted into the template and used for immunization to generate polyclonal antibodies. To demonstrate the feasibility of this approach, 15 residues of the yeast transcription factor GCN4 were inserted into this template, and the resultant antibodies were screened for conformational specificity. Peptide antigens that contain the same surface-exposed residues but differ in structure were used as competitors in a competition assay. Direct competition between the capture peptide immobilized on a biosensor chip, the peptide antigens, and the antibodies generated by the template demonstrated that the antibodies were specific for helical structure in the native coiled-coil (synthetic GCN4 residues 250–280). These antibodies were unable to recognize the same inserted sequence in an unstructured analog. The helix-specific antibodies were also able to identify native GCN4 (31.3 kDa) from yeast whole cell extracts.
      HPLC
      high performance liquid chromatography
      BB
      4-benzoylbenzoic acid
      BSA
      bovine serum albumin
      ELISA
      enzyme-linked immunosorbent assay
      PBS
      phosphate-buffered saline
      RPC
      reversed-phase chromatography
      TFE
      trifluoroethanol
      The immune system is a remarkable system of protein-protein interactions capable of recognizing an extraordinary number of antigens from a limited pool of genetic segments. While the immune system is of great importance for protecting an organism from foreign proteins, toxins, and bacterial infections, a great deal of work has also been devoted to research and diagnostic uses of antibodies. Development of antibodies as recognition molecules for bioassays and affinity purification is now commonplace.
      Immunization with native proteins, domains, or segments is well within the scope of a typical laboratory using recombinant expression techniques. However, immunization with proteins typically yield epitopes directed toward surface loops (
      • Davies D.R.
      • Cohen G.H.
      ), e.g. nearly the entire surface of hen egg white lysozyme has been shown to be antigenic (
      • Newman M.A.
      • Mairhart C.R.
      • Mallett C.P.
      • Lovoie T.B.
      • Smith-Gill S.J.
      ). Generation of monoclonal antibody libraries directed toward a protein target is also a common approach. The binding epitope of each clone is identified by mapping techniques (
      • Westwood O.M.R.
      • Hay F.C.
      ) using combinatorial peptide or phage display libraries (Refs.
      • Pinilla C.
      • Martin R.
      • Gran B.
      • Appel J.R.
      • Boggiana C.
      • Wilson D.B.
      • Houghten R.A.
      and
      • Irving M.B.
      • Pan O.
      • Scott J.K.
      and references therein). Alternatively, the entire protein sequence of the immunogen can be screened for binding epitopes with the PEPSCAN approach (
      • Geysen H.M.
      • Rodda S.J.
      • Mason T.J.
      • Tribbick G.
      • Schoofs P.G.
      ). These epitope mapping techniques typically use short contiguous sequences and map linear epitopes. However, epitopes are often discontinuous. Nevertheless, these methods allow for the generation of protein-specific antibodies, although the epitope is not explicitly delineated during immunization.
      As an alternative, synthetic peptide chemistry has also advanced such that peptides of moderate length can now be routinely made in the typical laboratory. Thus, it is possible to synthesize chemically selected protein segments of interest for immunization. Short peptides are poorly immunogenic on their own, but this drawback is circumvented by conjugation to a carrier protein (
      • Aslam M.
      • Dent A.
      ). Polyclonal antibodies can be used in lieu of preparing monoclonal antibodies, because only a single epitope is typically used for immunization, and therefore only a single specificity is generated. Recent mutagenesis studies of antigen-antibody interactions have shown that although many residues are often identified by structural means as comprising an epitope, relatively few residues contribute the majority of binding energy (
      • Jin L.
      • Wells J.A.
      ,
      • Benjamin D.C.
      • Perdue S.S.
      ). Therefore, it is possible to minimize epitopes if key interactions are maintained.
      The usefulness of synthetic peptides to represent protein domains is limited by conformational issues (
      • Van Regenmortel M.H.V.
      ). The protein segment of interest is stabilized by secondary and tertiary interactions in the native protein, but the corresponding peptide on its own in solution will typically be a random coil. Without structural restraints, the flexibility of peptides will lead to a diverse set of conformations presented to the immune system, most of which are non-native (
      • Dyson H.J.
      • Wright P.E.
      ). For example, a 19-residue peptide corresponding to a helical segment of myohemerythrin was shown to be bound to an antibody as a β-turn (
      • Stanfield R.L.
      • Fieser T.M.
      • Lerner R.A.
      • Wilson I.A.
      ), whereas an overlapping peptide was later shown to be bound as a helix (
      • Tsang P.
      • Rance M.
      • Fieser T.M.
      • Ostresh J.M.
