Title: Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs
Abstract: Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins. Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins. Designed ankyrin repeat proteins (DARPins) are a class of antibody mimetics that have been conceived and developed about two decades ago at the University of Zurich (1Forrer P. Stumpp M.T. Binz H.K. Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins.FEBS Lett. 2003; 539: 2-6Google Scholar, 2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar, 3Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. High-affinity binders selected from designed ankyrin repeat protein libraries.Nat. Biotechnol. 2004; 22: 575-582Google Scholar). Their application as research tool and protein therapeutic was recently reviewed (4Boersma Y.L. Protein scaffolds, design, synthesis, and applications.Methods Mol. Biol. 2018; 1798: 307-327Google Scholar). Originally devised as an alternative to immunoglobulins (“antibodies”), the potential of DARPins in protein engineering, directed evolution of binders, and drug development became obvious immediately at inception. Importantly, this potential extends beyond areas of applications that have classically been “occupied” by recombinant immunoglobulins. The DARPin scaffold was shown to serve as an alternative (5Foord E. Klynning C. Schoutrop E. Förster J.M. Krieg J. Mörtberg A. Müller M.R. Herzog C. Schiegg D. Villemagne D. Fiedler U. Snell D. Kebble B. Mattsson J. Levitsky V. Uhlin M. Profound functional suppression of tumor-infiltrating T-cells in ovarian cancer patients can be reversed using PD-1-blocking antibodies or DARPin proteins.J. Immunol. Res. 2020; 2020: 7375947Google Scholar), as a complementation, (6Akbari V. Chou C.P. Abedi D. New insights into affinity proteins for HER2-targeted therapy: Beyond trastuzumab.Biochim. Biophys. Acta Rev. Cancer. 2020; 1874: 188448Google Scholar) and as an expansion of what is possible with binders derived from immunoglobulins (7Münch R.C. Muth A. Muik A. Friedel T. Schmatz J. Dreier B. Trkola A. Plückthun A. Büning H. Buchholz C.J. Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors.Nat. Commun. 2015; 6: 6246Google Scholar, 8Plückthun A. Designed ankyrin repeat proteins (DARPins): Binding proteins for research, diagnostics, and therapy.Annu. Rev. Pharmacol. Toxicol. 2015; 55: 489-511Google Scholar). Translation of academic research in DARPin technology toward pharmaceutical benefits has been predominantly steered by Molecular Partners, who provided the fundamental clinical validation of the scaffold (9Stumpp M.T. Dawson K.M. Binz H.K. Beyond antibodies: The DARPin drug platform.BioDrugs. 2020; 34: 423-433Google Scholar). However, in light of the long generation cycles in drug development—especially in the case of biologics that typically require 10 years from concept to drug approval—the DARPin technology can still be regarded as young and emerging, and the full potential of DARPins as a class of biologics has yet to be realized. The recent development of ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar), a multi-specific anti-SARS-CoV-2 DARPin, which has entered clinical trials in November 2020 in less than 9 months after initial research and development activities had commenced, reinforces this high potential (11Walser M. Rothenberger S. Hurdiss D.L. Schlegel A. Calabro V. Fontaine S. Villemagne D. Paladino M. Hospodarsch T. Neculcea A. Cornelius A. Schildknecht P. Matzner M. Hänggi M. Franchini M. et al.Highly potent anti-SARS-CoV-2 multi-DARPin therapeutic candidates.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.08.25.256339Google Scholar, 12Rothenberger S. Walser M. Malvezzi F. Mayor J. Ryter S. Moreno H. Liechti N. Hälg S. Bosshart A. Iss C. Calabro V. Cornelius A. Hospodarsch T. Neculcea A. Looser T. et al.Multispecific DARPin therapeutics demonstrate very high potency against SARS-CoV-2 variants in vitro.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.02.03.429164Google Scholar). DARPins are based on natural ankyrin repeat proteins (13Li J. Mahajan A. Tsai M.D. Ankyrin repeat: A unique motif mediating protein-protein interactions.Biochemistry. 