Unusual amino acid composition of MaSp NTDs

In search for the mechanism of conformational change we analyzed the amino acid composition of aligned sequences of MaSp NTDs, published previously8. The amino acid composition shows striking peculiarities. Alanine and serine are most abundant with a combined content of ~30%. Only very few charged side chains are present and the domain is unusually rich in Met. We focussed specifically on the NTD of MaSp1 from the nursery web spider E. australis because this representative has been intensely investigated in the past8,11,12,13,14,17. The sequence contains 12.4% alanine, which is more than the average 8.1% alanine found in proteins from all three domains of life (Bacteria, Archaea and Eukaryota)18. Alanine is known to stabilize helical secondary structure19 and is presumably important for the structural integrity of the five-helix bundle. The NTD contains only 9% charged side chains, which is little compared to the average content of 25% charged side chains typically found in proteins18. Water-solubility of proteins is commonly provided by charged and hydrophilic side chains located on a surface. Water-solubility of the NTD appears to be facilitated by its unusually high content of 16.1% serine, which is a hydrophilic amino acid that can compensate the lack of charges. The average content of serine in bacterial, archaeal and eukaryotic proteins, by contrast, is only 6.2%18.

Met is commonly an infrequent amino acid with an average content of only 2.5% in proteins from across all phyla of life18. The content of Met in dragline silk is even lower (0.2–0.4%)20. It therefore took us by surprise to find that NTDs from MaSps contain a substantial number of Met residues (content of 7.4% Met in the sequence of MaSp1 NTD from E. australis, Fig. 1a). Met is unevenly distributed along a spidroin sequence and accumulates in the NTD. We analyzed the location of Met side chains in the NTD using sequence alignment and homology of available structural data8,14,15,16. We found that the majority of Met residues are buried in the hydrophobic core and/or involved in tertiary interactions of helices. Only few Met residues were solvent-exposed (Fig. 1a). The high abundance of Met in the domain core indicated an unknown structural and/or functional role of this side chain.

Fig. 1 Met content in spidroin NTDs and solution structure of L6-NTD. a Number of Met residues in homologous, ~135-residue long NTD sequences of MaSp1 and MaSp2 from spider species Ea Euprosthenops australis, Lg Latrodectus geometricus, Lh Latrodectus hesperus, Nc Nephila clavipes, At Argiope trifasciata, and Nim Nephila inaurata madagascariensis (black bars). The number of Met residues in core position and/or involved in tertiary interaction are shown as gray bars. b Far-UV CD spectrum of WT-NTD (blue) and of L6-NTD (orange). c Solution NMR structure of L6-NTD (PDB ID: 6QJY) (orange) aligned with the structure of WT-NTD (PDB ID: 2LPJ) (blue). The side chains of the six Met residues that are in core position and/or involved in tertiary interaction in WT-NTD and are mutated to Leu in L6-NTD are highlighted as blue and orange sticks. The side chain of the conserved Trp (W10), helices 1–5 (H1-H5) and N-terminus (N) and C-terminus (C) are indicated. Source data of Fig. 1b are provided as Source Data file Full size image

Solution structure of a Met-depleted spidroin NTD

We investigated the structural role of Met in the NTD of MaSp1 from E. australis. Visual inspection of the structure (PDB IDs: 2LPJ and 3LR2)8,14 showed that six of a total of ten Met side chains of the 137-residue domain are located in the core and/or are involved in tertiary interactions. The branched aliphatic side chain of Leu has a similar hydrophobicity and size as Met and can therefore serve as a suitable replacement21,22. To investigate the potential structural, dynamic and energetic effects of the six core Met we replaced them simultaneously by Leu, yielding a construct that we termed L6-NTD. Even though mutations in protein cores are generally not well tolerated and often act destabilizing23, we found that L6-NTD expressed well and at high yield. Far-UV circular dichroism (CD) spectroscopy showed that the helical secondary structure of the wild-type NTD (WT-NTD) was retained in the mutant (Fig. 1b). Using NMR spectroscopy, we determined the atomic-resolution solution structure of L6-NTD at pH 7.0, i.e., under conditions where the domain is a monomer (Fig. 1c, Supplementary Fig. 1, Supplementary Table 1, PDB ID: 6QJY). The solution structure showed that L6-NTD is a well-folded, five-helix bundle with no significant deviations from the structure of the WT-NTD. The helices of L6-NTD and WT-NTD have the same respective lengths and form the same tertiary interactions. The all-atom alignment of residues 9–129 (omitting the flexible, unstructured C-terminal and N-terminal tails) of the lowest-energy conformer of L6-NTD with that of the WT-NTD14 yielded a heavy-atom RMSD of 1.2 Å. The backbone heavy-atom RMSD was 1.2 Å. Helix orientations and positions of mutated side chains superimposed well. The conformation of the conserved Trp, which is wedged in the center of the helix bundle14, was also preserved. We concluded that replacement of the six core Met by Leu had no impact on the structure of the NTD.

