Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope‐scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope‐scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody‐binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope‐scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV‐1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope‐scaffold that bound 2F5 with subnanomolar affinity (K_D = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope‐scaffold represents a successful example of rational protein backbone engineering and protein–protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity. Proteins 2014; 82:2770–2782. © 2014 Wiley Periodicals, Inc.     

Computational protein design using flexible backbone remodeling and resurfacing: case studies in structure-based antigen design

Computational protein design has promise for vaccine design and other applications. We previously transplanted the HIV 4E10 epitope onto non-HIV protein scaffolds for structural stabilization and immune presentation. Here, we developed two methods to optimize the structure of an antigen, flexible backbone remodeling and resurfacing, and we applied these methods to a 4E10 scaffold. In flexible-backbone remodeling, an existing backbone segment is replaced by a de novo designed segment of prespecified length and secondary structure. With remodeling, we replaced a potentially immunodominant domain on the scaffold with a helix-loop segment that made intimate contact to the protein core. All three domain trim designs tested experimentally had improved thermal stability and similar binding affinity for the 4E10 antibody compared to the parent scaffold. A crystal structure of one design had a 0.8 Å backbone RMSD to the computational model in the rebuilt region. Comparison of parent and trimmed scaffold reactivity to anti-parent sera confirmed the deletion of an immunodominant domain. In resurfacing, the surface of an antigen outside a target epitope is redesigned to obtain variants that maintain only the target epitope. Resurfaced variants of two scaffolds were designed in which 50 positions amounting to 40% of the protein sequences were mutated. Surface-patch analyses indicated that most potential antibody footprints outside the 4E10 epitope were altered. The resurfaced variants maintained thermal stability and binding affinity. These results indicate that flexible-backbone remodeling and resurfacing are useful tools for antigen optimization and protein engineering generally.     

Grafting of protein-protein interaction epitope

Transferring the biological function of one protein to another is a key issue in understanding the structure and function relationship of proteins. We have developed a strategy for grafting protein-protein interaction epitopes. As a first step, residues at the interface of the ligand protein which strongly interact with the receptor protein were identified. Then protein scaffolds were docked onto receptor protein based on geometric complementarity. Only high docking score matches were saved. For each saved match, the scaffold protein was accepted if it had suitable positions for grafting key interaction residues of the ligand protein. These candidate residues were mutated to corresponding residues in the ligand protein at each relevant position and the mutated scaffold protein was co-minimized with receptor protein. Finally, the minimized complexes were evaluated by a scoring function deduced from statistical analysis of rigid binding data sets. As a test case, the binding epitope of barstar, the inhibitor of barnase, was grafted onto smaller proteins. Pheromone Er-1 (PDB entry 1erc) has been found to be a good scaffold. The calculated binding free energy for mutated Pheromone Er-1 is equivalent to that of barstar.     

Heterologous epitope-scaffold prime:boosting immuno-focuses B cell responses to the HIV-1 gp41 2F5 neutralization determinant

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime:boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime:boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime:boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime:boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.     

DNA vaccine with discontinuous T‐cell epitope insertions into HSP65 scaffold as a potential means to improve immunogenicity of multi‐epitope Mycobacterium tuberculosis vaccine

DNA‐based vaccine is a promising candidate for immunization and induction of a T‐cell‐focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi‐functional T response against multi‐TB antigens, a multi‐epitope DNA vaccine and a ‘protein backbone grafting’ design method is adopted to graft five discontinuous T‐cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T‐cell epitopes derived from ESAT‐6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope‐scaffold construct was significantly more potent to induce IFN‐γ⁺ T response to five T‐cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi‐functional‐CD4⁺ T response. It also enhanced granzyme B⁺ CTL and IL‐2⁺ CD8⁺ T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi‐epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.     

蛋白质骨架库的构建及其在功能蛋白质设计中的应用

利用活性位点嫁接设计新的功能蛋白质的方法需要功能模体和蛋白质骨架两种构件。出于活性位点嫁接的需要,我们构建了一个蛋白质骨架库,且提供搜索与功能模体相配骨架的工具及其评估方法。并用两个活性位点的嫁接进行了检验。     

Structure-affinity relationships in the gp41 ELDKWA epitope for the HIV-1 neutralizing monoclonal antibody 2F5: effects of side-chain and backbone modifications and conformational constraints.

