1

Insights into G-quadruplex specific recognition by the DEAH-box helicase

RHAU: Solution structure of a peptide-quadruplex complex

Brahim Heddi

†, Vee Vee Cheong

†, Herry Martadinata,

and Anh Tuân Phan*

School of Physical and Mathematical Sciences, Nanyang Technological University,

Singapore

*Correspondence to: phantuan@ntu.edu.sg †These authors contributed equally

Supporting Information (SI) Appendix

METHODS

Protein expression and purification

Recombinant plasmid coding for the His6-TEV-Rhau53 sequence was supplied by the Protein

Production Platform at School of Biological Sciences (SBS), Nanyang Technological

University (NTU), Singapore (https://www.proteins.sg).

Unlabeled, 15

N-labeled and 15

N,13

C-labeled Rhau53 protein production was performed as

described below. Cells were grown at 37 °C till the optical density (OD) at 600 nm reached

0.6 – 0.8, then they were induced by adding 0.5 mM IPTG, followed by 20 h incubation at 18

°C. The cells were collected by centrifugation at 10,000 rpm, 4 °C for 15 min, and re-

suspended in a buffer containing 20 mM KPi, 0.5 M KCl, 10 mM imidazole, 10 % (vol/vol)

glycerol, protease inhibitor cocktail (Roche), pH 7.5, and lysed via sonication. The crude

lysate was centrifuged at 30,000 rpm, 4 °C for 15 min; the supernatant was loaded onto

HisTrap HP column (GE Healthcare), pre-equilibrated with 20 mM KPi, 0.5 M KCl, 10 %

(vol/vol) glycerol, pH 7.5. The column was washed extensively at 10 mM and 25 mM

imidazole; the protein was eluted at 100 mM imidazole. The His6-tag was cleaved off from

Rhau53 after overnight digestion at 4 °C by Tobacco Etch Virus (TEV) protease, and

removed via cation chromatography using HiTrap SP HP column (GE Healthcare), pre-

equilibrated with 20 mM KPi, 10 % (vol/vol) glycerol, pH 7.5. The protein was further

purified through gel filtration chromatography using Superdex 75 10/300 GL column (GE

Healthcare), pre-equilibrated with 20 mM KPi, 70 mM KCl, 10 % (vol/vol) glycerol, pH 7.5

to obtain protein of ≥ 95% purity. Fractions containing Rhau53 were combined and

concentrated to 0.5 – 1 mL using 3000 MWCO centricon (Millipore), washed with a buffer

containing 20 mM KPi, 70 mM KCl, 10 % D2O, pH 6.6 before flash-freezing with liquid

nitrogen. 0.6 – 0.8 mg of unlabeled/labeled protein was produced per liter of Terrific Broth

(TB) medium.

The gene encoding for Rhau18 with an additional N-terminal TEV cleavage site was sub-

cloned from Rhau53 plasmid into pET32 Ek/LIC vector (Merck). This vector encoded for an

N-terminal thioredoxin (Trx-His6) fusion tag. The recombinant plasmid was transformed into

Rosetta T1R competent cells (Protein Production Platform), which were grown in TB media

or M9 minimal media as described above. Trx-tagged Rhau18, 15

N-Rhau18 and 15

N,13

C-

Rhau18 were produced and purified as described in the purification method of Rhau53 with

the following modifications: Trx-tagged Rhau18 was applied to HisTrap HP column (GE

2

Healthcare), washed at 10 mM and 20 mM imidazole prior to elution at 200 mM imidazole.

Trx tag was cut from Trx-tagged Rhau18 by TEV protease and separated from the peptide

pool using cation exchange chromatography. Rhau18 peptide was pooled and loaded onto

Superdex 75 10/300 GL column for further purification. Buffer exchange of the Rhau18 was

carried out with 20 mM KPi, 70 mM KCl, 10 % D2O, pH 6.6 using 2000 MWCO centricon

(Sartorius). Rhau18 was flash-frozen with liquid nitrogen and stored at -80 °C. Rhau18 was

characterized by MALDI-TOF and found to be ≥ 95 % pure. 5 – 10 mg of unlabeled/labeled

peptide was obtained per liter of culture.