L6-NTD folds at the speed limit

In order to investigate the influence of the Met-to-Leu mutations on the mechanism and energetics of folding, we performed chemical and thermal denaturation experiments of L6-NTD under pH 7.0 solution conditions where the NTD is a monomer. Equilibrium denaturation data revealed a dramatic increase of stability of L6-NTD compared to WT-NTD (Fig. 2a–c, Table 1). The free energy of folding increased from 5.6 ± 0.1 kcal/mol of WT-NTD to 8.0 ± 0.3 kcal/mol of L6-NTD (mean value ± s.d. of denaturation data measured by CD and fluorescence). The equilibrium m-value (m D-N ) of L6-NTD was slightly reduced compared to the WT value (Table 1). Changes in m-value are known to correlate with changes in accessible surface area between native and denatured states24. Since the structures of native WT-NTD and L6-NTD overlay (Fig. 1c), the slightly lower m-value measured for L6-NTD indicates that its denatured state is slightly more compact than that of WT-NTD. The melting temperature T m increased from originally 61.0 ± 0.1 °C of WT-NTD to 81.0 ± 0.2 °C of L6-NTD (±s.e. from regression analysis). To test whether stabilization resulted from a single mutation or was an additive effect25 we generated six cumulative point mutants that build up L6-NTD (L1: M20L; L2:M20L/M24L; L3: M20L/M24L/M41L; L4: M20L/M24L/M41L/M48L; L5: M20L/M24L/M41L/M48L/M77L; L6: M20L/M24L/M41L/M48L/M77L/M101L) and analyzed their thermal stabilities. We found a cumulative increase of melting temperature, which showed that the observed effect of stabilization was additive (Fig. 2c). Mutation M48L (L4) had little effect on stability of L3 and thus behaved differently (Fig. 2c, inset). The structure shows that the side chain of residue M48 is squeezed between helix 2 and helix 3, and is located close to the protein surface. M48 appears to form a less well consolidated van-der-Waals interaction network than the other Met side chains probed, which may explain the lack of effect upon mutating this residue. On the other hand, Met is reported26 to interact specifically with Trp side chains, which may cause a stabilizing effect. Removing this stabilizing interaction through mutation may be balanced by the added stability increment from substitution by Leu.

Fig. 2 Equilibrium denaturation and folding kinetics of L6-NTD at pH 7.0. a Chemical denaturation of L6-NTD (orange) and WT-NTD (blue) measured using far-UV CD spectroscopy (light open circles) and Trp fluorescence at 325 nm (closed squares). Black lines are fits to the data using a thermodynamic model for a two-state equilibrium. b Data shown in a but normalized to the fraction of folded protein. Same symbol and color code applies. c Thermal denaturation of six cumulative Met-to-Leu mutants (blue to orange) measured using far-UV CD spectroscopy. Data are normalized to the fraction of folded protein. The increase of transition midpoint (T m ) rank-orders with the cumulative number of mutations (inset). d Kinetic transients of folding (cyan, violet, blue; 5.0, 5.5, 6.0 M urea, respectively) and of unfolding (green, orange, magenta; 6.5; 7.5, 8.5 M urea, respectively) of L6-NTD measured using stopped-flow Trp fluorescence spectroscopy. Signal amplitudes were normalized for reasons of clarity. Data were fitted using a mono-exponential decay function (black lines). e Observed rate constants (WT-NTD: blue, L6-NTD: orange) plotted versus concentration of urea (chevron plot). Data were fitted using a model for a barrier-limited two-state transition (black lines). f Free-energy profile of folding of WT-NTD (blue) and L6-NTD (orange) inferred from equilibrium and kinetic denaturation data. D Denatured state, TS transition state, and N native state are indicated. The free energy of the native state of WT-NTD was set as a reference state to zero kcal/mol. Source data are provided as Source Data file Full size image