The human monoclonal antibody, mAb 2F5, has broad HIV-1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high-affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme-linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end-capped, linear nonapeptide, Ac-LELDKWASL-amide, was sufficient to attain maximal affinity within the set of native gp41-sequence peptides assayed. Scanning single-residue alanine and d-residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high-affinity binding of the linear nonapeptide. Further studies of side-chain and backbone-modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu(3) and Trp(6) residues. The nine-residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15-residue, disulfide-bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C-peptide helix immediately adjacent to the N-terminal end of the ELDKWA epitope were examined through the synthesis of helix-initiated analogs. Circular dichroism (CD) studies indicated that the alpha-helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV-neutralizing actions of 2F5.     

Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope

Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K_d of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.     

Complex of a protective antibody with its Ebola virus GP peptide epitope: unusual features of a V lambda x light chain

13F6-1-2 is a murine monoclonal antibody that recognizes the heavily glycosylated mucin-like domain of the Ebola virus virion-attached glycoprotein (GP) and protects animals against lethal viral challenge. Here we present the crystal structure, at 2.0 Å, of 13F6-1-2 in complex with its Ebola virus GP peptide epitope. The GP peptide binds in an extended conformation, anchored primarily by interactions with the heavy chain. Two GP residues, Gln P406 and Arg P409, make extensive side-chain hydrogen bond and electrostatic interactions with the antibody and are likely critical for recognition and affinity. The 13F6-1-2 antibody utilizes a rare Vλ_x light chain. The three light-chain complementarity-determining regions do not adopt canonical conformations and represent new classes of structures distinct from Vκ and other Vλ light chains. In addition, although Vλ_x had been thought to confer specificity, all light-chain contacts are mediated through germ-line-encoded residues. This structure of an antibody that protects against the Ebola virus now provides a framework for humanization and development of a postexposure immunotherapeutic.     

Epitope grafting, re-creating a conformational Bet v 1 antibody epitope on the surface of the homologous apple allergen Mal d 1

Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen from Betula verrucosa (Bet v 1). Still, to what extent two molecular surfaces need to be similar for clinically relevant antibody cross-reactivity to occur is unknown. Here, we describe the grafting of a defined conformational antibody epitope from Bet v 1 onto the surface of the homologous apple allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction to Bet v 1 and to the mutated Mal d 1 variant, respectively, were assessed by Biacore experiments demonstrating indistinguishable binding kinetics. This demonstrates that a conformational epitope defined by a high affinity antibody-allergen interaction can successfully be grafted onto a homologous scaffold molecule without loss of epitope functionality. Furthermore, we show that increasing surface similarity to Bet v 1 of Mal d 1 variants by substitution of 6-8 residues increased the ability to trigger basophil histamine release with blood from birch-allergic patients not responding to natural Mal d 1. Conversely, reducing surface similarity to Bet v 1 of a Mal d 1 variant by substitution of three residues abolished histamine release in one patient reacting to Mal d 1.     

DYNLL/LC8: a light chain subunit of the dynein motor complex and beyond

The LC8 family members of dynein light chains (DYNLL1 and DYNLL2 in vertebrates) are highly conserved ubiquitous eukaryotic homodimer proteins that interact, besides dynein and myosin 5a motor proteins, with a large (and still incomplete) number of proteins involved in diverse biological functions. Despite an earlier suggestion that LC8 light chains function as cargo adapters of the above molecular motors, they are now recognized as regulatory hub proteins that interact with short linear motifs located in intrinsically disordered protein segments. The most prominent LC8 function is to promote dimerization of their binding partners that are often scaffold proteins of various complexes, including the intermediate chains of the dynein motor complex. Structural and functional aspects of this intriguing hub protein will be highlighted in this minireview.     

Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.     

Multiple display of peptides and proteins on a macromolecular scaffold derived from a multienzyme complex

The acyltransferase components (E2) from the family of 2-oxo acid dehydrogenase multienzyme complexes form large protein scaffolds, to which multiple copies of peripheral enzymes bind tightly but non-covalently. Sixty copies of the E2 polypeptide from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus assemble to form a pentagonal dodecahedral scaffold with icosahedral symmetry. This protein scaffold can be modified to present foreign peptides and proteins on its surface. We show that it is possible to display two epitopes (MAL1 and MAL2) from the circumsporozoite CS proteins of Plasmodium falciparum and Plasmodium berghei, respectively, and a green fluorescent protein (EGFP), on the E2 surface. Immunization with an E2 scaffold displaying the MAL1 epitope elicited MAL1-specific antibodies in rabbits. EGFP (25 kDa) displayed as an N-terminal fusion in each of the 60 copies of the E2 chain folded into its active form, as judged by its fluorescence and detection in localized foci in Escherichia coli cells in vivo. Simultaneous display of a hexahistidine affinity tag, the MAL1 epitope and the green fluorescent protein, all on the same E2 scaffold, could be achieved by reversible denaturation and reassembly of mixtures of appropriately modified E2 chains. This new methodology offers several important advantages over other current display technologies, not least in the size of insert that can be accommodated and the multiplicity of display that can be achieved.     

