Ligand Controlled Assembly of Hexamers, Dihexamers, and Linear Multihexamer Structures by the Engineered, Acylated, Insulin Degludec

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1 Supplementary material Ligand Controlled Assembly of Hexamers, Dihexamers, and Linear Multihexamer Structures by the Engineered, Acylated, Insulin Degludec D. B. Steensgaard*, G. Schluckebier, Holger M. Strauss, M. Norrman, J. K. Thomsen, A. V. Friderichsen, S. Havelund, and I. Jonassen. Diabetes Protein Engineering, Novo Nordisk A/S, DK2760 Maaloev, Denmark ToC S1. Sedimentation velocity of zinc free IDeg. S2. Sedimentation equilibrium of zinc free IDeg. S3. 1D 1H NMR spectroscopy of zinc free IDeg. S4. Conformational change of HI in the presence of resorcinol. S5. Simulation of circular dichroism data based on species distribution. S6. FTIR spectroscopy of films of zinc complexes of IDeg with and without phenol. S7. Sedimentation velocity of IDeg multihexamers. S8. Sedimentation equilibrium of IDeg multihexamers. S9. Circular dichroism of phenol deprived zinc complexes of IDeg. 1 of 12

2 S1. Sedimentation velocity of zinc free IDeg. Sedimentation velocity experiments on a series of different concentrations of IDeg (0.02 mm 0.32 mm displayed slightly increasing average sedimentation coefficients with increasing concentrations. Extrapolation of the reciprocal sedimentation coefficients to infinite dilution and correction to standard conditions resulted in s 0 20,w = 0.98 S for IDeg. The corresponding diffusion coefficients were independently measured in artificial boundary experiments and amounted to D020,w = 14.8 cm 2 /s for IDeg /(s* av ) [S -1 ] concentration [mm] x 10-7 [cm 2 /s] Figure S1: Sedimentation velocity, extrapolation to infinite dilution of zinc free IDeg. The black line represents a linear extrapolation to infinite dilution of sedimentation coefficients measured at different concentrations (black dots, lower scale). The red line represents a linear extrapolation to infinite dilution of diffusion coefficients measured at different concentrations in artificial boundary cells (red dots, upper scale). Values at infinite dilution are given in the text. 2 of 12

3 S2. Sedimentation equilibrium of zinc free IDeg. Sedimentation equilibrium gradients of zinc free IDeg observed at different rotational speeds were best described by a monomer-dimer-hexamer equilibrium, with a fitted molar mass of 6.23 ( ) kg/mole) and values for K D1-2 of 8.7 ( ) x 10-4 M and K D1-6 of 1.3 ( ) x M 5. Fixing the molar mass parameter to the expected value resulted in a statistically equivalent fit with slightly shifted values for K D1-2 and K D r.i. 5 0 Y exp -Y fit (x 10 2 ) radius [cm] Figure S2: Sedimentation equilibrium of zinc free IDeg. Upper panel shows equilibrium gradients for a single concentration (out of five) of IDeg, containing 5 Zn 2+ /IDeg hexamer and 30 mm Phenol at 20 krpm, 30 krpm and 40 krpm (black, red and green open circles, respectively). Only every 10 th data point is shown. The corresponding lines represent simulated data for a monomer-dimer-hexamer equilibrium. The lower panel shows the deviation between the experimental and simulated data (every data point is shown and offset for clarity). The overall rmsd is 7.4 mfringes. 3 of 12

4 S3. 1D 1H NMR spectroscopy of zinc free IDeg. For a qualitative evaluation of the insulin self-association IDeg was compared to human insulin in a series of 1D 1 H NMR spectra. As expected the NMR spectra of human insulin display relatively poor resolution and broad lines for all concentrations measured from 4.0 mm to mm (figure S3, A-D). However, there is an increase in relative intensity, and narrower NMR signals are observed at the lower concentrations which indicate a tendency to dissociation at the lowest insulin concentration. For IDeg very broad lines are also observed at the highest insulin concentration of 4.0 mm (Figure S3, E). A direct comparison between the NMR spectra of IDeg and human insulin at an insulin concentration of 0.5 mm shows higher NMR signal intensity and narrower line widths from IDeg peptide signals (Figure S3, B and F). At the lowest IDeg concentrations the relative NMR signal intensities increase and the line widths decrease slightly. In conclusion the NMR fingerprint spectra of IDeg show that this insulin has a much stronger tendency to dissociation than human insulin. Figure S3: 1D 1H NMR spectra showing aliphatic signals between 0.3 and 1.1 ppm as a function of protein concentration for human insulin (A-D) and IDeg (E-H). Insulin concentrations were as follows: panels A and E; 4.0 mm, B and F 0.5 mm, C and G mm, and D and H mm protein in 8 mm phosphate buffer at ph 7.4. The data were recorded and plotted in order to make a direct comparison of NMR signal intensity from the protein possible. All 1H 1D NMR spectra were recorded at 22 C on a 700 MHz Bruker magnet equipped with a Bruker AvanceTM III console and a Bruker 5 mm TCI CryoProbe using 3 mm SampleJet NMR tubes. All samples were prepared in 8 mm Na 2 HPO4/NaH 2 PO4 ph 7.4 in 95/5% H 2 O/D 2 O. The NMR data were processed and plotted using Bruker Topspin software. 4 of 12