Table 1 Thermodynamic and kinetic data of folding of L6-NTD compared to WT-NTD Full size table

To gain insight into the origin of the enhanced stability of L6-NTD, we measured its kinetics of folding using stopped-flow Trp fluorescence spectroscopy (Fig. 2d, e). Our previous kinetic experiments performed on a set of homologous spidroin NTDs shows that WT-NTDs fold rapidly on a time scale of ~100 µs through a barrier-limited two-state transition11. Here we found that L6-NTD folded 40 times faster than the WT-NTD (Table 1). The rate constant of folding of L6-NTD, extrapolated to zero denaturant, was k f = 519,000 ± 213,000 s−1 (±s.e. from regression analysis). Synergy of theory and experiment predicts that the speed of folding of a generic N-residue single-domain protein is limited to τ f = N/100 µs, which is referred to as the speed limit of folding27. Following this theory, the speed limit of folding of the 137-residue NTD is ~1.4 µs. For L6-NTD we determined τ f = 1/k f = 1.9 ± 0.8 µs, which corresponds to the theoretical speed limit. Besides the observed increase of k f we found that L6-NTD was stabilized by a 15-fold decrease of unfolding rate constant compared to the WT-NTD (Table 1). From k f and k u of WT-NTD and L6-NTD we estimated a decrease of the free energy barrier between the denatured and the transition state by ~2.2 kcal/mol, and an increase of the free energy barrier between the native and the transition state by ~1.6 kcal/mol, of L6-NTD compared to WT-NTD (Fig. 2f).

Met-depletion of the NTD core impairs dimerization

Conservation of structure and increase of stability suggested that the L6 mutant should retain functionality. Functionality of NTDs can be tested by measuring their ability to undergo pH-induced dimerization, in analogy to what happens in a spider’s spinning duct4,9,12,13,28. NTDs at pH 6.0 are tight dimers that exhibit dissociation constants (K d ) in the low nanomolar range11. We characterized the equilibrium dimerization of L6-NTD and its response to changing solution conditions using size-exclusion chromatography in combination with multi-angle light scattering spectroscopy (SEC-MALS). At pH 7.0 and at 200 mM ionic strength, L6-NTD eluted homogenously as a monomer, as expected, similar to WT-NTD (expected molecular weight, M w = 14 kDa; measured M w = 14 kDa). At pH 6.0 and 60 mM ionic strength, however, the average molecular mass of L6-NTD measured by SEC-MALS was between that of a monomer and a dimer (expected M w = 28 kDa; measured M w = 20 kDa; Fig. 3a). The detected intermediate mass can be interpreted as that of a system in rapid, dynamic equilibrium between a monomer and a dimer. The intermediate M W indicated an elevated K d of L6-NTD compared to WT-NTD, which fell in the µM sample concentration range applied in this experiment. WT-NTD measured under the same conditions showed a molecular mass close to the value expected for a dimer (expected M w : 28 kDa; measured M w : 26 kDa, Fig. 3a). The measured value was only 7% below the expected value for a dimer. The discrepancy was little above the precision of the measurement (±3–5%) and may be explained by small amounts of salt (60 mM ionic strength) present in SEC experiments, which were required to reduce sticking of protein material to the column. Ions in solution shield electrostatics and induce dissociation of the WT-NTD9,10,12. High concentrations of salt lead to Debye–Hückel29 screening of attractive electrostatic forces in the dimerization interface. This explains residual population of monomer in the dimer elution band and consequently a lower detected molecular mass. In order to strengthen dimerization we reduced the ionic strength of the pH 6.0 buffer to 8 mM. Under these conditions, the measured molecular mass of L6-NTD was closer to that of a dimer (Fig. 3b). Dimerization of L6-NTD was thus apparently stabilized by electrostatic forces that are screened at high solution ionic strength, similar as previously observed for WT-NTD9,10,12. Upon progressive reduction of concentration of L6-NTD samples in SEC-MALS experiments we measured a decrease of molecular weight and an increase of elution volume (V E ), confirming that, despite low ionic strength, dimerization was still weak. The K d of L6-NTD was in the concentration range probed during measurement (i.e., K d in the µM range) (Fig. 3b). However, we were not able to obtain a full binding isotherm required to quantify K d because MALS detection was not sensitive enough to probe sub-µM protein concentrations. We therefore measured V E of dilute L6-NTD samples in pH 6.0 buffer using high-resolution SEC in combination with fluorescence detection, which had sub-µM sensitivity. An increase of L6-NTD concentration shifted the V E to lower values indicating formation of dimers (Fig. 3c). From concentration-dependent data we obtained a binding isotherm that fitted well to a thermodynamic model of dimerization, yielding a K d of 1.1 ± 0.2 µM (±s.e. from regression analysis) (Fig. 3d). This K d was three orders of magnitude higher than the value reported for WT-NTD (K d = 1.1 ± 0.1 nM)11. V E of WT-NTD at pH 6.0 did not change significantly over the probed protein concentration range because it was far above K d 11 (Fig. 3d). However, there was a slight increase of V E of WT-NTD probed in the 10-nM concentration range, which may indicate the onset of dimer dissociation. The V E of the WT-NTD dimer was significantly higher than the value of the L6-NTD dimer, which indicated that the L6-NTD dimer had larger dimensions. Expansion of the L6-NTD dimer can be explained by loose association of subunits. To test if high-resolution SEC data reported reliably on K d of the dimer, we conducted thermophoresis experiments as an alternative probe for dimerization (Fig. 3e). Using thermophoresis we obtained a K d of 3.6 ± 1.8 µM, which was in reasonable agreement with the value obtained by high-resolution SEC.