Uniform isotopic-labeling of all proteins were achieved by growing cells in M9 minimal

media containing 0.5 g/L 15

NH4Cl and/or 2 g/L [13

C6]-D-glucose. Due to the TEV cleavage

site, all the proteins expressed in this work contained two extra amino acids (SM) at the N-

terminal.

Quantitative gel binding analysis

The gel binding data between the peptide and different DNA conformations was fitted using

the following equation:

𝛼 =(𝐾𝑑 + 𝑎 + 𝑏) − [(𝐾𝑑 + 𝑎 + 𝑏)2 − 4𝑎𝑏]1/2

2𝑎

where a represents the DNA concentration, b - the peptide concentration, - the fraction of

bound DNA, and Kd - the dissociation constant for DNA-peptide interaction (DNA + peptide

complex).

NMR spectroscopy

Triple-resonance HNCO, HNCACB, and CBCA(CO)NH experiments were used to assign the

backbone resonances of Rhau53 and Rhau18. For the free and bound Rhau53, 82.8 % and

80.2% of backbone 15

N, 1HN,

1H,

13C, and

13C’ resonance assignments have been

achieved, respectively. For Rhau18 and Rhau18-T95-2T, 89.3 % and 71.4 % of backbone 15

N, 1HN,

1H,

13C, and

13C’ resonance assignments have been achieved.

13

C-HSQC, CC(CO)NH, HCC(CO)NH, and HCCH-TOCSY were used to assign side-chain

resonances of Rhau18 and Rhau18-T95-2T. The side-chain assignments of aromatic residues

were further assisted by HNHA, 13

C-edited NOESY, 15

N-edited NOESY and 2D

(HB)CB(CGCD)HD and (HB)CB(CGCDCE)HE experiments (1).

DNA protons were assigned using a sample at the DNA/peptide ratio 1:0.5. Chemical

exchange cross-peaks were observed in NOESY spectra between peaks of the free and bound

form of T95-2T. The assignments were completed using 2D 13

C-15

N-filtered NOESY

experiments.

NOE constraints for the free Rhau18 were assigned and given distances using the automated

procedure form CYANA. The consistency of NOEs was visually cross-checked on the

NOESY spectra. NOE constraints for T95-2T were taken from the previously published

structure (2), except for those involving the two first thymine residues (T1 and T2).

Intramolecular NOE constraints for the bound Rhau18 were manually assigned to be strong

(1.50 – 4.00 Å), medium (1.50 – 5.00 Å) and weak (2.50 – 6.00 Å) from 13

C- and 15

N-edited

NOESY spectra. Intermolecular NOEs were assigned to be (2.50 – 7.00 Å) using 13

C-half

filtered NOESY (in which only the NOE peaks between the labeled protein and unlabeled

3

DNA are visible), 2D NOESY, and 15

N-edited NOESY spectra (for NOE peaks involving

well-resolved imino protons of DNA).

Dihedral φ/ψ angle constraints of Rhau18-T95-2T were derived from TALOS+ (3) and an

HNHA experiment.

Spectra were analyse using SpinWorks (http://home.cc.umanitoba.ca/~wolowiec/spinworks)

and Sparky software (4).

NMR-restrained structure calculation

The structure of the free and bound Rhau18 was calculated using the CYANA (5) and

XPLOR-NIH (6) programs. The computed structures were displayed using the PyMOL

viewer program (7). An initial extended conformation of T95-2T and Rhau18 was generated

using the XPLOR program and separated by ~30 Å. The system was then subjected to

distance geometry simulated annealing by incorporating the hydrogen-bond, distance and

planarity restraints. 100 structures were generated and subjected to further refinements as

previously described (8).