      • Houghten R.A.
      • Lerner R.A.
      • Wright P.E.
      ). Additional complications arrive from the induced fit of the antigen to the antibody recognition site for ligands such as peptides (
      • Stanfield R.L.
      • Wilson I.A.
      ). Thus, a need is evident for structure-stabilized immunogens for the generation of antibodies that recognize a specific conformation, like α-helices in a native protein.
      There are many means of stabilizing helical structure described in the literature. For instance, lactam bridges between Glu and Lys side chains in the i to i+4 positions can be incorporated to stabilize helical structure (
      • Houston Jr.M.E.
      • Gannon C.L.
      • Kay C.M.
      • Hodges R.S.
      ). Helical flanking sequences have been used to stabilize intervening sequences into helical structure (
      • Cooper J.A.
      • Hayman W.
      • Reed C.
      • Kagawa H.
      • Good M.F.
      • Saul A.
      ) for use as both an epitope mapping library and helical-stabilized immunogens. De novo design of stabilized helical proteins has also been reported (
      • Barthe P.
      • Rochette S.
      • Vita C.
      • Roumestand C.
      ,
      • Starovasnik M.A.
      • Braisted A.C.
      • Wells J.A.
      ,
      • Walsh S.T.R.
      • Cheng H.
      • Bryson J.W.
      • Roder H.
      • DeGrado W.F.
      ,
      • Schafmeister C.E.
      • LaPorte S.L.
      • Miercke L.J.W.
      • Stroud R.M.
      ). One particular motif, the coiled-coil stem loop, an anti-parallel coiled-coil connected by an intervening loop, was shown to be an effective and compact scaffold for presentation of epitopes as either loops or helices (
      • Miceli R.
      • Myszka D.
      • Mao J.
      • Sathe G.
      • Chaiken I.
      ). Here, we focus on a method for the design of stabilized α-helical immunogens as a means of generating antibodies specific for helical protein segments using the two-stranded α-helical parallel coiled-coil as a template. The advantages of this template will be presented.
      This laboratory has extensively studied the de novo design of coiled-coils, an excellent model of helical structure (
      • Zhou N.E.
      • Zhu B.Y.
      • Kay C.M.
      • Hodges R.S.
      ,
      • Hodges R.S.
      ,
      • Kohn W.D.
      • Hodges R.S.
      ,
      • Wagschal K.
      • Tripet B.
      • Hodges R.S.
      ). The coiled-coil motif is comprised of two right-handed α-helices that interact intricately with each other in a slight left-handed superhelical twist. This motif has been identified in many muscle proteins and leucine zipper dimerization domains (Ref.
      • Lupas A.
      and references therein). It is characterized by a heptad repeat (abcdefg) n in which the a andd positions are typically hydrophobic residues. This repeat pattern creates a hydrophobic face that provides a large thermodynamic driving force for dimerization. The remaining residues are solvent-exposed and often used in de novo design applications. One key advantage of using coiled-coils as a model of helical structure is that stability can be increased by relatively few changes at the a and d heptad positions. The effect of substitution of all 20 naturally occurring amino acids on stability within a model coiled-coil was studied at the aand d positions (
      • Wagschal K.
      • Tripet B.
      • Lavigne P.
      • Mant C.
      • Hodges R.S.
      ,
      • Tripet B.
      • Wagschal K.
      • Lagvine P.
      • Mant C.T.
      • Hodges R.S.
      ).
      To demonstrate the feasibility of using a de novo designed coiled-coil as a helically stabilized immunogen, residues 254–273 of the transcription factor GCN4 (general control of amino acid biosynthesis protein N4) from yeast were designated as an epitope target, and we designed a peptide immunogen in which the surface residues were placed into our helical template (see Fig. 1). GCN4 is a well studied example of a coiled-coil (and hence helical) in which its crystal structure (
      • O'Shea E.K.
      • Klemm J.D.
      • Kim P.S.
      • Alber T.
      ), oligomerization state (
      • Harbury P.B.
      • Zhang T.
      • Kim P.S.
      • Alber T.
      ), and the thermodynamic properties (
      • Thompson K.S.
      • Vinson C.R.
      • Freire E.
      ) of GCN4 and its variants have been reported. To evaluate the conformational specificity of the resultant helix-specific antibodies generated from the helical template, several peptides were also designed as antigens for competition experiments. Surface plasmon resonance (i.e. Biacore) was used as a convenient means of measuring antibody-antigen interaction (
      • Van Regenmortel M.H.V.
      • Altschuh D.
      • Chatellier J.
      • Christensen L.
      • Rauffer-Bruyere N.
      • Richalet-Secordel P.
