Structural variation and immune recognition of the P1.2 subtype meningococcal antigen

Neisseria meningitidis is a globally important cause of bacterial meningitis and septicemia. No comprehensive antimeningococcal vaccine is available, largely as a consequence of the high sequence diversity of those surface proteins that could function as components of a vaccine. One such component is the protein PorA, a major surface porin of this Gram-negative organism that has been used in a number of experimental and licensed vaccines. Here we describe a series of experiments designed to investigate the consequences for antibody recognition of sequence diversity within a PorA antigen. The binding of a 14-residue peptide, corresponding to the P1.2 subtype antigen, to the MN16C13F4 monoclonal antibody was sensitive to mutation of five out of the six residues within the epitope sequence. The crystal structure of the antibody Fab fragment, determined in complex with the peptide antigen, shows a remarkably hydrophobic binding site and interactions between the antigen and antibody are dominated by apolar residues. Nine intrachain hydrogen bonds are formed within the antigen which maintain the beta-hairpin conformation of the peptide. These hydrogen bonds involve residues that are highly conserved amongst different P1.2 sequence variants, suggesting that some positions may be conserved for structural reasons in these highly polymorphic regions. The sensitivity of antibody recognition of the antigen towards mutation provides a structural explanation for the widespread sequence variation seen in different PorA sequences in this region. Single point mutations are sufficient to remove binding capability, providing a rationale for the manner in which different meningococcal PorA escape variants arise.     

Molecular structural and functional characterization of tumor suppressive anti-ErbB-2 monoclonal antibody by phage display system

To investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with (87)AHNQVRQVPLQR(98) in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.     

Molecular recognition of a peptide mimic of the Lewis Y antigen by an anti-Lewis Y antibody

Peptides as mimics of carbohydrates display a distinct advantage in vaccine design because of ease of synthesis and their inherent T cell-dependent nature as immunogens. While peptides that mimic carbohydrates have been described, it is not clear how they do so. To further our insight into structural relationships between peptide-mimics and carbohydrate structures, we have analyzed a potential recognition scheme between the murine monoclonal antibody, B3, directed against the tumor-associated antigen Lewis Y oligosaccharide and a peptide identified from phage display screening with B3. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. The B3 antibody binds to the peptide sequence APWLYGPA in which the putative sequence APWLY is critical for binding to the antibody. Not having experimental structural information for B3, the crystal structure of another anti-Lewis Y antibody, BR96, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, provides a molecular basis for LeY antigen recognition and specificity, and how this binding relates to peptide binding. As a guide to place the APWLY motif in the B3 combining site, a fragment library was searched for analogous compounds that have the potential to bind to B3. Our modeling study shows that the B3-peptide complex shares similar recognition features for the difucosylated type 2 lactoseries' structure. This analysis provides a molecular perspective for peptide mimicry of a carbohydrate epitope. © 1998 John Wiley & Sons, Ltd.     

Structural consensus among antibodies defines the antigen binding site

The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.     

Dissecting contact potentials for proteins: relative contributions of individual amino acids

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.     

Molecular recognition by a binary code

Functional antibodies were obtained from a library of antigen-binding sites generated by a binary code restricted to tyrosine and serine. An antibody raised against human vascular endothelial growth factor recognized the antigen with high affinity (K(D)=60 nM) and high specificity in cell-based assays. The crystal structure of another antigen binding fragment in complex with its antigen (human death receptor DR5) revealed the structural basis for this minimalist mode of molecular recognition. Natural antigen-binding sites are enriched for tyrosine and serine, and we show that these amino acid residues are intrinsically well suited for molecular recognition. Furthermore, these results demonstrate that molecular recognition can evolve from even the simplest chemical diversity.     

An nmr conformational analysis of a synthetic peptide Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from the New World Centruroides noxius 2 (Cn2) scorpion toxin: comparison of the structure with those of the Centruroides scorpion toxins

The solution structure of a synthetic peptide, Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from toxin 2 (Cn2) of the New World scorpion Centruroides noxius was determined using nmr and molecular dynamics calculations. The peptide has no significant secondary structure such as an alpha-helix or a beta-sheet, yet it has a fixed conformation for the first chain. The backbone secondary structure involving residues 6-12 in this peptide shows an excellent overlap with the structures of natural neurotoxins from Centruroides sculpturatus Ewing. Residues 6-9 form a distorted type I beta-turn and residues 10-12 form a gamma-turn. As residues 7-10 in the Centruroides toxins correspond to one of the regions of highest sequence variability, it may account for the species specificity and/or selectivity of toxic action. The conformation of this region evidently plays an important role in receptor recognition and in binding to the neutralizing monoclonal antibody BCF2 raised against the intact toxin.     

