Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum ( Pf A-M1) and Plasmodium vivax ( Pv A-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets Pf A-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on Pf A-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of Pf A-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

On-target, dual aminopeptidase inhibition provides cross-species antimalarial activity.

To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum, is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817-resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817, and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial.

Structure-based development of potent Plasmodium falciparum M1 and M17 aminopeptidase selective and dual inhibitors via S1'-region optimisation.

Malaria remains a global health threat and growing resistance to artemisinin-based therapies calls for therapeutic agents with novel mechanisms of action. The Plasmodium spp M1 and M17 metalloaminopeptidases have been identified as attractive new antimalarial drug targets as inhibition of these enzymes results in antiplasmodial activity. Previously identified novel hydroxamic acid 2 as a moderate inhibitor of PfA-M1 and PfA-M17 and a potent inhibitor of P. falciparum. This study has sought to improve the enzymatic inhibitory properties in addition to increasing the drug-likeness of this scaffold by introducing polar moieties into the S1' region of the active site. Structural biology studies on the co-crystallised structures of potent dual-inhibitor 9aa bound to PfA-M1 and PfA-M17 have revealed that there are few direct interactions between the inhibitor and the S1' domain of these enzymes. Structure-based compound design led to the identification of a variety of novel hydroxamic acids that show improved inhibitory activity against PfA-M1 and PfA-M17, in addition to displaying antiplasmodial activity. Notably, compounds with substitutions on the aniline ring resulted in a loss of potency (Ki > 500 nM) toward PfA-M1 and PfA-M17. ioisosteric replacement of the S1-region biaryl ring system with a bromophenyl moiety resulted in increased potency compared to parent 9aa. Elaboration of 9aa to bioisosterically replace the S1 moiety with an aryl bromide, combined with substituted anilines has resulted in potent selective PfA-M1 inhibitors which show strong activity against Pf-3D7, with meta- and para-fluoroaniline groups of 15ag and 15ah forming hydrogen-bonds with residues within the active site. These findings establish the importance of the previously under-utilised S1' domain and will aid the design of future PfA-M1 and PfA-M17 inhibitors.

A metal ion-dependent conformational switch modulates activity of the Plasmodium M17 aminopeptidase.

The metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. This large enzyme family is characterized by a conserved binuclear metal center and a distinctive homohexameric arrangement. Recently, we showed that hexamer formation in Plasmodium M17 aminopeptidases was controlled by the metal ion environment, although the functional necessity for hexamer formation is still unclear. To further understand the mechanistic role of the hexameric assembly, here we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from Plasmodium falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket, rendering the protein inactive. Molecular dynamics simulations and biochemical analyses of PfA-M17 variants demonstrated that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is metal dependent and part of a dynamic regulatory mechanism. Key to the mechanism is a remodeling of the binuclear metal center, which occurs in response to a signal from the neighboring active site and serves to moderate the rate of proteolysis under different environmental conditions. In conclusion, this work identifies a precise mechanism by which oligomerization contributes to PfA-M17 function. Furthermore, it describes a novel role for metal cofactors in the regulation of enzymes, with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center, including DNA processing enzymes and metalloproteases.

Mapping the substrate specificity of the Plasmodium M1 and M17 aminopeptidases.

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. Overall, the P. vivax and P. berghei enzymes had a faster substrate turnover rate than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

High avidity drives the interaction between the streptococcal C1 phage endolysin, PlyC, with the cell surface carbohydrates of Group A Streptococcus.

Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC is known to bind the Group A streptococcal cell wall, but the specific molecular target or the binding site within PlyC remain uncharacterized. Here we report for the first time, that the polyrhamnose backbone of the Group A streptococcal cell wall is the binding target of PlyC. We have also characterized the putative rhamnose binding groove of PlyC and found four key residues that were critical to either the folding or the cell wall binding action of PlyC. Based on our results, we suggest that the interaction between PlyC and the cell wall may not be a high-affinity interaction as previously proposed, but rather a high avidity one, allowing for PlyC's remarkable lytic activity. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop the antibacterial agents with new modes of action. A detailed understanding of this potent endolysin may facilitate future developments of PlyC as a tool against the rise of antibiotic resistance.

A Structure-Activity Relationship Study of Novel Hydroxamic Acid Inhibitors around the S1 Subsite of Human Aminopeptidase N.

Aminopeptidase N (APN/CD13) is a zinc-dependent ubiquitous transmembrane ectoenzyme that is widely present in different types of cells. APN is one of the most extensively studied metalloaminopeptidases as an anti-cancer target due to its significant role in the regulation of metastasis and angiogenesis. Previously, we identified a potent and selective APN inhibitor, N-(2-(Hydroxyamino)-2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide (3). Herein, we report the further modifications performed to explore SAR around the S1 subsite of APN and to improve the physicochemical properties. A series of hydroxamic acid analogues were synthesised, and the pharmacological activities were evaluated in vitro. N-(1-(3'-Fluoro-[1,1'-biphenyl]-4-yl)-2-(hydroxyamino)-2-oxoethyl)-4-(methylsulfonamido)benzamide (6 f) was found to display an extremely potent inhibitory activity in the sub-nanomolar range.

