Abstract

Introduction

Trypsin-like serine protases (TPLs) play a pivotal role in a wide range of biological functions by mediating cellular signalling through highly regulated and limited proteolysis^1–3. Proteases with the trypsin fold are defined by their hallmark double β-barrel topology connected by several surface-exposed loops of distinct functional relevance (Fig. 1A)^4,5. While the N- and C-terminal β-barrels are highly conserved and serve as the scaffold for active-site triad placement and substrate cleft formation, functional diversity and specificity of substrate interactions are mainly mediated by the surface-exposed loops^2,6. These loops have been adapted extensively in length and amino acid composition (Fig. 1B and Sup Fig. 1) through the TLP linage, allowing for a high level of evolutionary divergence within the family^3,7,8. This has facilitated diversified functions for TLPs ranging from immunity and coagulation to metabolism, with an impressive diversity in regulatory mechanisms^2,9 governed by a number of co-factors and exo-sites interactions^9–11. A prime example is blood coagulation, where a number of TLPs act in a co-factor-dependent enzymatic cascade on the membrane surface to halt bleeding in an organized fashion^12–15.

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Figure 1

FVII Structural Features and the Evolution of Surface-Exposed Loops. (A) Crystal structure of FVIIa:sTF (PDB ID:1dan, grey ²¹), shown in standard representation. (B) Alignment of serine proteases from various stages of blood coagulation compared with the ancestral protease trypsin, together with the surface-exposed loops of FVIIa from various species (see supplemental Fig. ^S1). Conserved residues are shown in red, non-conserved in grey and the loop extension region in black. Both numbering based on the human Factor VII sequence and on the consensus Chymotrypsin numbering is shown. (C) The different conformations of the 170 loop in FVIIa, FVIIa-Y_T (4z6a, wheat ²⁵) and trypsin (3ptn, blue ⁷⁴) with deletion scanned residues in red and an N-glycan attachment site. (D) Deletion strategy in FVIIa with scanned residues in red and start positions for each deletion window marked with grey. A total of 39 variants were generated by moving the deletion windows through the loop.

Remarkable insights into the detailed structure–function relationship of TLPs has been gathered over the last decades increasing our appreciation of how surface-exposed loops on the protease domain can lead to specific functional characteristics. For coagulation factor VIIa (FVIIa), the 170 loop has proven itself to be of predominant importance in the co-factor-mediated initiating of coagulation and as such has received considerable attention^16–20. Several crystal structures are available which^21–26, in combination with extensive molecular dynamics simulations, has been used to explore the TLP domain ensemble and different loop conformations^27–35, shedding light on allostery, substrate recognition and co-factor interaction. Past work has substantiated that the conformation of the 170 loop 1) plays a central role in co-factor mediated allosteric regulation of FVIIa activity by Tissue Factor (TF) 2) is involved in maintaining free FVIIa in its low activity state and 3) influences the initiation of coagulation upon TF binding¹⁶. From an evolutionary perspective, the 170 loop of human FVIIa has been extended by 3–5 residues and a N-glycan has been added when compared to ancestral trypsin and paralogous TLPs in the coagulation cascade (Fig. 1B and ​andCC)³⁶. This loop extension is likely to have occurred after the divergence of the mammalian line as it is not present in lower vertebrates such as fish or bird (Fig. 1B)^37,38, and may have developed due to selection pressure for a more tightly controlled coagulation system³⁹.

Elucidation of such detailed understanding of surface loop functions in TLPs from structural evidence requires cumbersome crystallization trials and data collection. Even if crystallization trials are successful, the loops may not be resolved in the density due to their flexible nature or they may be distorted by crystal contacts, reducing the insights from such studies^23,40–43. The insights derived from extensive studies using grafting methods and various point mutation are unquestionably valuable^44–48; however, such results can be confounded and their interpretation unclear since both the position of a residue in relation to the remainder of the loop and relative to the protease body may affect activity and specificity. More extensive or high-throughput approaches to investigate the function of TLPs⁴⁹, or re-engineer new functions, often rely on random or direct mutagenesis, but due to the likely deleterious effects of introducing truncations above 2–3 amino acids at many locations, such methods only sample a limited space when it comes to the effects of loop length on protease function. Furthermore, local deletion mutations may be considered more destructive than a corresponding insertion at the same position as the protein backbone is more constrained; insertions can sometimes be accommodated by “looping out” segments⁵⁰. Other approaches that work on global protein truncations, such as incremental truncation and combinatorial recombination⁵¹, may be less useful for engineering TLP function since the palette of characteristics are associated with the behaviour of distinct surface-exposed loops^44,52–54.