Synthesis and NMR solution structure of an alpha-helical hairpin stapled with two disulfide bridges

Helical coiled-coils and bundles are some of the most common structural motifs found in proteins. Design and synthesis of alpha-helical motifs may provide interesting scaffolds that can be useful as host structures to display functional sites, thus allowing the engineering of novel functional miniproteins. We have synthesized a 38-amino acid peptide, alpha2p8, encompassing the alpha-helical hairpin present in the structure of p8MTCP1, as an alpha-helical scaffold particularly promising for its stability and permissiveness of sequence mutations. The three-dimensional structure of this peptide has been solved using homonuclear two-dimensional NMR techniques at 600 MHz. After sequence specific assignment, a total of 285 distance and 29 dihedral restraints were collected. The solution structure of alpha2p8 is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics, using simulated annealing protocol with the AMBER force field. The RMSD values for the backbone and all heavy atoms are 0.65+/-0.25 and 1.51+/-0.21 A, respectively. Excised from its protein context, the alpha-hairpin keeps its native structure: an alpha-helical coiled-coil, similar to that found in superhelical structures, with two helices spanning residues 4-16 and 25-36, and linked by a short loop. This motif is stabilized by two interhelical disulfide bridges and several hydrophobic interactions at the helix interface, leaving most of its solvent-exposed surface available for mutation. This alpha-helical hairpin, easily amenable to synthetic chemistry and biological expression system, may represent a stable and versatile scaffold to display new functional sites and peptide libraries.     

HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.     

Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity

The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize 'FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1-peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K(d)s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.     

Comparison of designed and randomly generated catalysts for simple chemical reactions

There has been recent success in designing enzymes for simple chemical reactions using a two-step protocol. In the first step, a geometric matching algorithm is used to identify naturally occurring protein scaffolds at which predefined idealized active sites can be realized. In the second step, the residues surrounding the transition state model are optimized to increase transition state binding affinity and to bolster the primary catalytic side chains. To improve the design methodology, we investigated how the set of solutions identified by the design calculations relate to the overall set of solutions for two different chemical reactions. Using a TIM barrel scaffold in which catalytically active Kemp eliminase and retroaldolase designs were obtained previously, we carried out activity screens of random libraries made to be compositionally similar to active designs. A small number of active catalysts were found in screens of 10(3) variants for each of the two reactions, which differ from the computational designs in that they reuse charged residues already present in the native scaffold. The results suggest that computational design considerably increases the frequency of catalyst generation for active sites involving newly introduced catalytic residues, highlighting the importance of interaction cooperativity in enzyme active sites.     

Computational studies on the backbone-dependent side-chain orientation induced by the (S,S)-CXC motif

Disulfide cyclization is a well-known procedure to impose conformational restriction to peptides undergoing backbone flexibility. Rigid conformations are induced only for small rings with a specific combination of amino acids. In this work, we present a computational search of the backbone and backbone-dependent side-chain orientation of two series of linear and cyclic peptide analogs. The -C[XY]C- scaffold (where X,Y is arginine, aspartic acid or alanine residue) in its open and (S,S) cyclic form was used for the design of the studied analogs. Thirty-six compounds, resulting from the extension with one residue at either the N- or the C-terminus were studied with classical MD. The local backbone conformation and the relative orientation of the X and Y side chains induced by either cyclization and/or the presence of the charged residues are discussed. From the present study it is concluded that cyclization has a great impact on the synplanar orientation of the X and Y side chains in the (S,S)Ac-XCYC-NH2 series of compounds while charge-charge interaction has only a weak synergic effect. On the contrary, the antiplanar orientation is favored in the case of (S,S)Ac-CXCY-NH2.     

Exploring new strategies for grafting binding peptides onto protein loops using a consensus‐designed tetratricopeptide repeat scaffold

Peptide display approaches, in which peptide epitopes of known binding activities are grafted onto stable protein scaffolds, have been developed to constrain the peptide in its bioactive conformation and to enhance its stability. However, peptide grafting can be a lengthy process requiring extensive computational modeling and/or optimisation by directed evolution techniques. In this study, we show that ultra‐stable consensus‐designed tetratricopeptide repeat (CTPR) proteins are amenable to the grafting of peptides that bind the Kelch‐like ECH‐associated protein 1 (Keap1) onto the loop between adjacent repeats. We explore simple strategies to optimize the grafting process and show that modest improvements in Keap1‐binding affinity can be obtained by changing the composition of the linker sequence flanking either side of the binding peptide.     

Assessment of the robustness of a serendipitous zinc binding fold: mutagenesis and protein grafting

Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.