Active site metals mediate an oligomeric equilibrium in Plasmodium M17 aminopeptidases.

M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and Plasmodium vivax (Pv-M17) function as catalytically active hexamers to generate free amino acids from human hemoglobin and are drug targets for the design of novel antimalarial agents. However, the molecular basis for oligomeric assembly is not fully understood. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species that can be controlled by manipulating the identity and concentration of metals available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of Pv-M17 by cryoelectron microscopy and X-ray crystallography together with solution studies revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of these studies, we propose a dynamic equilibrium between monomer ↔ dimer ↔ tetramer ↔ hexamer, which becomes directional toward the large oligomeric states with the addition of metal ions. This sophisticated metal-dependent dynamic equilibrium may apply to other M17 aminopeptidases and underpin the moonlighting capabilities of this enzyme family.

X-ray crystal structure and specificity of the Toxoplasma gondii ME49 TgAPN2.

Toxoplasmosis is a parasitic disease caused by infection with Toxoplasma gondii that currently has few therapeutic options. The M1 aminopeptidase enzymes have been shown to be attractive targets for anti-parasitic agents and/or vaccine candidates, suggesting potential to re-purpose inhibitors between parasite M1 aminopeptidase targets. The M1 aminopeptidase TgAPN2 has been suggested to be a potential new drug target for toxoplasmosis. Here we investigate the structure and function of TgAPN2, a homologue of the antimalarial drug target PfA-M1, and evaluate the capacity to use inhibitors that target PfA-M1 against TgAPN2. The results show that despite a similar overall fold, the TgAPN2 has a unique substrate specificity and inhibition profile. Sequence and structure differences are investigated and show how comparative structure-activity relationships may provide a route to obtaining potent inhibitors of TgAPN2.

Cephamycins inhibit pathogen sporulation and effectively treat recurrent Clostridioides difficile infection.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.

Novel Human Aminopeptidase N Inhibitors: Discovery and Optimization of Subsite Binding Interactions.

Aminopeptidase N (APN/CD13) is a zinc-dependent M1 aminopeptidase that contributes to cancer progression by promoting angiogenesis, metastasis, and tumor invasion. We have previously identified hydroxamic acid-containing analogues that are potent inhibitors of the APN homologue from the malarial parasite Plasmodium falciparum M1 aminopeptidase (PfA-M1). Herein, we describe the rationale that underpins the repurposing of PfA-M1 inhibitors as novel APN inhibitors. A series of novel hydroxamic acid analogues were developed using a structure-based design approach and evaluated their inhibition activities against APN. N-(2-(Hydroxyamino)-2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide (6ad) proved to be an extremely potent inhibitor of APN activity in vitro, selective against other zinc-dependent enzymes such as matrix metalloproteases, and possessed limited cytotoxicity against Ad293 cells and favorable physicochemical and metabolic stability properties. The combined results indicate that compound 6ad may be a useful lead for the development of anticancer agents.

Identification of the Binding Site of Apical Membrane Antigen†1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

M17 aminopeptidases diversify function by moderating their macromolecular assemblies and active site environment.

The family of M17 aminopeptidases (alias 'leucine aminopeptidases', M17-LAPs) utilize a highly conserved hexameric structure and a binuclear metal center to selectively remove N-terminal amino acids from short peptides. However, M17-LAPs are responsible for a wide variety of functions that are seemingly unrelated to proteolysis. Herein, we aimed to investigate the myriad of functions attributed to M17. Further, we attempted to differentiate between the different molecular mechanisms that allow the conserved hexameric structure of an M17-LAP to mediate such diverse functions. We have provided an overview of research that identifies precise physiological roles of M17-LAPs, and the distinct mechanisms by which the enzymes moderate those roles. The review shows that the conserved hexameric structure of the M17-LAPs has an extraordinary capability to moderate different molecular mechanisms. We have broadly categorized these mechanisms as 'aminopeptidase-based', which include the characteristic proteolysis reactions, and 'association-driven', which involves moderation of the molecule's macromolecular assembly and higher order complexation events. The different molecular mechanisms are capable of eliciting very different cellular outcomes, and must be regarded as distinct when the physiological roles of this large and important family are considered.

Hydroxamic Acid Inhibitors Provide Cross-Species Inhibition of Plasmodium M1 and M17 Aminopeptidases.

There is an urgent clinical need for antimalarial compounds that target malaria caused by both Plasmodium falciparum and Plasmodium vivax. The M1 and M17 metalloexopeptidases play key roles in Plasmodium hemoglobin digestion and are validated drug targets. We used a multitarget strategy to rationally design inhibitors capable of potent inhibition of the M1 and M17 aminopeptidases from both P. falciparum ( Pf-M1 and Pf-M17) and P. vivax ( Pv-M1 and Pv-M17). The novel chemical series contains a hydroxamic acid zinc binding group to coordinate catalytic zinc ion/s, and a variety of hydrophobic groups to probe the S1' pockets of the four target enzymes. Structural characterization by cocrystallization showed that selected compounds utilize new and unexpected binding modes; most notably, compounds substituted with bulky hydrophobic substituents displace the Pf-M17 catalytic zinc ion. Excitingly, key compounds of the series potently inhibit all four molecular targets and show antimalarial activity comparable to current clinical candidates.