Here, we investigate the applicability of a systematic approach inspired by the evolutionary “staircase” (whereby the length of a given loop can vary greatly between species in distinct “steps”) as observed for TPLs and even between paralogs within the same signalling cascade (Fig. 1 B). The 170 loop from FVIIa was chosen as a model system due to its crucial involvement in activity, specificity and allosteric control of this protease. Using a deletion window size of 1 to 7 residues through positions 313{c170A} to 321{c170I} (FVIIa full-length numbering is used in the remaining part of the article; chymotrypsin numbers are indicated in curly brackets) incrementally proceeding through the entire loop from the N to C-terminus direction, generated a total of 39 variants that were fully reconstructed in silico, recombinantly produced and experimentally characterized in the present study (Fig. 1D). We found that the combined methodology of experimental characterization and in-silico loop reconstruction with the Next-generation Kinematic Closure (NGK) protocol⁵⁵ allowed for detailed structure–function insights. Importantly, our investigation recapitulates the main features described previously for 170 loop function in FVIIa. In addition, the approach revealed that the variant with highest amidolytic activity was achieved by a six-amino acid truncation – i.e. shorter than the shortest naturally appearing 170 loop variant – and as such would not have been detected with the sampling space of directed/random mutagenesis.

Experimental procedures

Results

Functional effects of 170 loop truncation in FVIIa

To evaluate the effects of the truncated loops on protease function, we probed the primary specificity pocket (S1), the extended substrate pockets (S2-S4), as well as the full complement of interactions with the natural substrate by performing binding assays using the small-molecule inhibitor pABA, the tri-peptidyl substrate mimic (S-2288), and the full length Factor X, respectively. It is evident from these functional data that both the size of the deletion window and its position affect the activity of a given FVIIa variant in the absence of sTF (Fig. 2). However, any mutational effects are negligible at saturating levels of sTF (supplemental Fig. ^S2). Indeed, only one variant (no. 36, supplemental Fig. ^S2) appeared to have an improved amidolytic activity (~twofold compared to FVIIa-WT) in the presence of sTF, suggesting that the native, extended 170 loop in FVIIa is optimal for the activity of the FVIIa:TF complex. In the absence of sTF, two distinct groups of variants are observed that exhibits both high activity towards S-2288 and a strong pABA binding: The first group consists of variants having a 2–3 residue truncation in the 315{c170C}-319{c170G} positions (no. 12–14 and 20–22, Fig. 2 and ​and3),3), while the second group comprises the 6–7 residue truncated variants in the 313{c170A}-320{c170H} positions (no. 36, 37 and 39, Figs. ​Figs.22 and ​and3).3). The most active variant in the absence of sTF (no. 36) was located in the second group and it showed a 12.8-fold increase in amidolytic activity and a 38-fold increased pABA affinity relative to FVIIa. The FX activation of variant no. 36 was, however, only increased twofold, whereas variants found in the first group did show somewhat higher FX activation levels (3–fivefold) when using S-2765 activity as a read-out. Similar findings were found when visualising FX activation by generation of the multichain chain FXa molecule using SDS-PAGE (supplemental Fig. ^S8).

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Figure 2

Heatmap of Functional FVIIa Variant Characterization. Soluble tissue factor (sTF) binding as K_d [nM], amidolytic activity as K_M [mM], k_cat [s⁻¹] and percentage k_cat/K_M compared to FVIIa, pABA inhibition as percentage K_i of FVIIa and Factor X activation as percentage of FVIIa. More active or stronger binding variants are shown in blue and less active or weaker binding variants in red. All data is shown as the mean of duplicate runs without sTF. Variants are sorted by loop length and deletion position (in red) with the start of each sliding window underlined and the resulting loop length listed. (right bold numbering), the number of deleted residues is show on the left.