      • Witz J.
      • Zeder-Lutz G.
      ). Direct competition with peptides of native sequence demonstrate that the generated antibodies specifically recognize helical epitopes derived from the inserted sequence.
      Figure thumbnail gr1
      Figure 1Sequences of peptides used for immunization, antibody capture and as competitive antigens. A template for helical epitopes is shown at the top. Hydrophobic core residues are indicated by the shaded boxes, whereasasterisks represent residues derived from the helical epitope of the target protein. The template allows for the insertion of 18 surface-exposed residues indicated by the asterisks. In this particular example, 15 surface-exposed residues from the coiled-coil region of GCN4 (residues 254–273) were inserted into the template, and the remaining three open surface positions were filled with alanine residues (underlined). The immunogens and capture molecules are shown in the middle section. The peptide antigens used in the competition experiments are shown in thebottom section. GCN4(p1-md) is the native antigen, where p1 denotes the native sequence residues 250–280 (
      • O'Shea E.K.
      • Klemm J.D.
      • Kim P.S.
      • Alber T.
      ), and md denotes that this peptide forms a two-stranded coiled-coil that is concentration-dependent (i.e. monomer-dimer equilibrium). The antigen GCN4(p1-db) denotes the disulfide-bridged (db) coiled-coil of the native p1 sequence. The double underline denotes the target region for template antibodies prepared from the immunogens defined above. The helical heptad positions are indicated in italics.

      DISCUSSION

      The specificity of antibodies for detecting specific regions within a protein can be invaluable in protein structure/function studies. A minimalist approach is often pursued in generating antibodies with desired specificity where a peptide corresponding to a segment of interest is used as an immunogen. The selection of potential surface-exposed linear epitopes on proteins from amino acid sequence information (
      • Parker J.M.R.
      • Guo D.
      • Hodges R.S.
      ) and the presentation of polyclonal antibodies that recognize these regions in the native protein have been widely successful (
      • Strynadka N.C.J.
      • Redmond M.J.
      • Parker J.M.R.
      • Scraba D.G.
      • Hodges R.S.
      ). This approach works well for small linear sequences that are surface-exposed, unstructured loops, or β-turn containing regions. However, a method to generate antibodies that recognize helical segments in proteins when using short synthetic peptides is needed. Because short peptides of helical sequences are unstructured when removed from the protein, there is a need to constrain these sequences in an α-helical conformation. We designed de novo a parallel two-stranded α-helical coiled-coil as a stabilized template suitable for presentation of helical epitopes. The design of the peptide involves relatively small changes to the native protein segment (only two residues/heptad). By substituting residues on the hydrophobic face, a stable hydrophobic core can be formed, thus driving the association of the two helices into a coiled-coil. Although we have described a homodimer in this work, this technique could easily be applied to create a heterodimer for immunization with two different epitopes.
      The approach described requires the identification of a helical protein segment of interest and its orientation. The typical helix found in a globular protein is short (less than 20 residues) and easily accommodated in our template. Alanine residues can be included to fill any open surface positions. Because ∼50% of all α-helices in proteins are amphipathic, the solvent-exposed face of the helix is readily defined. The spacing of hydrophobic residues will follow a three-four or four-three residue hydrophobic repeat. In this case, the design of a helical epitope immunogen simply involves placing the helical sequence into the template in Fig. 1 in an appropriate register such that hydrophobic residues in the amphipathic helix are replaced by the hydrophobic residues in the template. The oxidized coiled-coil template will have the same solvent-exposed positions as that in the amphipathic helix from the protein of interest. If the helix of interest is not clearly amphipathic, there are seven possible helical orientations in which a given peptide sequence can be inserted into the template and, thus, seven sets of a/d core residues. The contribution of each a/d residue in each set could be simply summed using the data of Refs.
      • Wagschal K.
      • Tripet B.
      • Lavigne P.
      • Mant C.
      • Hodges R.S.
      and
      • Tripet B.
      • Wagschal K.
      • Lagvine P.
      • Mant C.T.
      • Hodges R.S.
      , and the frame that yields the greatest stability is selected as the most probable orientation of surface-exposed and hydrophobic core residues. In cases where several orientations are equally plausible, multiple immunogens could be prepared, and the resultant antibodies could be used to probe the correct surface accessible residues in the helix of the native protein.