2006; 45: 15168-15178Google Scholar) that have evolved to mediate various kinds of protein-protein interactions in all kingdoms of life (14Mosavi L.K. Cammett T.J. Desrosiers D.C. Peng Z.Y. The ankyrin repeat as molecular architecture for protein recognition.Protein Sci. 2004; 13: 1435-1448Google Scholar). Their structure is simpler than that of immunoglobulins. Immunoglobulins naturally consist of four polypeptide chains and unite more than four chains in recombinant formats like T-cell bispecifics (15Klein C. Schaefer W. Regula J.T. Dumontet C. Brinkmann U. Bacac M. Umaña P. Engineering therapeutic bispecific antibodies using CrossMab technology.Methods. 2019; 154: 21-31Google Scholar), whereas a single polypeptide chain is sufficient to form a multispecific DARPin (16Binz H.K. Bakker T.R. Phillips D.J. Cornelius A. Zitt C. Göttler T. Sigrist G. Fiedler U. Ekawardhani S. Dolado I. Saliba J.A. Tresch G. Proba K. Stumpp M.T. Design and characterization of MP0250, a tri-specific anti-HGF/anti-VEGF DARPin drug candidate.MAbs. 2017; 9: 1262-1269Google Scholar). For example, ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar) combines five DARPin domains on a single polypeptide chain, in which two domains bind human serum albumin (HSA) and three domains associate with the SARS-CoV-2 spike protein (11Walser M. Rothenberger S. Hurdiss D.L. Schlegel A. Calabro V. Fontaine S. Villemagne D. Paladino M. Hospodarsch T. Neculcea A. Cornelius A. Schildknecht P. Matzner M. Hänggi M. Franchini M. et al.Highly potent anti-SARS-CoV-2 multi-DARPin therapeutic candidates.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.08.25.256339Google Scholar). DARPins are built from solenoid protein domains, which possess a modular architecture that was derived by a consensus design approach (2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar, 17Mosavi L.K. Minor D.L. Peng Z.Y. Consensus-derived structural determinants of the ankyrin repeat motif.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16029-16034Google Scholar, 18Forrer P. Binz H.K. Stumpp M.T. Plückthun A. Consensus design of repeat proteins.ChemBioChem. 2004; 5: 183-189Google Scholar): a stack of internal ankyrin repeats, each composed of 33 amino acids, flanked by N- and C-terminal capping repeats (N- and C-Caps) that function to seal the hydrophobic core of the protein domain (Fig. 1). Together, these structural units form an elongated ankyrin repeat domain. Amino acids present at defined positions at the surface of the internal repeats form a paratope, enabling the binding to target proteins with high affinity and specificity (3Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. High-affinity binders selected from designed ankyrin repeat protein libraries.Nat. Biotechnol. 2004; 22: 575-582Google Scholar, 19Cheung L.S. Kanwar M. Ostermeier M. Konstantopoulos K. A hot-spot motif characterizes the interface between a designed ankyrin-repeat protein and its target ligand.Biophys. J. 2012; 102: 407-416Google Scholar, 20Zahnd C. Wyler E. Schwenk J.M. Steiner D. Lawrence M.C. McKern N.M. Pecorari F. Ward C.W. Joos T.O. Plückthun A. A designed ankyrin repeat protein evolved to picomolar affinity to Her2.J. Mol. Biol. 2007; 369: 1015-1028Google Scholar). These positions are randomized in DARPin libraries, which are used as starting point for in vitro selection, most prominently by means of ribosome display (21Hanes J. Plückthun A. In vitro selection and evolution of functional proteins by using ribosome display.