Precise and reversible protein microtubule-like structure

Supporting Information

of

Precise and reversible protein microtubule-like structure with

helicity driven by dual supramolecular interactions

Guang Yanga, Xiang Zhang

d, Zdravko Kochovski

b,c, Yufei Zhang

a, Bin Dai

d, Fuji Sakai

a,

Lin Jiangf, Yan Lu

b, Matthias Ballauff

b, Xueming Li

e, Cong Liu

d*, Guosong Chen

a*,

Ming Jianga

aThe State Key Laboratory of Molecular Engineering of Polymers and Department of

Macromolecular Science, Fudan University, Shanghai 200433, China

bSoft Matter and Functional Materials, Helmholtz-Zentrum Berlin für Materialien und

Energie, 14109 Berlin, Germany cTEM Group, Institute of Physics, Humboldt-Universität zu Berlin, 12489 Berlin,

Germany dInterdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of

Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China eMinistry of Education Key Laboratory of Protein Science, Center for Structural Biology,

Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua

University, Beijing 100084, China fDepartment of Neurology, Easton Center for Alzheimer’s Disease Research, David

Geffen School of Medicine, University of California, Los Angeles, California 90095,

USA

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Sample preparation

The small molecules were synthesized and characterized as described in supporting

information (Scheme S1 and Figure S22-42). SBA protein was purchased from Sigma-

Adrich. All chemicals and proteins are used as received. The buffer solution was

prepared with HEPES {4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid} buffer

containing 20 mM HEPES, 5 mM CaCl2, 5 mM MnCl2 and 40 mM NaCl. The SBA

solution was prepared by dissolving SBA (lyophilized powder) in buffer and was stored

over 2 h at 5 °C. R3GN or RnG was also dissolved in buffer separately. The solutions

were filtered through a Millipore 0.45 µm membrane before mixing. The SBA/R3GN

protein tube was prepared by mixing SBA and R3GN together in buffer solution, fixing

the concentrition of SBA and R3GN both at 0.2 mM and then the mixture was stored at

4 °C for 24 h. The protein tube at this condition can be stable for more than one year. The

SBA/RnG (n = 2, 3, 4) protein tubes were prepared under the same condition.

Characterization

Nuclear magnetic resonance (NMR) was taken by AVANCE III HD 400 MHz of

Bruker BioSpin International. Matrix Assisted Laser Desorption Ionization-Time Of

Flight (Maldi-TOF) Mass Spectrum was taken on a AB SCIEX 5800 instrument.

Ultraviolet–vis (UV-vis) absorption spectra were recorded by a Shimadzu UV-2550

spectrophotometer with a 1 mm cuvette. Circular dicroism spectra was taken by a

JASCO-815 instrument with a 1 mm cuvette. Small Angle X-ray Scattering expeiment

was conducted on a NanoStar U SAXS System. Isothermal titration calorimetry (ITC)

experiments were conducted on a MicroCal VP-ITC system at 20.00±0.01°C. Dynamic

light scattering (DLS) was taken by Zetasizer Nano ZS90 from Malvern Instruments

(UK).

Sample preparation and data collection by electron microscopy

For the preparation of negatively stained samples, a drop of the mixture solution was

applied onto a copper grid and the excess solvent was blotted away. Samples were

subsequently stained with 1 wt% uranyl acetate. Samples for Cryo-EM were prepared by

applying 4 µL drop of mixture solution to holey carbon grids (Quantifoil R2/1) and

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plunge-frozen into liquid ethane with an FEI vitrobot Mark IV set at 4°C and 95%

humidity. In the case of samples for Cryo-ET, 3 µL drop of 10 nm colloidal gold solution

(Aurion) was applied to the grids and allowed to dry before plunge freezing. Vitrified

grids were either transferred directly to the microscope cryoholder or stored in liquid

nitrogen. All grids were glow-discharged before use.