      With our current design criteria, we maximized the stability of the template (Fig. 5) by incorporating an interchain disulfide bridge and maximizing the hydrophobicity of the a and dresidues in the hydrophobic core. Although the incorporation of the disulfide bridge increases stability (compare GCN4(p1-md) and GCN4(p1-db)), the additional increase in stability by optimizing the hydrophobic core was substantially greater (compare GCN4(p1-db) and GCN4 coiled-coil template). Thus, the stability of our template design ensures that antibodies are generated to an α-helical structure regardless of the residues inserted into the template. The observed increase in stability of the template could be predicted from previous research on model coiled-coils showing that the major contributing factor to coiled-coil stability is the hydrophobic core positionsa and d. Substitution of three Val residues by Ile in the a position was shown to increase the guanidine hydrochloride midpoint of a model coiled-coil by 1.8 m(
      • Zhu B.Y.
      • Zhou N.E.
      • Kay C.M.
      • Hodges R.S.
      ). An Asn to Ile substitution in the center of another model coiled-coil, also at the a position, was also found to increase the guanidine denaturation midpoint by 1.7 m (
      • Wagschal K.
      • Tripet B.
      • Lavigne P.
      • Mant C.
      • Hodges R.S.
      ). The disulfide bridge contribution to coiled-coil stability is also very significant, even when replacing a large hydrophobe like Leu in the hydrophobic core. The increase in the guanidine hydrochloride midpoint was ∼2.5 m (
      • Zhou N.E.
      • Kay C.M.
      • Hodges R.S.
      ). The observed increase in stability of the GCN4 disulfide-bridged coiled-coil template over the GCN4(p1-db) peptide is predicted by the sum of these changes.
      Several coiled-coils have been shown to exist in higher order oligomers (
      • Tripet B.
      • Wagschal K.
      • Lagvine P.
      • Mant C.T.
      • Hodges R.S.
      ,
      • Harbury P.B.
      • Zhang T.
      • Kim P.S.
      • Alber T.
      ,
      • Betz S.
      • Fairman R.
      • O'Niel K.
      • Lear J.
      • DeGrado W.F.
      ), which may be a problem in presenting the surface residues properly. However, it is unlikely that sequences in the-bc-efg positions of helices can override the oligomerization state of this extremely stable disulfide-bridged coiled-coil. Each template molecule after conjugation will present two helical epitopes per coiled-coil with the hydrophobic core residues buried and not presented.
      Purified sera were qualitatively screened using ELISAs, but we opted to use the Biacore instrument for comparative analysis of specificity. The same interaction surface is used for the entire peptide dilution series and is amenable to automation. Real time data collection allows for estimation of association and dissociation rates and hence equilibrium binding constants. In addition to the peptide antigen competition experiments shown in Fig. 7, the binding affinity of the two antibodies for their respective capture peptides was determined by an antibody dilution series (data not shown). The affinity of the helix-specific antibody (L298) for the coiled-coil template capture peptide was estimated to be ∼1 × 107m−1 using the bivalent analyte model, whereas that of nonhelix-specific antibody (L392) for the random coil analog capture peptide was ∼3 × 105m−1. This is in good agreement with our peptide competition results (Table II). Helix-specific antibody (L298) recognizes not only the coiled-coil domain (peptide GCN4p1-md) but also the entire full-length native 31.3-kDa protein through immunoprecipitation and Western blotting (data not shown). This result reiterates the utility of using a designed epitope specific for a small functional domain to generate antibodies that successfully recognize the native full-length protein. Additionally, only the surface-exposed residues (once identified) are required for this approach.
      The direct competition between the capture peptide (on the biosensor chip surface) and peptide antigens (in solution) for antibody binding allows for a relative ranking of binding affinity. The helical specificity of antibodies generated with the coiled-coil template was assessed by direct competition experiments (Fig. 7) with the series of peptide antigens shown in Fig. 1. Each peptide differs only by the residues in the a and d heptad positions and contains the same surface-exposed residues in the cassette region. The GCN4(p1-db) disulfide-bridged peptide is the most effective inhibitor of the helix-specific antibody (L298) as expected because it is a stabilized coiled-coil and closely resembles the immunogen. Because this antibody is effectively captured by the coiled-coil template immobilized on the chip surface and effectively competed by GCN4(p1-db) peptide, this demonstrates that the inserted sequence is specifically recognized. In addition, the extremely high stability of the coiled-coil template ensures that only the helical conformation is presented to the immune system, and therefore it would be expected that this polyclonal serum will have only helical specificity. The GCN4(Ala core) peptide is also recognized by our helix-specific antibody, albeit 100-fold weaker than the GCN4(p1-db) coiled-coil. This implies that the antigen-binding site is capable of inducing helical conformation in peptides with helical propensity. The GCN4(Gly core) peptide is poorly but measurably recognized by helix-specific antibody L298, suggesting weak induction of helical structure in this peptide antigen. Because these peptides contain the same surface-exposed residues in the same register as the other peptides in the series, specificity for helical structure is also demonstrated. The helicity of the GCN4(p1-md) peptide was shown to decrease in a concentration-dependent manner (Fig. 4). We expect that induction of helical structure as seen with GCN4(Ala core) will also occur with the GCN4(p1-md) monomer. At lower concentrations, the monomer-dimer equilibrium shifts toward predominately unfolded monomer, and therefore the GCN4(p1-md) peptide in solution becomes less effective as an inhibitor.