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Figure 3

Functional FVIIa Data as a Function of Loop Length Normalized to FVIIa-WT in the absence of sTF (top) or presence of 3 μM sTF (bottom). (A) Functional data for hydrolysis of S-2288, (B) inhibition of FVIIa variants by pABA and (C) Activation of factor X. Group 1 consist of variants no. 12–14 and 20–22 while group 2 contains of variants no. 36, 37 and 39. Data is shown as the mean (n=2) with high-low error bars for S-2288 and pABA, with mean±SD (n=4) for FX activation. The grey shaded area represents one standard deviation for FVIIa-WT around the mean (n=6) from experiments conducted on different days and different plate positions. Loop length is the remaining number of residues in the 170-loop s for a give variant after a deletion, starting from the wild-type length of 14.

The deletion of residue P321{c170I} in variants shortened by 3 to 5 residues (no. 24, 30, and 35) had a pronounced negative effect on K_M and, to some extent, on k_cat for S-2288 hydrolysis in the absence of sTF. These effects were also observed as reduced pABA binding and FX activation levels (Fig. 2). Furthermore, deletions in the vicinity of the TF α-helix (residues 311{c169}-LQQS-314{c170B}) was poorly tolerated with a significant effect on the k_cat for hydrolysis of S-2288 for variants no. 10–11, 18–19, 25–26 and 31. In general, loops with 4–5 residue truncation showed poor amidolytic activity, even if the deletion did not include the two regions mentioned above, with the main effect observed on k_cat for these variants (no. 29, 33, and 34). An effect was also observed on the interaction with tissue factor, where variants with shorter loops tended to have a lowered binding affinity in good agreement with previous observations⁴⁷.

Structure–function relationship of loop shortening

To investigate the structural effects of 170 loop truncation and to support the functional observations, we built structural models of the 170 loop for all 39 variants using the Rosetta NGK protocol from residue Q313{c170A} to P321{c170I}. Two different starting structures were used to allow for the sampling of a larger conformational space that may better capture longer or short loop variants, with either the wide angle orientation of the TF-interacting α-helix (residues 307{c165}-313{c170A}) from FVIIa-WT²¹ or the more closed orientation determined in a FVIIa variant (FVIIa-Y_T) induced by the inclusion of the shorter 170 loop from trypsin²⁵ (Fig. 4A). The model scores (in Rosetta Energy Units; REU) were calculated in combination with the root-mean square deviation (RMSD) from the lowest scoring structure in order to evaluate clusters of low-energy structures (see supplemental Figs. ^S4 and ^S6 for all energy plots). The FVIIa-WT starting structure contained an additional auxiliary gamma-carboxyglutamic domain when compared to FVIIa-Y_T and baseline REU levels for the two structures are therefore not identical. Such differences do however not affect the scoring of the lowest energy structures within each generated ensemble. From the modelled structures and the fluctuations around the geometric mean, it was evident that the diversity of structures was dependent on the TF α-helix orientation (Fig. 4B). Loop lengths of 8–9 and 13–14 showed large variation when the structures were built from FVIIa as template, whereas a loop length of 7 or 10–12 in general provided a more restricted ensemble of loop conformations (Fig. 4B). The propensity to generate a narrow ensemble of structures at loop lengths of 11–12 when starting from FVIIa can be interpreted to agree with an increase in functional activity for variants belonging to group 1 (Fig. 4C). Shorter loops of 8–9 that were built on the FVIIa backbone tended to mostly occupy two conformations, one with the loop pointing towards the protease domain and the other pointing into the surrounding solvent (supplemental Fig. ^S3). Ensembles constructed with the narrower TF α-helix angle of 91° from FVIIa-Y_T showed high variability at long loop lengths that decreased as the loop got shorter (Fig. 4B). Stabilization for variants with a loop length of 11–12 was not seen, suggesting that the TF-helix angle in FVIIa allows a certain conformation that stabilizes the loop by favouring interactions with the protease domain. In general, the narrow angle was more compatible with the shorter loop variants (Fig. 4B-C and supplemental Fig. ^S5) as might be expected based on the tilt angle seen in trypsin (or FIXa and FXa) and FVIIa-Y_T (Fig. 1C).