Cryo-EM and negative stain micrographs were acquired at a number of

magnifications on a JEOL JEM-2100 equipped with a 4 k × 4 k CMOS digital camera

(TVIPS TemCam-F416), operated at 200 kV and on a Philips CM120 operated at 80

kV. Tomographic data were acquired on a JEOL JEM-2100 equipped with a 4 k × 4 k

CMOS digital camera (TVIPS TemCam-F416), operated at 200 kV. Tilt series were

collected to ± 60° with a 2° angular increment and a total dose of either 100 e−/Å2 or 300

e−/Å2 for vitrified or negatively stained samples respectively. In all cases tilt series were

collected at a magnification of 30,000 x, corresponding to a pixel size of 3.9 Å at the

specimen level.

Cryo-EM Data Acquisition for 3D reconstruction

Cryo-EM grids were prepared with Vitrobot Mark IV (FEI), using 8 °C and 100

percent humidity. 4 µL of sample were applied to glow-discharged Quantifoil Cu

R1.2/1.3 grids, blotted for 2.5 s, and plunged into liquid ethane cooled by liquid nitrogen.

Images were taken by an FEI Titan Krios electron microscope operating at 300 kV with a

nominal magnification of 22,500x. Images were recorded by a Gatan K2 Summit detector

(Gatan Company) with the super-resolution mode, and binned to a pixel size of 1.32 Å.

Defocus values varied from 1.1 to 2.2 µm. Each image was dose-fractionated to 32

frames with a dose rate of ~8 counts per second per physical pixel (~6 e−/sÅ2), a total

exposure time of 8 s, and 0.25 s per frame. UCSF Image41 was used for all data

collection.

Image Processing and single particle reconstruction

The images were aligned and summed using the whole image motion correction2. The

defocus value of each image was determined by ctffind33. Micrographs were selected

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based on the quality of the micrograph and protein microtube. Protein microtube quality

was defined by length and straightness. A total of 2303 protein microtubes were

segmented by using EMAN2′s e2helixboxer program with step size of 6.34 nm (10%

overlap), resulting in an image stack of 65035 images of 63.4 × 63.4 nm. The segement

images was binned to the pixel size of 2.64 Å for the further 3D alignment and

reconstruction.

The helical symmety was roughly measeasured based on the layer lines in the power

spectrum of a single tube. Then IHRSR4 was used for further refinement. A cylinder was

used as the initial model. After 100 cycles of IHRSR refinement, a more accurate helical

parameters was determined. The helical rotation per subunit is -36.90o and the helical rise

per subunit is 21.22 Å. The 3D reconsturction was then improved by using FREALIGN

v8.095 and finally reach the resolution of 7.9 Å. The final reconstruction was sharpened

by applying an empirical negative B-factor of -700 Å and low-pass filered to 7.9 Å with a

soft cosine edge.

Notice that although the final density map showed a left-handed twisted helical

microtube structure, we observed both left- and right-handed helical microtube from 2D

classification. However, the resolution remained relatively low when we used a right-

handed helical model, which might due to the hetergenous structures of the right-handed

SBA microtube co-exist in solution.

Model building of SBA protein microtube without ligand

The B-factor sharpened map were used for model building in UCSF Chimera. The

crystal structure of SBA from Glycine max (PDB accession code: 1SBE) was first

docked into the density map as a single rigid-body tetramer defined as the biological

assembly. Individual monomers within the tetramer were then locally fitted into the

density to maximize the correlation between the model and the map. Finally the fitted

tetramer was used as a building block to build the whole filament.