5 S4. Conformational change of HI in the presence of resorcinol. Upon titration with zinc the amplitude change from about -2.0 M -1 cm -1 to -8.0 M -1 cm -1 both in the absence and the presence of 150 mm NaCl. In the presence of 100 mm occurs at higher zinc ratios. The data show that in the presence of zinc and resorcinol HI readily adopt R6 conformation even in the absence of NaCl. 0 ε 251nm (M -1 cm -1 ) Zn/6Ins HI, Res HI, Res, NaCl HI, Res, Imz Figure S4: Conformational change as observed by elipticity at 251 nm as a function of zinc ratio for various conditions of ligands. Samples contain 0.6 mm human insulin, 30 mm resorcinol, ph 7.4 (triangle) with the addition of either 150 mm NaCl (circle) or 100 mm imidazole (square). 5 of 12

6 S5. Simulation of circular dichroism data based on species distribution. Simulation of the data in Figure 7 (B and C) by using the species distribution presented in Figure 3 and Figure 5 and assuming a molar elipticity at 251 nm for monomer -2.0 M -1 cm -1, hexamer -8.0 M -1 cm -1 and dihexamer -5.0 M -1 cm -1 and using these numbers for calculation of a linear combination of the species observed. The simulated data resembles the measured data presented in Figure 7 (B, C) which suggest that secondary structure and conformational state of the IDeg zinc complexes are tightly linked to the quaternary structure. Simulated CD Zn/6Ins Phenol Resorcinol Resorcinol, NaCl Resorcinol, Imz Figure S5: Simulated molar elipticity based on species distribution obtained by SEC. 6 of 12

7 S6. FTIR spectroscopy of films of zinc complexes of IDeg with and without phenol. The secondary structure of thin films of IDeg was evaluated from horizontal attenuated total reflection Fourier transform infrared spectroscopy (HATR FTIR). This method is particularly useful in this context since TEM captures images of dried protein solution on a copper grid, and HATR FTIR provides structural information of the dried protein obtained from the same solution. Secondly, FTIR is highly sensitive to the presence of amyloid fibrillation. Four spectral markers are known for native insulin fibrils (Nielsen et al (2001) J Pharm Sc, 90, 29-37, Webster et al (2011) J Phys. Chem. B 115, , and references herein): Amide Ia: ~1622 cm -1, C=O stretching, H-bonded intermolecular β-sheets Amide Ib: ~1635 cm -1, C=O stretching, H-bonded intermolecular β-sheets Amide II: ~1540 cm -1, C-N stretching + in-plane N-H bending Amide A: ~3285 cm -1, N-H stretching of which the Amide Ib and A are the most intense. Two samples of IDeg formulations were prepared, mm IDeg, 16 mm phenol, 16 mm m-cresol, 0.5 mm zinc acetate, 1.6% glycerol, 10 mm tris/hcl, ph Sample 1 was depleted for phenol by passing through a NAP10 column. Volumes of 20 µl were dispersed on a Ge HATR crystal mounted in a Nicolet 6700 FTIR spectrometer equipped with a liquid N 2 -cooled MCT/B detector, Michelson interferometer, KBr beamsplitter and ETC EverGlo source (Thermo Fisher Scientific Inc., Madison, WI), and dried under N 2 flow for 3 minutes. The reflection intensity was measured at 2 cm -1 spectral resolution in the range cm -1 and averaged from 200 scans. The resulting interferogram was Fourier transformed and the reflectance calculated as the ratio between sample and background reflection (clean crystal). The spectra were ATR-corrected (n sample =1.2, n Ge =4.0, # bounces: 12), for comparability to transmission data and converted to log(1/r) units. Finally the spectra were corrected for excipient interference by subtraction of the corresponding vehicles. Positive controls of fibrillated human insulin were prepared by agitation of solutions of human insulin, mm HI, 0.3 mm zinc acetate, 140 mm NaCl, 10 mm tris/hcl, ph mm HI, 0.5 mm zinc acetate, 10 mm tris/hcl, ph of 12