Fig. 3 NTD dimerization assays. a SEC-MALS of L6-NTD and WT-NTD. SEC elution bands (solid lines) and corresponding average values of M w (broken lines) measured by MALS are shown. L6-NTD and WT-NTD measured at pH 7.0 (200 mM ionic strength) are shown in orange and blue. L6-NTD and WT-NTD measured at pH 6.0 (60 mM ionic strength) are shown in red and cyan. b SEC-MALS of 12 µM (red), 4 µM (orange), 1.2 µM (magenta), and 0.4 µM (blue) L6-NTD measured at pH 6.0 (8 mM ionic strength). Signals of elution bands are normalized to peak intensities. c High-resolution SEC of 100 µM (cyan), 6 µM (red), 0.8 µM (blue), and 0.05 µM (violet) L6-NTD measured using Trp fluorescence detection. Signals of elution bands are normalized to peak intensities. d V E of L6-NTD (orange) and WT-NTD (blue) measured at pH 6.0 (20 mM ionic strength) and at various protein concentrations using a high-resolution SEC. The black line is a fit to the data using a thermodynamic model for a monomer/dimer equilibrium. e Thermophoresis ratio of fluorescently modified L6-NTD-Q50C in presence of increasing concentration of L6-NTD (orange). The black line is a fit using a model for a binding isotherm. Differences in absolute peak values of V E of elution bands shown in panels (a–c) originate from the different SEC columns used in these experiments. Source data are provided as Source Data file Full size image

Next, we investigated the influence of individual Met side chains on size and stability of the NTD dimer by measuring dimerization of six cumulative Met-to-Leu point mutants using high-resolution SEC (mutations are detailed above in denaturation experiments). The obtained isotherms are shown in Fig. 4a–e. The K d increased progressively with increasing number of mutations. At the same time, the V E of the dimer decreased with increasing number of mutations (Fig. 4f). This showed that the dimensions of the dimer increased progressively with increasing number of mutations, which indicated progressive loosening of the complex.