Computational modeling of designed ligands in the protein microtube

The structure of R3GN ligand was built into our 7.9 Å density map based on crystal

structure of the previously designed RhB dimer and the complex of SBA tetramer

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binding to the sugar ring GalNAc (PDB entries: 4P9W and 1SBE). The whole modelling

process consists of several iterative rounds of two optimization steps: 1) internal ligand

minimization: the conformation of R3GN pair is generated from the crystal structure of

RhB dimer. In order to fit the R3GN pair into the density map, we miminized the pair

while kept the rigid body orientation between two GalNAc match with the sugar binding

sites of two adjacent SBA tetramer in the density map (Figure S12). The small molecule

modelling and minimization was using the Clean feature of WebLab Viewer Pro. 2)

protein-ligand docking: The R3GN pair was then docked into two adjacent SBA tetramer

in the density map by Rosetta6. During docking, the SBA sidechain conformation at

binding site was repacked and small pertubation of rigid body degree of freedom was

refined. Several iterative optimization rounds of internal ligand minimization and

protein-ligand docking were carried until there is no steric clash between SBA tube and

R3GN ligand. Finally the R3GN ligands were fitted into the SBA tube density map to

ensure reasonable interactions at the dimer interface of R3GN pair.

Atomic force microscopy (AFM)

AFM was operated in air on a Bruker Multimode VIII SPM equipped with a J

scanner. Experiments were performed in tapping mode with NSC11 tip (spring constant

48 N·m-1, MikroMasch). Sample (5 µL) was placed on a freshly cleaved mica for AFM

test under dry conditions. Sample solution was allowed to adsorb for 5 min and then it

was washed gently with 1 mL buffer followed by air drying.

Small Angle X-ray Scattering (SAXS)

0.2 mM SBA and equimolar R3GN was mixed together in buffer solution, and the

mixture was stored at 4 °C for 48 h, then the mixture was freeze-dried to obtain red

powder for SAXS experiment. Small Angle X-ray Scattering (SAXS) results were

performed on a Nanostar U small-angle X-ray scattering system (Germany) by using Cu

Ka radiation (40 kV, 35 mA) at room temperature.

Cell experiment

RAW264.7 macrophage cell line was cultured in RPMI 1640, supplemented with

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10% fetal bovine serum (GIBCO) and 1% antibiotic antimycotic solution (GIBCO).

RAW264.7 cells were seeded in 24 well plate with conc. of 0.1 million/well. Then cells

were treated with SBA, R3GN, E3GN or their fiber for 24 h at 37 °C . Supernatant was

collected for being detected with ELISA for cytokine secretion.

ELISA protocol

The concentrations of IL-6 in culture supernatant was measured by ELISA kit

(eBioscience) following the manufacturer's procedures. In brief, the captured antibody

was coated at 4°C overnight in 96 well plate with 100 µL/well. Then the wells were

aspirated and wahsed for 3 times with wash buffer. The wells were blocked with 200

µL/well of 1X ELISA Diluent. Then the wells were aspirated and washed 3 times again.

The samples (100 µL/well) were added to the appropriate wells at room temperature for 2

h followed by aspiration and washing of wells. Then 100 µL/well of detection antibody

was added at room temperature for 1 h, followed by aspiration and washing of wells.

Then 100 µL/well of Avidin-HRP was added at room temperature, followed by aspiration

and washing of wells for 5 times. Finally, 100 µL TMB was used for color development.

Optic densities at 450 nm were determined using a Microplate Reader (Model ELx800,

BioTek) within the linear regression. Each sample was measured in triplicates. Data was

presented as means ± SEM. Data was analyzed with one-way ANOVA. The statistical

significance level was setted as *p<0.05.

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Scheme S1. Synthetic procedure of small molecules used in this paper.

Figure S1. ITC data of titration of 1.2 mM E3GN into 0.1 mM SBA (calculated as

monomer) at 20°C in aqueous solution.

Figure S2. DLS results of SBA and SBA/R3GN mixture after 72 h incubation at 4 °C.

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Figure S3. The diameter distribution of SBA/R3GN microtubes observed under cryo-

EM.