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4937-4942Google Scholar), to generate highly specific target-binding molecules. Originally, the N- and C-Caps of DARPins were taken from the human guanine-adenine-binding protein (hGABP_beta1) as they could be adapted to fit to the consensus-designed internal repeats (2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar). Such original N- and C-Caps are also present in the first DARPin that became clinically validated abicipar pegol. Despite the clinical validation, most of the amino acid sequence of these original caps was not optimized, indicating that there may be room for further improvements, in particular, for those that increase DARPin thermostability. In general, we identify three major motivations that fuel the quest for increased thermostability of biologics: (i) reduction of aggregation and thus reduction of immunogenicity risk, (ii) simplification of chemistry, manufacturing, and controls processes, thus bringing down the manufacturing costs and reduction of cold chain drug storage requirements, and (iii) increase of degrees of freedom for protein engineering to allow for mode-of-action design, for example, advanced multispecificity (11Walser M. Rothenberger S. Hurdiss D.L. Schlegel A. Calabro V. Fontaine S. Villemagne D. Paladino M. Hospodarsch T. Neculcea A. Cornelius A. Schildknecht P. Matzner M. Hänggi M. Franchini M. et al.Highly potent anti-SARS-CoV-2 multi-DARPin therapeutic candidates.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.08.25.256339Google Scholar), receptor fine-tuning (22Mohan K. Ueda G. Kim A.R. Jude K.M. Fallas J.A. Guo Y. Hafer M. Miao Y. Saxton R.A. Piehler J. Sankaran V.G. Baker D. Garcia K.C. Topological control of cytokine receptor signaling induces differential effects in hematopoiesis.Science. 2019; 364eaav7532Google Scholar), and proximity-based activation (23Lajoie M.J. Boyken S.E. Salter A.I. Bruffey J. Rajan A. Langan R.A. Olshefsky A. Muhunthan V. Bick M.J. Gewe M. Quijano-Rubio A. Johnson J. Lenz G. Nguyen A. Pun S. Correnti C.E. Riddell S.R. Baker D. Designed protein logic to target cells with precise combinations of surface antigens.Science. 2020; 369: 1637-1643Google Scholar). For the thermostability of DARPins, the importance of the capping repeats—particularly the C-Cap—was first shown by Interlandi et al. (24Interlandi G. Wetzel S.K. Settanni G. Plückthun A. Caflisch A. Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.J. Mol. Biol. 2008; 375: 837-854Google Scholar). Seven point mutations, five of which are located at the interface to the preceding internal repeat and two at the very C-terminus, were introduced to optimize the C-Cap and were shown to increase the Tm of a model DARPin, consisting of an N-Cap, a single full-consensus repeat and a C-Cap by about 17 °C; that is, from 60 °C (wt) to 77 °C (the respective C-Cap variant was referred to as the “mut5” C-Cap) (24Interlandi G. Wetzel S.K. Settanni G. Plückthun A. Caflisch A. Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.J. Mol. Biol. 2008; 375: 837-854Google Scholar). When this improved mut5 C-Cap was compared with the original C-Cap (2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar), a rigid-body movement of the C-Cap toward the internal repeat was observed, as evidenced by crystallographic data (25Kramer M.A. Wetzel S.K. Plückthun A. Mittl P.R. Grütter M.G. Structural determinants for improved stability of designed ankyrin repeat proteins with a redesigned C-capping module.J. Mol. Biol. 2010; 404: 381-391Google Scholar). This movement results in an increased buried surface area and a superior complementarity of the interface between the internal repeat and the C-Cap, which explains the improved thermostability. Although the C-Cap of DARPins was thoroughly investigated, we are not aware of any scientific article describing a corresponding analysis or thermostability improvement of the original N-Cap (denoted as “N01” N-Cap in the following, (Fig. 2)), which is still predominantly used by the research community. Nevertheless, thermostability improvements of the N-Cap have been published in the patent application WO2012/069655 (WO′655) and are clinically validated in the aHSA domains of ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar). The corresponding N-Cap of WO′655 is denoted as “N02” in the following (Fig. 2). Comparing N02 with the original N-Cap, N01, resulted in a Tm increase of approximately 7 °C. It is likely that this improvement mainly arises from the Met24Leu-mutation present in N02, which removes the only methionine (besides the methionine encoded by the start codon) from the original DARPin sequence, and thereby also removes this hotspot for oxidation. The