Cell Reports, Volume 14

Supplemental Information

Structural and Functional Insights

into the Unwinding Mechanism of Bacteroides sp Pif1

Xianglian Zhou, Wendan Ren, Sakshibeedu R. Bharath, Xuhua Tang, Yang He, ChenChen, Zhou Liu, Dewang Li, and Haiwei Song

Supplementary information

Structural and functional insights into the unwinding mechanism of Bacteroides sp Pif1

Xianglian Zhou, Wendan Ren, Sakshibeedu R Bharath, Xuhua Tang, Yang He, Chen Chen,

Zhou Liu, Dewang Li and Haiwei Song

Supplementary experimental procedures

Cloning, overexpression and purification of hPif1-HD

The N-terminal truncated form of hPif1 corresponding to residues, 200-641 (hPif1-

HD) was cloned into pET-28a-sumo vector. The resultant plasmids were transformed into E.

coli BL21 (DE3) strain and protein was overexpressed for 12 hours at 18 °C after induction

with 0.1 mM IPTG. Cells were harvested and later resuspended in lysis buffer A containing

20 mM HEPES pH 7.5, 500 mM NaCl and 2 mM β-ME. The cell suspension was lysed by

sonication followed by centrifugation to segregate the cell debris from the supernatant

containing hPif1-HD. The soluble fraction was loaded on a 5 ml Talon resin charged with

Co2+. The column was washed with three column volumes of buffer A supplemented with 10

mM imidazole. Bound proteins were eluted using a linear gradient of buffer A supplemented

with 200 mM imidazole. Fractions containing hPif1-HD were pooled; desalted against buffer

A and the sumo tag was removed by enzymatic digestion using ULP1. The resultant tag free

hPif1-HD was purified to homogeneity using successive affinity and gel filtration

chromatographic steps. Fractions containing pure hPif1-HD were pooled, concentrated, fast

frozen in liquid nitrogen and stored at –80°C in buffer B consisting of 20mM HEPES pH 7.5,

500 mM NaCl, 2 mM DTT, and 10% glycerol.

Supplementary Figures and Legends

 

Figure S1. Sequence alignment of BaPif1, hPif1 and RecD2 The secondary structural elements of BaPif1 are marked on top of the sequence alignment. The helicase sequence motifs I, Ia, II, III, IV, V and VI are shown along with the Pif1-like sequence motifs A, B and C. The Pif1-signature motif is colored gold while the wedge region is shown in red. Residues mutated in the current study, Thr66, His68, Ser69, Phe75, Arg83, Glu85, Lys87, Phe88, Lys92, Ile118, Arg123, Val149, Gly249, Asn296, Ala355, Thr359 and Phe379 are marked with a green star. See also Figures 1 and 3.

 

 

Figure S2. Structures of Pif1-like helicases (A) Structural superposition of Pif1-like helicases, RecD2 and Dda bound to oligo-dT highlight the position of β-hairpin like structures implicated in unwinding dsDNA substrates. The domains 1A, 2A and 2B of RecD2 are colored green, salmon and blue while; Dda is shown in grey. The pin-like structures in RecD2 and Dda are colored magenta and green, respectively. The bound oligonucleotides in RecD2 and Dda are shown in yellow and grey, respectively and their orientations are marked. (B) Structures of BaPif1-ADF and hPif1-HD are superimposed at domain 1A. BaPif1-ADF is colored as in A, while hPif1-HD is in grey. (C) Structural comparison of BaPif1-ADF with BaPif1 show that the structures are identical to each other except for the bound ADP⋅AlF4

–. Domains 1A, 2A and 2B of BaPif1-ADF are colored in green, salmon and blue, respectively while BaPif1 is in grey. See also Figures 1 and 5.

  Figure S3. Functional characterization of BaPif1 and its mutants Isothermal calorimetric titrations of BaPif1 to dT10 in the presence (A) and absence (B) of ADP⋅AlF4

–. The upper panels show the binding isotherms and the lower panels show the integrated heat for each injection fitted to a single-site model. (C) and (E) Mutational effects of BaPif1 on the binding of a 20-nucleotide oligo-dT and unwinding of a tailed dsDNA. The relative DNA binding and unwinding activities of BaPif1 and its variants are averaged from three measurements and plotted in (D) and (F). The error bars indicate standard deviation of each set of three measurements. DNAH refers to the tailed dsDNA obtained by annealing of oligo-dH with its complementary strand DNAc. SDS-PAGE profile of the tested mutants along with the wildtype BaPif1 is presented underneath the panel E. See also Figure 2.

 

 

 

Figure S4. Electron density of bound ssDNA in BaPif1 (A) and (B) Electron densities corresponding to the bound ssDNA in BaPif1-dT and BaPif1-dH are shown along with oligo-dT and oligo-dH, respectively. Electron density corresponding to T3-T8 in BaPif1-dT is similar to the density around T3-C8 in BaPif1-dH. The additional nucleotides observed in BaPif1-dH belong to the duplex region of oligo-dH. Bending of the phosphate backbone is clearly visible between nucleotides, C8 and C9. Furthermore, C9 and G10 are oriented in a direction opposite to those of bases, T3-C8. See also Figure 2.