Figure S4. 3-nm-thick xy, xz and yz orthogonal slices through the 3D tomographic

reconstruction of frozen-hydrated protein microtubes. The red cross marks the same

voxel in all slices. Inset: enlarged version of marked area. Scale bars: 100 nm in main

panel and 50 nm in inset.

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Figure S5. (a) TEM micrograph of negatively stained protein microtubes. (b) The

diameter distribution of the collapsed SBA/R3GN microtubules. c) AFM image of

collapsed microtubules and a representation of the microtubules collapsing to unilamellar

structure during drying. d) the height profile along the blue line of image c.

Figure S6666. 0.8-nm-thick xy, xz and yz orthogonal slices through the 3D tomographic

reconstruction of a negatively stained protein microtube. The red cross marks the same

voxel in all slices. Scale bar: 50 nm.

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Figure S7. SAXS result after freeze-drying of the microtubes.

Figure S8. (a) Cryo-EM micrograph of protein microtubes with curvature. (b) TEM

micrograph of negatively stained protein microtubes with curvature.

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Figure S9. Cryo-EM micrographs of SBA (0.2 mM) mixing with equivalent molar of (a)

R2G, (b) R3G and (c) R4G at 5 °C after 24 h incubation. (d) The diameter distribution of

(d) SBA/R2G, (e) SBA/R3G, and (f) SBA/R4G microtubes.

Figure S10. The Fourier shell correction (FSC) between two independently-reconstructed

volumes indicates that the resolution of the 3D reconstruction is ~ 7.9 Å with a 0.143

cutoff and is ~ 9.6 Å with a 0.5 cutoff.

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Figure S11. Superposition of the tetrameric SBA structure derived from the tube (Cyan)

and free tetrameric SBA structure in solution (Magenta, structure derived from crystal

structure, PDB accession code: 1SBE). The RMSD values for backbone atoms between

the two structures do not exceed 0.712 Å. Four 3,6-pentagalactose substrates7 are colored

in yellow.

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Figure S12. Demonstration of the electron density of R3GN within the tube. (a) The

SBA microtube strucutural model fitted in the electron density map with the R3GN

molecule highlighted in orange circle. (b) The R3GN molecules were omitted, so the

corresponding electron density of R3GN circled in orange can be directly visualized.

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Figure S13 Demonstration of the inter and intra-molecular interactions in R3GN dimeric

structure. The π-π interaction between trizole group and RhB within the same R3GN is

highlighted in red. The π-π interaction between neighbooring RhB is colored in blue.

Figure S14. Structural comparison between the R3GN molecule derived from the tube

model and the crystal structures of related molecules. (a) The structure of GalNAc-EG3-

RhB dimer is overlayed with SBA tetramer-GAL complex (PDB accession code: 1SBE).

The GAL molecule is colored in cyan. The R3GN model matches well with the SBA

tetramer-GAL interface in crystals. (b) The R3GN molecule recapitulates the RhB dimer

π-π interface observed in the crystal structure of ConA-RhB complex (PDB accession

code: 4P9W, green).

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Figure S15. (a) The UV-vis absorbance of SBA/R3GN (0.2 mM) mixture before and

after 72 h incubation in buffer at 5 °C. (b) The RhB dimer scheme according to the

literature8,9. (c) The enlarged UV-vis absorbance of SBA/R3GN (0.2 mM) mixture in (a).

(d) The corresponding CD spectrum of SBA/R3GN (0.2 mM) mixture before and after

72 h incubation in buffer at 5 °C.

Figure S16. Cryo-EM micrographs of SBA/R3GN mixture (0.2 mM) at different

incubation time interval (a) 2 h, (b) 5 h, (c) 24 h at 5 °C.

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Figure S17. Cryo-EM micrographs of SBA (0.2 mM)/R3GN mixture with different

concentrations of R3GN. (a) [R3GN] = 0.2 mM, (b) [R3GN] = 0.3 mM, (c) [R3GN] =

0.4 mM.