N-Cap analysis of WO′655 was limited to the RILMAN sequence motif around Met24 of N01. Here, we set out to improve the thermostability of DARPins through engineering the N-Cap. Through in silico analyses and high-temperature unfolding experiments at equilibrium, we identify N-Cap Asp17 as an Achilles heel of DARPin domains. Molecular dynamics (MD) simulations and MD trajectory analysis provide an explanation for the significantly increased Tm values observed upon Asp17 replacement in DARPin domains. We chose a three repeat DARPin domain (denoted as N1C in the following) consisting of one full-consensus internal repeat (IR) flanked by an N- and C-Cap as a model DARPin to screen for improved thermostability (see Table 1 for an overview of the different DARPin domains used, as well as Table S1 for their respective sequences). The choice of this DARPin model has a 3-fold motivation. First, it has the minimal DARPin architecture consisting of only three repeats with two repeat interfaces, one between the N-Cap and the IR and one between the IR and the C-Cap. Second, using a consensus IR that represents an “average structure” of all the natural ankyrin repeats should eliminate interferences originating from amino acids at randomized positions or possible framework mutations, that may only be present in particular sequences (18Forrer P. Binz H.K. Stumpp M.T. Plückthun A. Consensus design of repeat proteins.ChemBioChem. 2004; 5: 183-189Google Scholar). Third, because DARPins get more stable with increasing number of IRs, we choose to have only one internal repeat and thus a low starting thermostability (26Wetzel S.K. Settanni G. Kenig M. Binz H.K. Plückthun A. Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins.J. Mol. Biol. 2008; 376: 241-257Google Scholar) such that stability improvements are readily observable.Table 1Description of DARPin domains used in the present study with varying N- and C-Caps, as detailed in the textDomain nameDescriptionN1CDARPin domain with one full consensus IR corresponding to NI1C of Interlandi et al. (24Interlandi G. Wetzel S.K. Settanni G. Plückthun A. Caflisch A. Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.J. Mol. Biol. 2008; 375: 837-854Google Scholar) & Wetzel et al. (26Wetzel S.K. Settanni G. Kenig M. Binz H.K. Plückthun A. Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins.J. Mol. Biol. 2008; 376: 241-257Google Scholar).aHER2DARPin domain based on an anti-HER2 DARPin domain corresponding to H10-2-G3 (“G3”; Zahnd et al. (20Zahnd C. Wyler E. Schwenk J.M. Steiner D. Lawrence M.C. McKern N.M. Pecorari F. Ward C.W. Joos T.O. Plückthun A. A designed ankyrin repeat protein evolved to picomolar affinity to Her2.J. Mol. Biol. 2007; 369: 1015-1028Google Scholar)).aVEGFDARPin domain based on an anti-VEGF-A DARPin domain of the protein moiety of abicipar pegol (28abiciparum pegolum: WHO Drug Information. Vol. 27. WHO, Geneva, Switzerland2013: 276Google Scholar).aHSADARPin domain based on the anti-HSA DARPin domain of ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar). It possesses an improved C-Cap similar to the mut5 C-Cap described by Interlandi et al. (24Interlandi G. Wetzel S.K. Settanni G. Plückthun A. Caflisch A. Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.J. Mol. Biol. 2008; 375: 837-854Google Scholar).For a comprehensive list of amino acid sequences of all domain variants used, see Table S1. Open table in a new tab For a comprehensive list of amino acid sequences of all domain variants used, see Table S1. By visual analysis of the DARPin structure, we identified four residues within the N-Cap to be of potential importance for the repeat stacking and thereby also for the overall domain stability. These residues are either at the edge (Leu4, Gly5, and Asp17) of the N-Cap or buried (Met24) at the interface between N-Cap and the adjacent IR (Fig. 3). As WO′655 already demonstrated that a Met24Leu mutation strongly improves the thermostability of DARPins, we focused our analysis on Leu4, Gly5, and Asp17 on an N1C background comprising the N02 N-Cap to find out if it is possible to further improve the most stable N-Cap known to