4

Figure S1. Native gel electrophoresis of DNA in the absence and presence of Rhau53: (A)

Htelo2, (B) Htelo3 (C) Htelo4 in dilute solution (left) and crowding condition (right).

Concentration of DNA and Rhau53 was 100 nM and 1000 nM, respectively.

5

Figure S2. Gel binding assays between different DNA G-quadruplexes and increasing

concentrations of Rhau53: (A) Htelo1, (B) Htelo1 under crowding condition, and (C) T95-2T.

Concentration of DNA was 100 nM; concentration of Rhau53 was 0, 100, 250, 500, 700,

1000, 2500, 5000, 10000, 25000 nM. The folding topology of each sequence is shown next to

the sequence name. Anti and syn guanines are colored cyan and magenta, respectively.

6

Figure S3. NMR imino proton spectra of DNA and RNA sequences used in this study (Table

2). Sequence name is labeled above each spectrum. The folding topology is shown next to

each spectrum. Anti and syn guanines are colored in cyan and magenta, respectively;

cytosines are colored in brown; bases in DNA duplexes are colored in red.

7

Figure S4. Native gel electrophoresis of different DNA and RNA in the absence and

presence of Rhau53: (A) parallel G4 DNA, (B) parallel G4 RNA, (C) non-parallel G4, and

(D) DNA duplexes. Sequence names are indicated above the lanes. The nucleic acids/Rhau53

ratio is indicated on top of each lane. DNA and RNA concentrations were fixed at 100 μM.

The gel was revealed using UV-shadowing.

8

Figure S5. (A) Folding topology of the sequence HT and T95-2T. (B) NMR imino proton

spectra of a mixture of HT (red open triangle) and T95-2T (black dot), in the absence

(bottom) and the presence (top) of Rhau53. The DNA/Rhau53 ratio is indicated next to each

spectrum.

9

Figure S6. HSQC spectra of Rhau53 free (black) and bound to T95-2T (red). Boxes represent

peaks observed at a lower threshold. Each dotted arrow shows the changes in chemical shifts

of a residue between the free and bound forms. Assignments of resonances in the Rhau53-

T95-2T complex are shown.

10

Figure S7. Native gel electrophoresis binding assay between T95-2T and different peptides.

Lanes 1 and 13, T95-2T; lanes 2-12: T95-2T in the presence of Rhau20m1, Rhau20m2,

Rhau5, Rhau9, Rhau12, Rhau14, Rhau16, Rhau20, Rhau18, Rhau29 and Rhau23. DNA

concentration was 100 μM. The DNA/peptide ratio was 1:5. DNA on the gel was revealed

using UV-shadowing.

11

Figure S8. NMR imino proton spectra of T95-2T in the absence and presence of Rhau53 (A),

Rhau18 (B) and Rhau16 (C). DNA concentration was 100 μM. The DNA/peptide ratio was

1:0.5. DNA Imino protons of DNA in the complex are marked by asterisks.

12

Figure S9. HSQC spectra of Rhau18 free (black) and bound to T95-2T (red). Each dotted

arrow shows the changes in chemical shifts of a residue between the free and bound form.

13

Figure S10. Native gel electrophoresis of DNA in the absence and presence of Rhau18. The

DNA/protein ratio is indicated on top of each lane. Sequence names are indicated above of

the lanes. The parallel conformation of HT was obtained under crowding condition.

14

Figure S11. (A) Cartoon representation of the ten superimposed lowest-energy structures of

Rhau18. (B) Side-view showing short distances between residues P4, L7, I12 and W15.

15

Figure S12. HNCACB/CBCACONH strips plots showing examples of the backbone

sequential connectivity of Rhau18 in complex with unlabeled T95-2T

16

Figure S13. NOE strip plots from the

13C-half filtered NOESY experiment showing the

intermolecular cross-peaks between the peptide and DNA.