8 at 960 rpm and 37 C for 20 hours. Their HATR spectra were subsequently measured and vehicle corrected as described above. All films were prepared and measured twice. Figure S6A shows the average, vehicle corrected, HATR spectra of samples 1-4 in the Amide I- III region, normalized to the Amide I peak maximum. The spectrum of sample 3 (red) exhibit a sharp peak at 1635 cm -1, characteristic of fibrillated insulin, whereas sample 4 is matches the profile of unstressed insulin (not shown). Both of the IDeg films have Amide I peak maxima at 1655 cm -1, characteristic of proteins rich in α-helix content. In particular, the IDeg film with no phenol (green) - simulating the TEM IDeg sample and multihexamer forming conditions - contains no spectral markers of fibrils and has, based on the similarity of the Amide I envelope, an overall secondary structure content close to that of human insulin (49% a-helix, 32% b-sheet, 8% turn, 12% unordered (Nielsen, Frøkjær, Carpenter and Brange (2001) J Pharm Sc, 90, 29-37). In the hydrogen stretching region (Figure S6B), a red-shift of the Amide A band from 3299 to 3284 cm -1 is observed in the fibrillated insulin sample (red), whereas the IDeg samples have Amide A bands maxima coinciding with native insulin (sample 1 omitted due to high noise level). IDeg is distinguished from HI in the aliphatic quartette as an increase in both the symmetric and asymmetric CH 2 /CH 3 intensity ratios at 2850 cm -1 /2870 cm -1 and 2931 cm - 1 /2961 cm -1, respectively, which we assign to the backbone of the hexadecanoic fatty acid chain. In conclusion, amyloid fibrils were not observed in dried samples of IDeg, neither with nor without phenol. This finding supports the conception that the elongated fiber-structures observed in TEM of phenol deprived IDeg bear no structural resemblance to amyloid fibrils. 8 of 12

9 Figure S6a: HATR spectra of IDeg formulation with (blue, sample 1) and without phenol (green, sample 2) and agitated samples of human insulin (red, sample 3; magenta, sample 4). Figure S6b: HATR spectra of IDeg formulation without phenol (green, sample 2) and agitated samples of human insulin (red, sample 3; magenta, sample 4). IDeg formulation with phenol omitted due to high noise level. 9 of 12

10 S7. Sedimentation velocity of IDeg multihexamers. Steep boundaries together with a considerable negative concentration dependence of the average sedimentation coefficient were observed for samples of IDeg containing 5 Zn 2+ /6Ins. Extrapolation to infinite dilution and correction to standard conditions yielded an s 0 20,w = 28.8 S /(s* av ) [S -1 ] concentration [mg/ml] Figure S7: Sedimentation velocity of IDeg multihexamers. The red line represents a linear extrapolation to infinite dilution of sedimentation coefficients measured at different concentrations of IDeg, containing 5 Zn 2+ /IDeg hexamer and no phenol. 10 of 12

11 S8. Sedimentation equilibrium of IDeg multihexamers. Sedimentation equilibrium experiments yielded an average molar mass at infinite dilution M0 = 59.7 x 10 3 kg/mol. The maximal, predicted sedimentation coefficient for a particle of the same molar mass is 524 S /(M x 10 3 ) [1/(kg/mole)] concentration [mg/ml] Figure S8: Sedimentation equilibrium of IDeg multihexamers. The red line represents a linear extrapolation to infinite dilution of molar masses measured at different concentrations of IDeg, containing 5 Zn 2+ /IDeg hexamer and no phenol. 11 of 12

12 S9. Circular dichroism of phenol deprived zinc complexes of IDeg. In order to examine the conformational state of the multihexamer of degludec we compared the elipticity at 251 nm indicative for the T-R conformational state of the zinc complexes with and without phenol. To mimic the change in ionic strength of the solvent and dissociation of phenol at the subcutaneous injection we dialyzed the preparations of degludec comprising zinc and phenol against a buffer without phenol but with 150 mm NaCl. Circular dichroism was measured before and after dialysis. The concentration of IDeg and zinc was determined and found to be virtually unchanged after dialysis. The data show that the multihexamers of IDeg adopt the T 6 conformation 0 ε@251nm (M -1 cm -1 ) Zn/6Ins before dialysis after dialysis Figure S9: Elipticity of degludec before and after removal of phenol by dialysis. 12 of 12