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Figure 4

α-helix Angle Determines the Effects of Loop Length on 170 Loop Flexibility. (A) Orientation of the TF-helix as defined by the angle between the bottom of the 170 loop and the outer left side of the TF-helix in FVIIa (PDB ID 1dan, grey) and FVIIa-Y_T (PDB ID 4z6a, wheat) Q313{c170A} to P321{c170I} was rebuild using the Rosetta NGK protocol. (B) Average RMSF for all atoms rebuild in Rosetta with remaining loop lengths colour coded as red (13), green (12), blue (11), orange (10), grey (9), purple (8) and black (7). (C) Group 1 and group 2 FVIIa variants build using Rosetta Generalized Kinematic Closure (see supplemental Fig. ^S3 and ^S5 for all 39 variants). The root-mean-square fluctuation (RMSF) is defined around the shown geometric mean (N=250) with low values shown in blue and high values in red.

TF-mimicking interactions facilitate increased functional activity

We investigated the variants from group 1 in more detail to shed light on the structural features underpinning the reduced sample space of loop conformation and its relation to the observed increased activity. Previous studies of FVIIa emphasized that a hydrogen bond located at the top of the 170 loop, between the backbone of R315{c170C} and G372{c223} (Fig. 5A), is involved in TF mediated activity enhancement⁶⁷. By measuring the distance between the backbone nitrogen of R315{c170C} (or the residue in its place) and the G372{c223} carbonyl oxygen for the variants, we found that a loop length of 11–12 results in a loop conformation with propensity for the correct orientation for hydrogen-bond formation (Fig. 5B). This effect was not observed in a control setup where the 14 amino acids of the wild type molecule were rebuilt. Interestingly, an increased propensity to sample an electrostatically favourable R315{c170C}-G372{c223} interaction was associated with increased activity for the variants in group 1 (Fig. 5C). Plots of model energy (in REU) against RMSD for the variants in this group (12–13, 20–22) showed a funnel-like shape (supplemental Fig. ^S4), consistent with the presence of an ensemble of energetically favourable structures for each variant (the 10 best scoring structures are depicted on Fig. 5C). Interestingly, this favourable interaction was not observed when constructing the loops based on the FVIIa-Y_T structure, suggesting that the~15° change in TF-helix orientation is unfavourable for this loop length (Fig. 5B).

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Figure 5

Variants with an Intermediate Loop Length Show TF-Induced-Like Interactions. (A) The 170 loop region of FVIIa (PDB ID, 1dan) with the hydrogen bond between R315{c170C} and G372{c223} shown in red, together with the C310{c168}-C329{c182} disulphide-bridge in yellow. (B) The mean distance between the residue corresponding to the R315{c170C} backbone nitrogen and G372{c223} oxygen plotted against the standard deviation of the bond length for the FVIIa-WT (left panel) or FVIIa-Y_T backbone (right panel). Bond lengths of 2.5–3.8 Å are marked in grey for loop conformations with possible stable hydrogen bonds including variants 12–13 and 20–22. (C) Overview of group 1 variants with high activity including 10 modelled structures with the lowest Rosetta Energy (see supplemental Fig. ^S4) with the average hydrogen-bond distance shown in red. Frequency histograms for variants with distances in the 2.5–4.0 Å range showing a favourable distribution for loop lengths 11–12. The corresponding S-2288 activity relative to FVIIa in the right top-corner and deleted amino acids are coloured in red on the shown sequence.

Trypsin-like interactions between the 170 loop and protease domain confers activity

A more detailed look at the computational models generated for variants with a loop length of 7–8 belonging to group 2 revealed that interactions between the 170 loop and the protease domain are analogous to those found in wild-type trypsin and FVIIa-Y_T (Fig. 6A-C). In variant no. 36 with the highest amidolytic activity, D319{c170G} is inserted into a pocket between Q366{c217} and F374{c225} in many modelled structures to form polar contacts with the backbone nitrogen of F374{c225} and an imidazole nitrogen of H373{c224} (Fig. 6B). This is in agreement with the significant positive effect on K_M for S-2288 hydrolysis (1.1 mM) when compared with FVIIa (7.2 mM) resulting in an overall 12.8-fold increase in activity when compared to FVIIa-WT. Only variant (no. 31) has D319{c170G} in the same position and it appears to exhibit a similar stabilizing effect, albeit to a lesser degree. This variant also showed an improved K_M (2.6 mM) as would be expected, but a significantly compromised k_cat. For the remaining seven variants (33–35 and 37–39) with a similar loop length, D319{c170G} is replaced with Q313{c170A}, resulting in a changed polar-contact pattern. In those variants, Q313{c170A} form polar contacts with either the backbone nitrogen or carbonyl of F374{c225}, but not both for variants 37–39 (Fig. 6C). This observation agrees with a modest improved S-2288 activity for all variants, except variant 38 that shows a reduced k_cat. These observations align well with the funnel-like shape of model energy (in REU) against RMSD plots (supplemental Fig. ^S6) with 15 energetically favourable models shown on Fig. 6B-C.