Cognitive features of continuous antigenic determinants

We sought to identify the features controlling the specificity of antibody recognition and thus gain insights into molecular recognition between proteins in general. A total of 103 epitopes within 63 well-defined antigenic peptides homologous with the relevant antigen sequence were identified. The contribution of each amino acid residue to the antibody binding activity of each epitope was investigated by ELISA testing of complete sets of peptide analogs containing single amino acid replacements. The data are summarized in a replaceability matrix. Some of the high frequency replaceabilities were expected, such as aspartate for glutamate, serine for threonine, etc., but unexpected relationships were also found, such as a high degree of acceptability of methionine as a replacement. Replaceability with a residue of opposite charge was rare. Glycine and tyrosine were frequently of low acceptability, except for glycine as a replacement for alanine. It was found that on average only about four to five amino acid residues in epitopes were required to determine specificity and provide binding energy. Specificity and binding energy were attributed to amino acid side chains rather than main chain atoms. Propensity factors for occurrence of amino acids in antigenic determinants were calculated. The prominence of certain hydrophobic residues as residues critical to recognition by antibody suggests that the molecular surface of an antigen in its combined form with antibody is altered from that occurring in the absence of antibody. Thus, antigenicity is not a static surface phenomenon but depends on the ability of the antigen to undergo rearrangement, supporting the induced fit concept.     

Principles and engineering of antibody folding and assembly

Antibodies are uniquely suited to serve essential roles in the human immune defense as they combine several specific functions in one hetero-oligomeric protein. Their constant regions activate effector functions and their variable domains provide a stable framework that allows incorporation of highly diverse loop sequences. The combination of non-germline DNA recombination and mutation together with heavy and light chain assembly allows developing variable regions that specifically recognize essentially any antigen they may encounter. However, this diversity also requires tailor-made mechanisms to guarantee that folding and association of antibodies is carefully this diversity also requires tailor-made mechanisms to guarantee that folding and association of antibodies is carefully controlled before the protein is secreted from a plasma cell. Accordingly, the generic immunoglobulin fold β-barrel structure of antibody domains has been fine-tuned during evolution to fit the different requirements. Work over the past decades has identified important aspects of the folding and assembly of antibody domains and chains revealing domain specific variations of a general scheme. The most striking is the folding of an intrinsically disordered antibody domain in the context of its partner domain as the basis for antibody assembly and its control on the molecular level in the cell. These insights have not only allowed a better understanding of the antibody folding process but also provide a wealth of opportunities for rational optimization of antibody molecules.     

Recognition of Mesothelin by the Therapeutic Antibody MORAb-009: STRUCTURAL AND MECHANISTIC INSIGHTS*

Mesothelin is a tumor differentiation antigen that is highly expressed in many epithelial cancers, with limited expression in normal human tissues. Binding of mesothelin on normal mesothelial cells lining the pleura or peritoneum to the tumor-associated cancer antigen 125 (CA-125) can lead to heterotypic cell adhesion and tumor metastasis within the pleural and peritoneal cavities. This binding can be prevented by MORAb-009, a humanized monoclonal antibody against mesothelin currently under clinical trials. We show here that MORAb-009 recognizes a non-linear epitope that is contained in the first 64-residue fragment of the mesothelin. We further demonstrate that the recognition is independent of glycosylation state of the protein but sensitive to the loss of a disulfide bond linking residues Cys-7 and Cys-31. The crystal structure of the complex between the mesothelin N-terminal fragment and Fab of MORAb-009 at 2.6 Å resolution reveals an epitope encompassing multiple secondary structural elements of the mesothelin, including residues from helix α1, the loops linking helices α1 and α2, and between helices α4 and α5. The mesothelin fragment has a compact, right-handed superhelix structure consisting of five short helices and connecting loops. A residue essential for complex formation has been identified as Phe-22, which projects its side chain into a hydrophobic niche formed on the antibody recognition surface upon antigen-antibody contact. The overlapping binding footprints of both the monoclonal antibody and the cancer antigen CA-125 explains the therapeutic effect and provides a basis for further antibody improvement.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall “humanness” was increased for both the light and heavy chain variable regions.     

Molecular Dynamics Simulations of the TSSPSAD Peptide Antigen in Free and Bound with CAMPATH-1H Fab Antibody States: The Importance of the β-Turn Conformation

Humanized CAMPATH-1H antibody has been found to have biological applications through the recognition of the CD52 antigen. A peptide mimotope of the CD52 antigen with the sequence T₁SSPSAD₇ has been co-crystallized with the CAMPATH-1H antibody. A plethora of hydrogen bond interactions were found to mediate antigen recognition. An important feature of peptide’s bound conformation was the type I β-turn found in the S₃PSA₆ peptide’s fragment. Paradoxically, this fact has been underestimated from other researchers. In order to further investigate the importance of this structural feature and its significance in antibody/antigen binding we have performed molecular dynamics simulations in explicit water of the T₁SSPSAD₇ peptide in both antibody free and bound states. We have found that the turn structure has been perfectly retained in the bound state but it was eliminated in the free state. This fact implies that the turn structure of the peptide is unstable in aqueous environment and it is induced upon antibody binding. Analysis of the trajectories revealed also several other important features of the antibody/antigen binding mode.     