date. Alanine scanning of residues four and five showed no improvement in thermostability, with Leu4Ala lowering the Tm value from 74.5 °C to 64.7 °C and Gly5Ala leaving the melting temperature unaltered at 74.3 °C. However, the Asp17Ala mutation showed a strong improvement of the Tm value from 74.5 °C to 82.4 °C (Fig. 4A). Consequently, we screened alternative amino acids at the N-Cap position 17 of N1C (Table 2 and Fig. 4B). All amino acid substitutions tested (excluding e.g., Cys, Trp, and Gly, that would not make sense from the point of protein engineering) resulted in a Tm increase, with the highest Tm increases being measured for the Asp17Val, Asp17Ile, and Asp17Leu variants (i.e., from 74.5 °C to 85.1 °C, 84.8 °C, and 84.6 °C, respectively). Overall, changing Asp17 in N1C to Val, Leu, Ile, Met, Ala, or Thr led to an increase of the respective Tm values between 8 °C to 10 °C. These results show that Asp is an exceptionally unfavorable amino acid at position 17, and that there are many alternative residues resulting in a strong thermostability gain. Of these alternative residues, Asp17Leu provided one of the largest improvements, which we investigated further.Figure 4Thermal unfolding of DARPin domains followed by CD spectroscopy between 40 °C and 95 °C; all variants were measured at a concentration of 10 μM in PBS. A, N1C_v01 to N1C_v04: The measured T m values were 74.5 °C for the control N1C (N1C_v01), 64.7 °C for the N-Cap Leu4Ala mutant (N1C_v02), 74.3 °C for the N-Cap Gly5Ala mutant (N1C_v03), and 82.4 °C for the N-Cap Asp17Ala mutant (N1C_v04). B, the Asp17Leu mutant (N1C_v05) has a T m of 84.6 °C and is strongly stabilized compared with N1C_v01 containing Asp17. DARPin, designed ankyrin repeat protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2Tm values of N1C variants having various amino acids at position 17NamePosition 17Tm (°C)N1C_v01D74.5N1C_v04A82.4N1C_v05L84.6N1C_v06V85.1N1C_v07M83.8N1C_v08I84.8N1C_v09T82.3N1C_v10S79.3N1C_v11N75.2N1C_v12Q77.4N1C_v13K77.9N1C_v14R78.3N1C_v15E79.2 Open table in a new tab To test if the improvements derived from mutating the N-Cap Asp17 are generic and independent of the N02 background, we transferred the Asp17Leu mutation onto the original N01 N-Cap and the N03 N-Cap that differs in nine amino acids from N02. The Asp17Leu mutation improved the thermostability of N1C also in the N01 and N03 backgrounds by more than 13 °C (Table 3). Further, we were interested in whether our observed stability improvement based on the N-Cap and the stability improvement based on the mut5 C-Cap (24Interlandi G. Wetzel S.K. Settanni G. Plückthun A. Caflisch A. Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.J. Mol. Biol. 2008; 375: 837-854Google Scholar) would be additive. We first found that replacing the wt C-Cap in N1C with a mut5 C-Cap results in a Tm increase of about 13 °C or 9 °C in an N01 or N02 background, respectively (Table 4), thus confirming the benefits of the mut5 C-Cap (24Interlandi G. Wetzel S.K. Settanni G. Plückthun A. Caflisch A. Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.J. Mol. Biol. 2008; 375: 837-854Google Scholar). In addition, the combination of the N02 N-Cap with the mut5 C-Cap in N1C_v22 proved that the individual improvements of each cap are additive and raised the Tm value to 84.2 °C in PBS, that is, by about 22 °C. With the additional substitution of Asp17Leu in N1C_v23 (N02, mut5 background), we did not observe any unfolding transition up to 95 °C when we measured the thermal unfolding of this molecule in PBS. Therefore, we repeated the measurements for N1C_v22 and N1C_v23 in a buffer containing 2 M GdmCl and obtained corresponding Tm values of 67.3 °C and 79.3 °C, respectively (Table 4). Thus, the already very thermostable N1C_v22, comprising N02 and the mut5 C-Cap, could be further stabilized by adding the Asp17Leu mutation to its N-Cap resulting in a Tm gain of about 12 °C in 2 M GdmCl. Overall, the Asp17Leu mutation adds about 9 °C to 14 °C to the Tm value of N1C independent of its concrete N- and/or C-Cap, indicating that this is a general improvement for DARPin domains.Table 3Tm values of N1C variants