 

 

 

Figure S5. Structural comparison of the ternary complexes of Pif1-like helicases (A) BaPif1-dT (blue) and RecD2-ssDNA-ADPNP (yellow) are superimposed at their 1A domains. The pin and wedge are colored magenta and red, respectively. The bound oligo-dTs in BaPif1 and RecD2 are colored grey and green. The ternary complexes of BaPif1 and RecD2 are similar. (B) Comparison of the ssDNA binding sites between BaPif1-dT and RecD2-ssDNA-ADPNP. The bound oligo-dTs in BaPif1-dT and RecD2-ssDNA-ADPNP are represented as grey and green sticks while residues contacting them are shown in blue and yellow sticks, respectively. See also Figure 6.

 

 

Figure S6. Insights into duplex unwinding activity of BaPif1 (A) Cartoon representation of BaPif1 (colored as in Figure 1) with the modeled duplex DNA (yellow). The first nucleotide of a regular B-form DNA helix was superposed with C8 of BaPif1-dH to mimic the state of DNAH in the initial binding mode with BaPif1. The duplex region would clash with the wedge region (red), therefore causing the bending of the phosphate backbone of the tracking strand as observed in BaPif1-dH. (B) Close-up view showing the detailed interactions of the wedge region with the modeled duplex (yellow). In the wedge region, the main chain of Ser89 would clash with the modeled duplex while the side chains of Glu85 and Lys87 would protrude to the base paired region of the duplex. The other residues from the wedge region mutated in the study, Phe75, Arg83 and Phe88 are also shown. See also Figures 4 and 7.

 

 

 Figure S7. DNA binding and unwinding activities of BaPif1 and its mutants (A) BaPif1 was tested for unwinding DNAH used during crystallization in presence of ATP and ADP⋅AlF4

-. The enzyme is active in the presence of ATP and inactive in the presence of transition state analog of ATP. (B) Mutational effects of the residues in the wedge region of BaPif1 on the duplex DNA unwinding. (C) The relative unwinding activities of BaPif1 and its variants shown in B are averaged from three measurements and plotted. The error bars indicate standard deviation of each set of three measurements. DNAH refers to the tailed dsDNA obtained by annealing of oligo-dH with its complementary strand DNAc. See also Figures 2 and 3.                      

Supplementary Table S1. Data collection and refinement statistics

BaPif1-Se BaPif1-apo BaPif1-ADF BaPif1-dT BaPif1-dH hPif1-HD Data collection Space group P41 P41 P212121 P3121 P212121 P3221

Cell Parameters

(Å)

a 136.0 123.5 67.1 87.4 48.5 204.8 b 136.0 123.5 75.3 87.4 149.6 204.8 c 68.6 66.6 111.4 149.8 185.1 77.8

Resolution (Å) 68.0-3.5 (3.62-3.50)

61.7- 2.0 (2.05 -2.00)

62.4-2.1 (2.16- 2.10)

33.8-2.9 (3.08-2.90)

30.0-2.0 (2.03-2.00)

71.2-3.5 (3.78-3.50)

aRmerge 0.12(0.54) 0.09(0.74) 0.06 (0.9) 0.12 (1.5) 0.11(0.51) 0.18(0.86) CC1/2 0.99 (0.90) 0.99 (0.51) 0.99 (0.52) 0.99 (0.87) 0.99 (0.90) 0.99 (0.62) I/sigI 13.6 (4.4) 9.2 (1.7) 20.2 (1.9) 14.6 (2.1) 27.7 (2.7) 8.3 (2.3) Completeness 100(100) 91.3(88.9) 98.9(92.5) 99.9(99.8) 99.2(97.0) 94.9(87.6) Redundancy 8.1 (8.3) 3.2 (3.2) 7.5 (6.3) 9.4 (10) 8.6 (7.7) 5.9(5.9) Refinement Resolution (Å) 20-2.0

(2.05-2.00) 20-2.1 (2.20-2.10)

20-2.9 (2.98-2.90)

20-2.0 (2.05-2.00)

20-3.6 (3.70-3.60)

No. reflections 58,685 (4164)

29,547 (2132)

14398 (1038)

87089 (6107)

19680 (1022)

bRwork/cRfree

(%) 19.1/24.1 19.9/24.2 20.4/25.5 16.9/19.3 32.9/36.9

R.m.s deviation Bond length (Å) 0.013 0.012 0.008 0.013 0.012 Bond angle (°) 1.51 1.53 1.36 1.27 1.82 No. of atoms 6793 3649 3632 7534 5600

Protein 5948 3476 3479 6725 5534 ssDNA - - 120 299 -

Ligands/ions - 32 33 66 66 Water 845 141 - 444 -

Values in parentheses correspond to those of highest resolution shell. aRmerge = ∑|Ij-<I>|/∑Ij, where Ij is the intensity of an individual reflection, and <I> is the average intensity of that reflection. bRwork = ∑||Fo| - |Fc||/∑|Fc|, where Fo denotes the observed structure factor amplitude, and Fc denotes the structure factor amplitude calculated from the model.cRfree is as for Rwork but calculated with 5.0% of randomly chosen reflections omitted from the refinement.