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Figure 6

Shorter Loop Lengths Allow for Trypsin-like Interactions with the Protease Domain. (A) Overview of trypsin-like interaction in a FVIIa-trypsin variant (PDB ID: 4z6a), with Y314 (172c) inserting into the protease domain, stabilizing Q366{c217} and F374{c225} by polar contacts (red) as observed in wild-type trypsin. (B) 10 models with lowest RMSD/REU of the most active variant (no 36.) from group 2, reconstructed using the NGK protocol. D319{c170G} makes polar contacts (red) with the backbone nitrogen of F374{c225} and sidechain of H373{c224} to stabilize the S1-pocket. A frequency histogram of the polar contacts between D319{c170G}-H373{c224} (grey) or D319-F374_N (red) highlights the favourability of the interaction (n=250) with S-2288 activity relative to FVIIa in the right top-corner and deleted amino acids are coloured in red on the shown sequence. (C) Overview of variants 37–39 with 3 groups of 5 modelled structures selected from RMSD groupings showing the lowest REU (see supplemental Fig. ^S6). Average hydrogen-bond distance is shown in red when the distance is<3.5 Å. Frequency histograms (n=250) of the polar contacts between Q313{c170A} -F374_N{c225} (red) or Q313{c170A}-F374_O{c225} (purple).

Discussion

We applied computational modelling combined with a systematic loop truncation approach with a sliding window size of 1–7 deletions, that in combination with functional studies, provides a powerful method for detailed structure–function elucidation of surface-exposed loops in trypsin-like protease. These results will complement structural studies using X-ray crystallography or cryo-EM but can also be a viable substitute when these are unavailable or co-factors/inhibitors that may have a distorting effect on surface exposed loops have to be employed to achieve high quality crystals. In addition, the extended space of considered truncations employed here allows for the detection of variants that would be ignored in approaches relying on random/direct mutagenesis, where 2–3 residues are truncated.

Using the sliding deletion window, 39 protease variants were generated. The functional characterization showed that high-activity variants tended to cluster in two groups with striking similarities to previous findings. The first group comprised loop lengths of 11–12 (2–3 residue deletions) when deleting outside the residues forming part of the TF α-helix (307{c165}-312{c170}) (Fig. 3). The structural models generated using the NGK approach with FVIIa as a template suggested a structural stabilization for these variants similar to that reported to be induced by TF binding⁶⁷, and favouring loops that had distances between R315{c170C} and G372{c223} that could accommodate favourable electrostatic interactions (Fig. 5B). This recapitulation of previous findings demonstrates the applicability of the method for examining the function of surface-exposed loops in TLPs and supports the notion of evolutionary truncation/variability to achieve specific functions in TLPs. If, however, a FVIIa variant with 170 loop from trypsin (FVIIa-Y_T) with a narrower TF α-helix angle was used as a template, the productive electrostatic interaction between R315{c170C} and G372{c223} was not favoured. Likely, this segment of the wild-type FVIIa is responsive to TF-mediated allosteric enhancement activity by stabilizing the S1-pocket^16,31, which is in good agreement with the increased functional activity observed here. Interestingly, variants with only a single deletion seemed to have a loop length that results in a high number of possible conformations for the reconstructed loops, consistent with a small loss or no change in activity. Taken together, these data support the notion that the full length 170 loop of FVIIa is too flexible to enable the R315{c170C}-G372{c223} hydrogen bond interaction that appears important for protease activity and that TF binding to FVIIa may tether the loop down and thereby stabilizing R315{c170C} for a productive interaction with G372{c223}^68,69. Another observation supporting this interpretation was that only a single variant (no. 36) had a higher amidolytic activity in the presence of sTF than FVIIa:sTF, suggesting that the extended 170 loop with a length of 14 residues is tailored for TF-mediated increased FVIIa activity.