Thermal sensitivity and flexibility of the Cε3 domains in immunoglobulin E.

Immunoglobulin E (IgE) is the antibody that plays a central role in the mechanisms of allergic diseases such as asthma. Interactions with its receptors, FcεRI on mast cells and CD23 on B cells, are mediated by the Fc region, a dimer of the Cε2, Cε3 and Cε4 domains. A sub-fragment lacking the Cε2 domains, Fcε3-4, also binds to both receptors, although receptor binding almost exclusively involves the Cε3 domains. This domain also contains the N-linked glycosylation site conserved in other isotypes. We report here the crystal structures of IgE-Fc and Fcε3-4 at the highest resolutions yet determined, 1.75Šand 2.0Šrespectively, revealing unprecedented detail regarding the carbohydrate and its interactions with protein domains. Analysis of the crystallographic B-factors of these, together with all earlier IgE-Fc and Fcε3-4 structures, shows that the Cε3 domains exhibit the greatest intrinsic flexibility and quaternary structural variation within IgE-Fc. Intriguingly, both well-ordered carbohydrate and disordered polypeptide can be seen within the same Cε3 domain. A simplified method for comparing the quaternary structures of the Cε3 domains in free and receptor-bound IgE-Fc structures is presented, which clearly delineates the FcεRI and CD23 bound states. Importantly, differential scanning fluorimetric analysis of IgE-Fc and Fcε3-4 identifies Cε3 as the domain most susceptible to thermally-induced unfolding, and responsible for the characteristically low melting temperature of IgE.

Crystal structure of a ß-aminopeptidase from an Australian Burkholderia sp.

ß-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal ß-amino acids from synthetic ß-peptides. ß-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of ß-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a ß-aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0 Šand showed a tetrameric assembly typical of the ß-aminopeptidases. Each monomer consists of an α-subunit (residues 1-238) and a ß-subunit (residues 239-367). Comparison of the structure of BcA5-BapA with those of other known ß-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.

Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2.

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.

M1 aminopeptidases as drug targets: broad applications or therapeutic niche?

M1 aminopeptidase enzymes are a diverse family of metalloenzymes characterized by conserved structure and reaction specificity. Excluding viruses, M1 aminopeptidases are distributed throughout all phyla, and have been implicated in a wide range of functions including cell maintenance, growth and development, and defense. The structure and catalytic mechanism of M1 aminopeptidases are well understood, and make them ideal candidates for the design of small-molecule inhibitors. As a result, many research groups have assessed their utility as therapeutic targets for both infectious and chronic diseases of humans, and many inhibitors with a range of target specificities and potential therapeutic applications have been developed. Herein, we have aimed to address these studies, to determine whether the family of M1 aminopeptidases does in fact present a universal target for the treatment of a diverse range of human diseases. Our analysis indicates that early validation of M1 aminopeptidases as therapeutic targets is often overlooked, which prevents the enzymes from being confirmed as drug targets. This validation cannot be neglected, and needs to include a thorough characterization of enzymes' specific roles within complex physiological pathways. Furthermore, any chemical probes used in target validation must be carefully designed to ensure that specificity over the closely related enzymes has been achieved. While many drug discovery programs that target M1 aminopeptidases remain in their infancy, certain inhibitors have shown promise for the treatment of a range of conditions including malaria, hypertension, and cancer.

X-ray crystal structure of cytochrome P450 monooxygenase CYP101J2 from Sphingobium yanoikuyae strain B2.

The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. © 2016 Wiley Periodicals, Inc.

Circumventing the stability-function trade-off in an engineered FN3 domain.

The favorable biophysical attributes of non-antibody scaffolds make them attractive alternatives to monoclonal antibodies. However, due to the well-known stability-function trade-off, these gains tend to be marginal after functional selection. A notable example is the fibronectin Type III (FN3) domain, FNfn10, which has been previously evolved to bind lysozyme with 1 pM affinity (FNfn10-α-lys), but suffers from poor thermodynamic and kinetic stability. To explore this stability-function compromise further, we grafted the lysozyme-binding loops from FNfn10-α-lys onto our previously engineered, ultra-stable FN3 scaffold, FN3con. The resulting variant (FN3con-α-lys) bound lysozyme with a markedly reduced affinity, but retained high levels of thermal stability. The crystal structure of FNfn10-α-lys in complex with lysozyme revealed unanticipated interactions at the protein-protein interface involving framework residues of FNfn10-α-lys, thus explaining the failure to transfer binding via loop grafting. Utilizing this structural information, we redesigned FN3con-α-lys and restored picomolar binding affinity to lysozyme, while maintaining thermodynamic stability (with a thermal melting temperature 2-fold higher than that of FNfn10-α-lys). FN3con therefore provides an exceptional window of stability to tolerate deleterious mutations, resulting in a substantial advantage for functional design. This study emphasizes the utility of consensus design for the generation of highly stable scaffolds for downstream protein engineering studies.