17

Figure S14. (A) Superposition of the free (red, PDB code 2LK7) and bound (blue) T95-2T.

(B) Superposition of the free (red) and bound (blue) Rhau18.

18

Figure S15. (A) Structure of the Rhau18-T95-2T complex. Dotted lines represent

intermolecular NOEs observed between the peptide and the 5’-end tetrad. (B) NOESY

spectrum at 15 ºC showing the intermolecular NOEs between different protons of Rhau18

and the guanines imino protons of T95-2T.

19

Figure S16. (A) NMR imino proton spectra of T95-2T with different amounts of Rhau18’

(Table 1). The DNA/protein ratio is indicated next to each spectrum. At the 1:0.5

DNA/peptide ratio NMR proton spectrum shows two sets of peaks for DNA and only one for

the peptide (imino protons in the complex are marked by asterisks); while at the 1:2

DNA/peptide ratio, there are only one set of peaks for DNA and two for the peptide (signal of

Tryptophan H1 is labeled W15). (B) Model for binding between T95-2T and Rhau18’

showing the formation of a first complex containing 1 peptide molecule (at the 1:0.5

DNA/peptide ratio) and the formation of a second complex containing 2 peptide molecules

(at the 1:2 DNA/peptide ratio). (C) Imino protons of DNA in the new second complex were

unambiguously assigned using site-specific 15

N-labeled samples. Assignments of guanines

imino protons of T95-2T in the presence of more than 2-fold of Rhau18 were obtained from 15

N-filtered experiments, run on samples containing ~4% 15

N-labeled at the indicated

position. Reference spectrum is shown on the top. (D) NOESY spectrum (mixing time, 350

ms; temperature, 37 ºC) of T95-2T in the presence of more than 2-fold of Rhau18, showing

intermolecular NOEs between different protons of Rhau18 and the guanines imino protons of

T95-2T at the two terminal G-tetrads (labeled in red for the top 5’-end G-tetrad and blue for

the bottom 3’-end G-tetrad respectively). NOE data indicate that the second peptide molecule

binds at the bottom 3’-end G-tetrad of T95-2T. The binding at the 3’-end occurs at a higher

peptide concentration than the binding at the 5’-end, indicating a lower affinity for the 3’

bottom site. Unresolved protons of the two peptide molecules are marked with asterisks.

20

REFERENCES

1. Yamazaki T, Formankay JD, & Kay LE (1993) Two-dimensional NMR experiments

for correlating 13

Cβ and 1Hδ/ε chemical-shifts of aromatic residues in

13C-labeled

proteins via scalar couplings. J. Am. Chem. Soc. 115(23):11054-11055.

2. Do NQ & Phan AT (2012) Monomer-dimer equilibrium for the 5'-5' stacking of

propeller-type parallel-stranded G-quadruplexes: NMR structural study. Chemistry

18(46):14752-14759.

3. Shen Y, Delaglio F, Cornilescu G, & Bax A (2009) TALOS+: a hybrid method for

predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol.

NMR 44(4):213-223.

4. Goddard TD & Kneller DG (SPARKY 3, University of California, San Francisco).

5. Herrmann T, Guntert P, & Wuthrich K (2002) Protein NMR structure determination

with automated NOE assignment using the new software CANDID and the torsion

angle dynamics algorithm DYANA. J. Mol. Biol. 319(1):209-227.

6. Schwieters CD, Kuszewski JJ, Tjandra N, & Clore GM (2003) The Xplor-NIH NMR

molecular structure determination package. J. Magn. Reson. 160(1):65-73.

7. Schrodinger, LLC (2010) The PyMOL Molecular Graphics System, Version 1.3r1.

8. Chung WJ, et al. (2013) Solution structure of an intramolecular (3 + 1) human

telomeric G-quadruplex bound to a telomestatin derivative. J. Am. Chem. Soc.

135(36):13495-13501.