The second group consisted of variants with 6–7 residue truncations, which included the most active variant in the library carrying a 12.8-fold increased amidolytic activity (no. 36). It was evident from the reconstructed loop models (using FVIIa-Y_T as a template) that the six-residue deletion in variant 36 moved residue D319{c170G} into a position to interact with the body of the protease domain. D319{c170G} appears to likewise stabilize the S1-pocket through interactions with activation-loop 3 at the backbone nitrogen of F374{c225} and the sidechain of H373{c224}. Surprisingly, this interaction is congruent to that observed for Y172c (Supplemental Fig ^S7) in trypsin⁷⁰ and a FVIIa variant with the 170 loop grafted from trypsin^25,47,71, supporting the notion that our approach can be used to identify “hot-spots” for further engineering. In addition, a number of other variants with similar loop lengths showed a corresponding placement and orientation of residue Q313{c170A}, but the frequency of occupying the stable conformation was much lower than that observed for variant no. 36. This is cause by the single hydrogen bond acceptor nature of Glutamine that only allows for production interaction with either F374{c225} or H373{c224}, correlating well with the modest increase in amidolytic activity. None of these interactions were observed when FVIIa-WT was used as a template for the loop reconstruction as the wider angle of the TF-helix shifted both D319{c170G} and Q313{c170A} out-of-range of where they could interact productively with the protease domain. It was furthermore observed that variants with suggested high propensity to form polar contacts seemed to exhibit a notable negative effect on sTF binding affinity (Fig. 2), in good agreement with previous observations for a FVIIa variant with the 170 loop from trypsin and corresponding tilt of the TF α-helix^25,47,71.

The narrow helix angle induced in FVIIa-Y_T and variants generated here are in good agreement with more intrinsically active proteases such as FXa or thrombin, where the 170 loop is truncated by 5 residues. In these proteases a structural water network, supported by residues from the 170 loop and the change in helix angle, is likely to be a foundation for the high intrinsic activity^25,71–73. The helix angle is even further narrowed in wild-type trypsin, allowing optimal insertion of Y172c to stabilize the S1 pocket and confer constitutive activity to the protease^5,70. Thus, for a general applicability toward TLPs where representative loop and loop-anchor conformations spanning the functional spectrum are not available, it may be necessary to sample a continuum of helix angles upon which computational loop reconstruction is performed.

Using the systematic deletion approach presented here, we were able to capture many of the important structural features of the 170 loop reported over the last several decades of work on FVIIa. In addition, it was revealed that the variant with highest activity was achieved by a six-amino acid truncation with a deletion-window shifted 3 residues towards the N-terminus when compared to the shorten loop of ancestral trypsin. Most likely, such a variant could not have been detected with the sampling space of traditional directed/random mutagenesis. Taken together these findings suggest that the methodology is useful as a screening tool for identifying “hot-spots” from a wide initial pool of in-silico designed variants that will significantly reduce the amount of experimental testing required to engineer highly active protease variants. The presented approach furthermore highlights the diversity afforded TLPs, all building on a remarkably similar beta-sandwich structure (“The Trypsin fold”) that combined with highly variable surface exposed loops, enables this class of vital enzymes to span the evolutionary need of constitutively active proteases (e.g., Trypsin and variant 36) to that of highly co-factor control homeostatic systems (e.g., FVIIa-WT with tissue factor).

Supplementary Information

Acknowledgements

Author contributions

A.B.S. designed the research, conducted experiments, supported the computational biology, analysed the functional and computational results, wrote and revised the manuscript. P.J.G. conducted the computational biology studies. P.J.G. and J.J.M. supported the analysis of the computational results and revised the manuscript. J.L., G.A., P.G.W.L. and A.A.P. conducted experiments and supported results analysis. P.S.G., M.T.O., H.Ø., and O.H.O. designed the research, reviewed the results, and revised the manuscript.

Competing interests

The authors declare no competing interests.

Footnotes

References