Figure S18. (a) Images of the protein microtube solution stored for (A) 2 m, (B) 6 m, (C)

1 y at 4°C. b) TEM micrograph with negative staining of the protein microtube after 6 m.

Figure S19. (a) CD spectra for control experiments with different ligands or incubation

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time. (b) CD spectra of R3GN/SBA mixture (after 72 h incubation) in the absence (black

line) and presence of β-cyclodextrin (red line).

Figure S20. (a) Cryo-EM micrograph of SBA/R3GN microtube solution after adding 1

mM β-CD at 4 ºC for 1 h. (b) Cryo-EM micrograph of SBA/R3GN/β-CD after adding 2

mM Ada for 48 h at 4°C.

Figure S21. (left) The UV-vis spectra of R3GN at different concentrations in aqueous

solution, the insets are the enlarged UV-vis spectra at different wavelength scale. (right)

The standard curves for R3GN (at 275 nm and 560 nm).

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Synthetic procedures and characterizations:

Synthesis of EG3N3. 4 g (26 mmol) triethylene glycol and 2.6 g (10 mmol) triethylamine

were mixed together in dry 60 mL THF and then 3 g (10 mmol) mesylate chloride (MsCl)

was added slowly to the stirred solution incubated in iced water, After stirring for 3 h at

room temperature, the precipitate was filtered out and the solution was evaporated. The

oily raw product without further purification was then directly dissolved in 80 mL

ethanol/ water (v:v = 5:1), and then 5.2 g (30 mmol) NaN3 was added. The solution was

refluxed about 24 h. Then excess NaN3 was removed by filtration and the solvent was

removed by rotavap. The raw product was purified by column chromatography with

Hexane/EtOAc = 1:1 (v/v) to give product (2.1 g, 46%) as colourless oil. 1H NMR (400

MHz, CDCl3): δ 3.71 (t, 2H), 3.69-3.64 (m, 6H), 3.61-3.58 (t, 2H), 3.41-3.36 (t, 2H), 2.48

(s,1H).

Synthesis of EG2N3 and EG4N3. The two compounds were synthesized according to the

same procedure.

Synthesis of Rh3N3. Rhodamine B carboxyloyl chloride (RhCl) was synthesized

according to literature10. 640 mg (1.4 mmol) RhCl and 141 mg (1.4 mmol) Et3N were

dissolved in 10 mL anhydrous DCM under Ar atomosphere, and then 193 mg (1.1 mmol)

EG3N3 in 2 mL anhydrous DCM was added into the solution dropwise and the mixture

was stirred at room temperature overnight. Then the solvent was removed by evaporation

and the raw product was purified by column chromatography with DCM/MeOH from

50:1 (v/v) to 10:1 (v/v) to give Rh3N3 (500 mg, 71%) as red oil. MALDI-TOF Mass

Spectrum (M/z): [Rh3N3-Cl]+ calcd. for C34H42N5O5, 600.31; found, 600.28. 1H NMR

(400 MHz, CD3OD): δ 8.38-8.36 (m, 1H), 7.89-7.83 (m, 2H), 7.47-7.45 (t, 1H), 7.17-

7.02(m, 6H), 4.14-4.12 (m, 2H), 3.74-3.68 (m, 8), 3.65-3.32 (m, 10H), 1.35-1.32 (t, 12H).

Synthesis of Rh2N3 and Rh4N3. The two compounds were synthesized according to the

same procedure.

Synthesis of AGalNAc. The propargyl N-acetylgalactosamine was synthesized according

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to the literature11. The ratio of α and β isomer was 9:1. 1H NMR of AGalNAc (400 MHz,

CD3OD-d4): δ 5.09-5.03 (m, 1H), 4.35-4.27 (m, 3H), 3.92-3.90 (t, 1H), 3.83-3.72 (m, 4H),

3.37 (s, 1H), 2.88-2.86 (t, 1H), 2.01(s, 3H).