having either Asp or Leu at position 17 of the N-Cap in N01, N02, or N03 backgroundsNameN-CapC-CapTm (°C)N1C_v01N02wt74.5N1C_v05N02_D17Lwt84.6N1C_v16N01wt62.1N1C_v17N01_D17Lwt75.2N1C_v19N03wt68.6N1C_v20N03_D17Lwt82.8 Open table in a new tab Table 4Tm values of N1C variants having either Asp or Leu at position 17 of the N-Cap in wt and mut5 C-Cap backgroundsNameN-CapC-CapTm (°C)TmaIndicates Tm measurements in 2 M GdmCl. (°C)N1C_v16N01wt62.1NDN1C_v25N01mut574.6NDN1C_v01N02wt74.5NDN1C_v22N02mut584.267.3N1C_v23N02_D17Lmut5ND79.3Abbreviation: ND, not determined.a Indicates Tm measurements in 2 M GdmCl. Open table in a new tab Abbreviation: ND, not determined. We performed MD simulations to investigate the structural implications for the N1C variants having either Asp or Leu at position 17 of their N-Caps. Starting from the X-ray diffraction structure of ankyrin repeat proteins of E3_5, NI1C-mut4, and NI3C-mut5 (PDB ID: 1MJ0 (27Kohl A. Binz H.K. Forrer P. Stumpp M.T. Plückthun A. Grütter M.G. Designed to be stable: Crystal structure of a consensus ankyrin repeat protein.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1700-1705Google Scholar), 2XEN (25Kramer M.A. Wetzel S.K. Plückthun A. Mittl P.R. Grütter M.G. Structural determinants for improved stability of designed ankyrin repeat proteins with a redesigned C-capping module.J. Mol. Biol. 2010; 404: 381-391Google Scholar), and 2XEE (25Kramer M.A. Wetzel S.K. Plückthun A. Mittl P.R. Grütter M.G. Structural determinants for improved stability of designed ankyrin repeat proteins with a redesigned C-capping module.J. Mol. Biol. 2010; 404: 381-391Google Scholar), respectively), we prepared six different homology models, each time comparing Asp17 with Leu17 constructs in the N01-background (N1C_v16 and N1C_v17, respectively), the N02-background (N1C_v01 and N1C_v05, respectively), and the N02-background combined with the mut5 C-Cap (N1C_v22 and N1C_v23, respectively) (see Table S1). Of note, the numbering in PDBs may be different for different DARPins, which is because of the fact that some PDB structures' counting might for example, include N-terminal tags like the MRGSH6-tag used for purification. In the above mentioned PDB ID entries, amino acid numbers corresponding to the N-Cap position 17 are #27 for 1MJ0 and #15 for 2XEN and 2XEE. Starting from the homology models, three independent simulations were carried out for each system, two at 350 K and one at 400 K, for a total sampling of 1.8 μs. Three conclusions can be drawn from the MD simulations (Fig. 5 and Table 5). First, the substitution of Asp at position 17 with Leu leads in all instances to improved interaction energies with the surrounding (Table 5). Second, the analysis focused on the protein flexibility at high temperatures (400 K) and on two different timescales (5 ns and 150 ns) revealed that the systems containing Leu at position 17 systematically show lower fluctuations than their Asp17 counterparts (Fig. 5). The effects of the Asp17Leu mutation are more pronounced on the 150-ns timescale (Fig. 5B) than on the 5-ns timescale (Fig. 5A). The profiles along the full protein sequences show the least fluctuations in the helical segments and the highest flexibility at the loops. The higher rigidity of the helical segments of the Leu17 mutants (lower fluctuations) is relevant for enthalpic stabilization. The differences in the fluctuations of the loops are less relevant as their flexibility contributes to entropic stabilization. The reduced flexibility of the Leu17 mutants as compared with Asp17 is in line with the increased thermostability of Asp17Leu DARPins observed in CD. Third, on the longer timescale, the Asp17Leu mutation reduces fluctuations in the N01-background (N1C_v16 versus N1C_v17) across the entire N-Cap and in one of the most flexible parts of a DARPin domain spanning from the end of the N-Cap (GADVNA motif, residues 27–32 (Fig. 2)) to the β-turn at the beginning of the internal repeat. In the N02-background (N1C_v01 versus N1C_v05) and the mut5 C-Cap (N1C_v22 versus N1C_v23), reduced fluctuations through the Asp17Leu mutation are more pronounced in the direct v