利用噬菌体展示技术筛选胰岛素模拟肽

为了寻找能够模拟胰岛素生物活性的小肽,以胰岛素多克隆抗体为靶标,筛选噬菌体展示随机C7C环肽库.3轮筛选后,通过ELISA方法挑取与靶分子特异性结合的15个阳性克隆,测序获得两条序列,分析所得序列并合成相应短肽.通过细胞生物学活性检测,小肽CPTSQANSC（ZJ1）能够竞争性的抑制胰岛素与其受体的结合,并对正常小鼠和四氧嘧啶诱导的糖尿病小鼠,都有明显的降血糖作用.上述结果表明,小肽CPTSQANSC具有胰岛素样生物学活性.而小肽CVQPSHSSC（ZJ2）表现出胰岛素拮抗活性,能引起正常小鼠血糖升高.这表明筛选到了能够模拟胰岛素表位的短肽CPTSQANSC,可能为治疗胰岛素依赖性糖尿病提供了新线索.     

Peptide design and structural characterization of a GPCR loop mimetic

G protein-coupled receptors (GPCRs) control fundamental aspects of human physiology and behaviors. Knowledge of their structures, especially for the loop regions, is limited and has principally been obtained from homology models, mutagenesis data, low resolution structural studies, and high resolution studies of peptide models of receptor segments. We developed an alternate methodology for structurally characterizing GPCR loops, using the human S1P(4) first extracellular loop (E1) as a model system. This methodology uses computational peptide designs based on transmembrane domain (TM) model structures in combination with CD and NMR spectroscopy. The characterized peptides contain segments that mimic the self-assembling extracellular ends of TM 2 and TM 3 separated by E1, including residues R3.28(121) and E3.29(122) that are required for sphingosine 1-phosphate (S1P) binding and receptor activation in the S1P(4) receptor. The S1P(4) loop mimetic peptide interacted specifically with an S1P headgroup analog, O-phosphoethanolamine (PEA), as evidenced by PEA-induced perturbation of disulfide cross-linked coiled-coil first extracellular loop mimetic (CCE1a) (1)H and (15)N backbone amide chemical shifts. CCE1a was capable of weakly binding PEA near biologically relevant residues R29 and E30, which correspond to R3.28 and E3.29 in the full-length S1P(4) receptor, confirming that it has adopted a biologically relevant conformation. We propose that the combination of coiled-coil TM replacement and conformational stabilization with an interhelical disulfide bond is a general design strategy that promotes native-like structure for loops derived from GPCRs.     

A Similar Pattern of Interaction for Different Antibodies with a Major Antigenic Site of Foot-and-Mouth Disease Virus: Implications for Intratypic Antigenic Variation

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C₁, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.     

Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.     

Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4?nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.     

Plasticity of S2-S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis

Many protein substrates of caspases are cleaved at noncanonical sites in comparison to the recognition motifs reported for the three caspase subgroups. To provide insight into the specificity and aid in the design of drugs to control cell death, crystal structures of caspase-7 were determined in complexes with six peptide analogs (Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho, Ac-WEHD-Cho) that span the major recognition motifs of the three subgroups. The crystal structures show that the S2 pocket of caspase-7 can accommodate diverse residues. Glu is not required at the P3 position because Ac-DMQD-Cho, Ac-DQMD-Cho and Ac-DNLD-Cho with varied P3 residues are almost as potent as the canonical Ac-DEVD-Cho. P4 Asp was present in the better inhibitors of caspase-7. However, the S4 pocket of executioner caspase-7 has alternate regions for binding of small branched aliphatic or polar residues similar to those of initiator caspase-8. The observed plasticity of the caspase subsites agrees very well with the reported cleavage of many proteins at noncanonical sites. The results imply that factors other than the P4-P1 sequence, such as exosites, contribute to the in vivo substrate specificity of caspases. The novel peptide binding site identified on the molecular surface of the current structures is suggested to be an exosite of caspase-7. These results should be considered in the design of selective small molecule inhibitors of this pharmacologically important protease.     

Chimeric Protein Engineering

Protein stability can be enhanced by the incorporation of non-natural amino acids and semi-rigid peptidomimetics to lower the entropic penalty upon protein folding through preorganization. An example is the incorporation of aminoisobutyric acid (Aib, α-methylalanine) into proteins to restrict the Φ and Ψ backbone angles adjacent to Aib to those associated with helix formation. Reverse-turn analogs were introduced into the sequences of HIV protease and ribonuclease A that enhanced their stability and retained their native enzymatic activity. In this work, a chimeric protein, design_4, was engineered, in silico, by replacing the C-terminal helix of full sequence design protein (FSD-1) with a semi-rigid helix mimetic. Residues 1–16 of FSD-1 was ligated in silico with the N-terminus of a phenylbipyridyl-based helix mimetic to form design_4. The designed chimeric protein was stable and maintained the designed fold in a 100-nanosecond molecular dynamics simulation at 280 K. Its β-hairpin adopted conformations that formed three additional hydrogen bonds. Compared to FSD-1, design_4 contained fewer peptide bonds and internal degrees of freedom; it should, therefore, be more resistant to proteolytic degradation and denaturation.