Synthesis of propargyl α-galactopyranoside and propargyl α-mannopyranoside. The two

compounds were synthesized according to the same procedure.

Synthesis of R3GN. 200 mg (0.31 mmol) Rh3N3, 55 mg (0.31 mmol) AGalNAc and 26

mg (0.15 mmol) N,N,N',N'',N''-Pentamethyldiethylenetriamine (PMDTA) were added to

5 mL DMF and then after bubbling Ar for 5 min, 11 mg (0.075 mmol) CuBr was added

accompanied by bubbling Ar for another 10 min. The mixture was then heated to 50°C

for 24 h. Then the solvent was removed by evaporation and the raw product was purified

by column chromatography with DCM/MeOH from 50:1 (v/v) to 10:1 (v/v) to give

R3GN (109 mg, 39%) as red solid. MALDI-TOF Mass Spectrum (M/z): [RGN – Cl]+

calcd. for C45H59N6O11, 859.42; found, 859.06. 1H NMR (400 MHz, CD3OD): δ 8.36-

8.34 (m, 1H), 7.97-7.89 (d, 1H), 7.87-7.83 (m, 2H), 7.46-7.44 (m, 1H), 7.15-76.99 (m,

6H), 5.00-4.99 (d, 2H), 4.54-4.51 (m, 2H), 4.13-4.08 (m, 4H), 3.84-3.80 (t, 3H), 3.72-

3.69 (m, 20H), 3.50-3.43 (m, 2H), 3.42-3.37 (m, 3H), 1.97-1.92 (d, 3H), 1.34-1.32 (t,

12H).

Synthesis of R2G, R3G and R4G. The three compounds were synthesized according the

same procedure as R3GN. RM was reported in our previous paper11.

Synthesis of E3GN. 100 mg (0.38 mmol) EG3N3, 70 mg (0.4 mmol) AGalNAc and 34.6

mg (0.2 mmol) N,N,N',N'',N''-Pentamethyldiethylenetriamine (PMDTA) in 5 mL DMF

was stirred after bubbling Ar for 5 min, then 14.3 mg (0.1 mmol) CuBr was added

accompanied by bubbling Ar for another 10 min. The mixture was heated to 50°C for 24

h. Then the solvent was removed by evaporation and the raw product was purified by

column chromatography with DCM/MeOH from 20:1 (v/v) to 10:1 (v/v) to give E3GN

(68 mg, 41%) as colourless solid. MALDI-TOF Mass Spectrum (M/z): [E3GN + Na]+

calcd. for C17H30N4O9Na, 457.20; found, 457.14. 1H NMR (400 MHz, CD3OD-d4): δ

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8.03 (s, 1H), 4.81 (d, 1H), 4.04-3.98 (d, 2H), 3.85-3.82 (m, 4H), 3.62-3.58 (t, 2), 3.55-

3.47 (m, 8H), 3.42-3.39 (m, 2H), 1.84 (s, 3H).

Figure S22. 1H NMR of EG3N3 in CDCl3.


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Figure S25. 1H NMR of AGalNAc in CD3OD.

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Figure S26. 1H NMR of propargyl n-galactose in CD3OD.

Figure S27. 1H NMR of Rh2N3 in CD3OD.

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Figure S30. 1H NMR of R2G in CD3OD.


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Figure S32. 1H NMR of R3GN in CD3OD.


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Figure S34. 1H NMR of E3GN in CD3OD.

Figure S35. The MALDI-TOF Mass Spectrum of AGalNAc.

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Figure36. The MALDI-TOF Mass Spectrum of Rh3N3.

Figure S37. The MALDI-TOF Mass Spectrum of Rh2N3.

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Figure S38. The MALDI-TOF Mass Spectrum of Rh4N3.

Figure S39. The MALDI-TOF Mass Spectrum of R2G.

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Figure S42. The MALDI-TOF Mass Spectrum of R3GN.

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Precise and reversible protein microtubule-like structure