      The same experiment was performed over the GCN4 single-stranded capture surface (Fig. 7 B) and yielded nearly identical results. In this single-stranded template strand, the hydrophobic residues are exposed, whereas they were buried in the coiled-coil template and presumably not accessible to the immune system. This suggests that the BSA-GCN4 single-stranded conjugate is easily induced to the helical conformation. The nearly identical peptide inhibition profile suggests that the surface-exposed residues that were inserted into the template are being specifically recognized and not the hydrophobic core residues.
      The target sequence was also inserted into a random coil analog of the coiled-coil template (Fig. 1). This random coil analog was designed to represent an unstructured peptide that contains the same epitope sequence. Glycine residues have poor helical propensity and a high degree of backbone flexibility. As such, many conformations are expected to be presented to the immune system, although the most probable conformation is expected to be that of the extended peptide chain. The antibody (L392) generated against this random coil analog is specific for the GCN4(Gly core) peptide, but the binding affinity appears to be weaker than that of antibody (L298) that was generated against the coiled-coil template (Table II). This illustrates the benefit of using a structurally stabilized template for immunization. Interestingly, the remaining three peptide antigens are very weakly recognized by this antibody (L392). The coiled-coil structure of the GCN4(p1-db) disulfide-bridged peptide is unlikely to adopt any nonhelical conformations. This suggests that there is a small population of antibodies in our polyclonal serum that are helix-specific. This minor component is also likely to be able to induce helical structure in the GCN4(p1-md) single-stranded (at low concentrations) and the GCN4(Ala core) peptides because these peptide antigens have high helical propensity. The concept of conformation adaptation of antigens as opposed to induced fit by the antibody was recently discussed (
      • Bosshard H.R.
      ).
      We have demonstrated that by using a de novo designed coiled-coil as a template for immunization, we are able to generate antibodies specific not only for the sequence of interest but also for the helical conformation. This will allow the facile generation of specific antibodies targeted against helical regions of proteins. In principle, once a helical segment of interest is identified (either by structural data or computer prediction), analogous peptides (inserted into this helical template) could be synthesized and used for immunization. In cases where structural data are not available, predicted helical segments could be used as epitopes as a means of mapping surface accessibility of different regions of the protein. Potential neutralizing antibodies could be generated in a similar fashion because many viral proteins are helical in nature (
      • Shu W.
      • Liu J., Ji, H.
      • Radigen L.
      • Jiang S.
      • Lu M.
      ,
      • Foster T.P.
      • Melancon J.M
      • Kousoulas K.G.
      ). Pathological protein precipitates (such as the prion protein) are also of great interest. Although the pathological form is the β-sheet, the cellular form is believed to be helical (
      • Zhang H.
      • Kaneko K.
      • Nguyen J.T.
      • Livshits T.L.
      • Baldwin M.A.
      • Cohen F.E.
      • James T.L.
      • Prusiner S.B.
      ). Recognition patterns by a panel of helix-specific antibodies could identify the regions that undergo structural change (
      • Yokoyama T.
      • Kimura K.M.
      • Ushiki Y.
      • Yamada S.
      • Morooka A.
      • Nakashiba T.
      • Sassa T.
      • Itohara S.
      ,
      • Leclerc E.
      • Peretz D.
      • Ball H.
      • Sakurai H.
      • Legname G.
      • Serban A.
      • Prusiner S.B.
      • Burton D.R.
      • Williamson R.A.
      ). We have demonstrated here the feasibility of using a de novo designed coiled-coil as a helical template for generation of helix-specific antibodies and will in the future apply this to a wide range of applications.

      Acknowledgments

      We thank the Protein Engineering Network of Centres of Excellence at the University of Alberta for peptide synthesis. We also acknowledge Dr. Paul Cachia for helpful discussions about immunization procedures and BIAcore analysis, Dr. Judith Jaehning and co-workers for providing yeast cells and assistance with Western blotting, Morris Aarbo for antibody purification, and Elsi Vacano for manuscript preparation. CD and Biacore experiments were performed at the Biophysics Core facility at the University of Colorado Health Sciences Center.

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