Fig. 4 Dimerization of cumulative Met-to-Leu mutants. a–e V E plotted versus protein concentration of cumulative Met-to-Leu mutants L1 to L5 measured using high-resolution SEC. The black lines are fits to the data using a thermodynamic model for a monomer/dimer equilibrium. f Values of K d (spheres) and end-point values of V E (squares) of cumulative Met-to-Leu mutants (L1 to L6) and of WT-NTD measured using high-resolution SEC. The K d of WT-NTD is the value determined from kinetics reported in ref. 11. The K D of L1 is an estimate because of the non-defined monomer baseline. Closed and open squares are end-point V E values of dimer and monomer, respectively. The end-point values of V E of the WT-NTD monomer and of the L1-NTD monomer could not be determined because their K d was too low to enter baseline region. Source data are provided as Source Data file Full size image

Met facilitates conformational changes upon dimerization

Dimerization of the NTD is known to be associated with conformational changes. Helices that form the dimerization interface tilt and the conserved Trp (W10) moves out of its binding pocket to solvent-exposed position14. This is evident from the quenching and red-shift of Trp fluorescence emission, effects that are conserved across NTDs from various glands and species8,9,11,13,14. We found that the L6-NTD lacked these fluorescence characteristics. There was no red-shift and only minor quenching of Trp fluorescence emission upon dimerization (Fig. 5a). The observation indicated that in L6-NTD Trp W10 remained in buried position both in the monomeric and in the dimeric state. Fluorescence characteristics changed gradually with increasing number of Met-to-Leu mutations (Fig. 5b), similar as observed for the change of stability and of K d (Figs. 2c and 4f). Full conformational change of Trp thus required the presence of several core Met.

Fig. 5 Native-state conformational changes of the NTD. a Trp fluorescence spectra of WT-NTD (blue) and L6-NTD (orange) measured at pH 7.0 and 200 mM ionic strength (solid lines), and at pH 6.0 and 8 mM ionic strength (dashed lines). b Quenching of Trp fluorescence emission of cumulative Met-to-Leu mutants upon change of solution condition from pH 7.0 (200 mM ionic strength) to pH 6.0 (8 mM ionic strength) (colored bars). The wavelengths of maximal fluorescence emission at pH 7.0 (gray squares) and at pH 6.0 (black squares) is shown for each mutant. c 1H, 15N-HSQC NMR spectra detailing the side chain amide signal of W10 of L6-NTD and WT-NTD in pH 7.0 buffered solution and 200 mM ionic strength (orange and blue, respectively) and in pH 6.0 and 8 mM ionic strength (light orange and cyan, respectively). d NMR chemical shift (CS) differences of assigned residues in the WT-NTD (blue) and L6-NTD (orange) upon change of solution pH from 7.0 to 6.0. e 1H, 15N-HSQC NMR signals of NTD loop residues. Close up of the HSQC spectral regions of glycine residues G59, G86, G110, and G32 located in loop segments connecting helices of WT-NTD and L6-NTD recorded at pH 7.0 and at pH 6.0 are shown. The location of residues on the aligned structures of WT (blue) and L6-NTD (orange) is highlighted as spheres. Source data are provided as Source Data file. Source data shown in panel e are provided in the BMRB (entry: 27683) Full size image

We measured and analyzed 1H, 15N HSQC NMR spectra of L6-NTD and WT-NTD recorded in both the monomeric and the dimeric states. The full 1H, 15N HSQC NMR data set is shown in Supplementary Fig. 2. In agreement with our results from fluorescence spectroscopy, 1H, 15N-Trp chemical shifts of L6-NTD measured by NMR spectroscopy showed only minute perturbations upon change of solution pH from 7.0 to 6.0, which was in stark contrast to what we observed for the WT-NTD (Fig. 5c). The NMR data showed that the environment of W10 in L6-NTD remained essentially unchanged upon dimerization, contrasting the substantial changes of the environment of W10 in WT-NTD. Figure 5d shows the respective differences in chemical shifts of all assigned residues in WT-NTD and L6-NTD between pH 7.0 and pH 6.0 (full HSQC spectra are provided as Supplementary Fig. 2). Residues of helices H1, H4, and H5 of L6-NTD could be reliably assigned at pH 6.0. However, assignment of helices H2 and H3 of L6-NTD was complicated by line broadening caused by dynamic exchange on the intermediate time scale, in agreement with the reduced dimer affinity. The observation supports our finding that mutation of Met to Leu in the domain core has profound consequences both for dynamics and function of the protein and prevents the dimer interface to adopt a conformation suitable for high-affinity dimerization. A loose L6-NTD dimer was found in the high-resolution SEC experiments described above (Fig. 4f). The loose dimer presumably shows rapid inter-molecular interaction dynamics between subunits that may enhance NMR line broadening. However, analysis of the many assigned residues showed that chemical shift changes upon dimerization covered the entire sequence and were in general substantially larger for WT-NTD compared to L6-NTD (Fig. 5d). The result was in agreement with strong dimer interactions and more extensive conformational changes in WT-NTD, which are apparently blocked in L6-NTD. In L6-NTD, the majority of chemical shift changes of residues that are part of the dimerization interface resulted from weak dimer interactions. Interestingly, we found pronounced chemical shift changes of resonances of glycine residues located in loops connecting helices of WT-NTD upon dimerization. These chemical shift changes were absent in L6-NTD (Fig. 5e). Since the glycine-rich NTD loops are not involved in dimerization, their significant chemical shift changes upon dimerization in WT but not in L6-NTD indicated that Met promotes conformational changes that are remote from the core and the dimerization interface.

Met drives native-state dynamics of the NTD monomer

Having established that Met enables structural changes required for dimerization, we set out to investigate the influence of core Met on native-state conformational dynamics of the NTD monomer. For insights into per-residue dynamics and changes in solvent-accessibility of residues in the L6-NTD structure in comparison to WT-NTD, we performed NMR-based hydrogen/deuterium exchange experiments (Fig. 6a). We found that proton-to-deuterium exchange in L6-NTD was on average ~10-fold slower compared to WT-NTD, i.e., L6-NTD was overall less dynamic and the exchange-competent conformations were less frequently accessible than in WT-NTD. For L6-NTD, H/D exchange was significantly slower not just at the positions of mutation but throughout all five helices, i.e., through residues 18–28, 41–51, 72–81, 98–107, and 118–128 (Fig. 6a, b). In contrast, the local backbone dynamics on the fast nanosecond-to-picosecond time scale were not influenced by the mutations, as indicated by heteronuclear NOE measurements (Fig. 6c, d). Protein dynamics on the nanosecond-to-picosecond time scale generally report on local events, while dynamics on time scales of microseconds (µs) and slower reflect collective conformational motions30. Met residues in the core of the NTD thus appeared to facilitate collective motions rather than local, uncoupled flexibility.

Fig. 6 Native-state dynamics of L6-NTD probed by NMR spectroscopy and PET-FCS. a NMR H/D exchange experiments. Amide H/D exchange time of L6-NTD (orange) and WT-NTD (blue) plotted versus residue number. Positions of helices and mutations are indicated on top of the panel. b Exchange times imposed on the structure of WT-NTD (PDB ID: 2LPJ) and of L6-NTD (PDB ID: 6QJY). c {1H},15N-hetNOE values of WT-NTD (blue) and L6-NTD (orange) backbone amides plotted versus residue number. Lower values indicate higher flexibility. d {1H},15N-hetNOE values imposed on the structure of WT-NTD (PDB ID: 2LPJ) and L6-NTD (PDB ID: 6QJY), respectively. e Reporter design for PET-FCS. Top view of the structure of L6-NTD (PDB ID: 6QJY) indicating the position of the AttoOxa11 fluorescence label (red sphere) attached to the N-terminus (G3C). The side chain of the conserved Trp W10 (blue sphere), which quenches fluorescence of AttoOxa11 upon solvent-exposure, is highlighted. Trp is buried in a network of tertiary interactions (side chains shown as sticks). f PET-FCS autocorrelation functions (G(τ)) recorded from WT-NTD and L6-NTD (blue and orange) modified with AttoOxa11 at position G3C. Data of L6-NTD modified with AttoOxa11 at position Q50C served as a control (cyan). Black lines are fits to the data using a model for translational diffusion of a globule containing two single-exponential relaxations. The control sample was described by a model for translational diffusion only. g Time constants of Trp moving out of the core (τ out , solid bars) and into the core (τ in , patterned bars) of WT-NTD (blue) and L6-NTD (orange), calculated from PET-FCS data. Error bars are propagated s.e. from regression analysis. The comic at the center of the panel illustrates the conformational change and assigns τ out and τ in . The NTD is illustrated as gray ellipsoid. Trp W10 (blue sphere) moves between buried and solvent-exposed positions. Source data are provided as Source Data file Full size image