.2022 Jun 10;14(6):1264.

doi: 10.3390/v14061264.

Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR

Smadar Kedem¹, Roni Rene Hassid¹, Yoav Shamir¹, Amir Goldbourt¹

Affiliations

PMID:35746735
PMCID: PMC9231167
DOI: 10.3390/v14061264

Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR

Smadar Kedem et al. Viruses.2022.

.2022 Jun 10;14(6):1264.

doi: 10.3390/v14061264.

Authors

Smadar Kedem¹, Roni Rene Hassid¹, Yoav Shamir¹, Amir Goldbourt¹

Affiliation

¹ School of Chemistry, Tel Aviv University, Ramat Aviv, Tel Aviv 6997801, Israel.

PMID:35746735
PMCID: PMC9231167
DOI: 10.3390/v14061264

Abstract

Gene V protein (gVp) of the bacteriophages of the Ff family is a non-specific single-stranded DNA (ssDNA) binding protein. gVp binds to viral DNA during phage replication inside hostEscherichia coli cells, thereby blocking further replication and signaling the assembly of new phage particles. gVp is a dimer in solution and in crystal form. A structural model of the complex between gVp and ssDNA was obtained via docking the free gVp to structures of short ssDNA segments and via the detection of residues involved in DNA binding in solution. Using solid-state NMR, we characterized structural features of the gVp in complex with full-length viral ssDNA. We show that gVp binds ssDNA with an average distance of 5.5 Å between the amino acid residues of the protein and the phosphate backbone of the DNA. Torsion angle predictions and chemical shift perturbations indicate that there were considerable structural changes throughout the protein upon complexation with ssDNA, with the most significant variations occurring at the ssDNA binding loop and the C-terminus. Our data suggests that the structure of gVp in complex with ssDNA differs significantly from the structure of gVp in the free form, presumably to allow for cooperative binding of dimers to form the filamentous phage particle.

Keywords: DNA binding protein; fd bacteriophage; gVp; protein–DNA interactions; solid-state NMR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Ribbon diagram of gVp structure as determined using solution NMR [16]. (A) The gVp dimer with a symmetry axis parallel to the plane of illustration. (B) View of the gVp monomer rotated around the Y-axis on panel A. The three major loops, the dyad loop (red), the complex loop (orange), and the DNA binding loop (blue), are indicated. The images were reproduced from data stored in the Protein Data Bank, pdb id 2GVB, using the software PyMOL [24].

**Figure 2**
Agarose gel analysis of complexation of gVp with the circular ssDNA from the fd phage. The numbers above lanes indicate the ratio of nucleotides to protein monomers. The gel was stained with SYBR^TM GOLD (Invitrogen, Waltham, MA, USA). ‘DNA’ indicates the lane with fd ssDNA in the absence of protein. ‘M’ indicates the lane with the DNA ladder (GeneDireX, Taoyuan, Taiwan) with bands corresponding from bottom to top to 0.250, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 8.0, and 10.0 kbp. The image was slightly processed to reduce brightness and increase visibility.

**Figure 3**
Spectral overlay of two 2D¹³C-¹³C DARR spectra of U-gVp/NA-DNA (orange) and of NA-gVp/U-DNA (blue). The mixing times were 5 ms and 100 ms, respectively. Signals of the unlabeled protein are observed on the diagonal of the blue spectrum, where many correlations are observed in the orange spectrum, including the aliphatic and carbonyl protein-unique regions. Signals due to correlations between ssDNA ribose sugar carbons (C1′–C5′) and of the thymine methyl group (TC7) with other base carbons (TC2, TC4, TC5, TC6) and with C4′ are marked. Weaker thymine base–sugar correlations with C1′/C4′ are also visible.

**Figure 4**
REDOR analysis of gVp-ssDNA complex. (A)³¹P S (black) and S₀ (red) REDOR signals of gVp-ssDNA complex that were obtained at a mixing time of 3.23 ms. (B)³¹P{¹³C} REDOR fraction curve and SIMPSON simulations showing the best fit at 5.5 ± 0.5 Å. Simulations were obtained for a four-spin system consisting of one phosphorous spin and three carbon spins.³¹P shifts are referenced relative to O-phospho-L-Serine at 0.3 ppm.

**Figure 5**
Four different spectra of U-gVp/NA-DNA complex. (A) The 2D¹³C-¹³C DARR spectrum acquired with a 5 ms mixing time (DARR5); (B) 2D (¹⁵N)¹³Cα¹³Cx projection from 3D NCACX25; (C) 2D¹⁵N-¹³CO; (D) 2D¹⁵N-¹³Cα; (E)¹⁵N(¹³Cα)¹³Cx projection from the 3D NCACX25 experiment. Sequential assignments of S67 and C33 are indicated using blue and red dashed lines, respectively. All spectra were processed with a sine-squared window function.

**Figure 6**
Representative strip plots of residues 9-15 from U-gVp/NA-DNA complex. Sequential assignments from the 3D spectra of NCACX (blue) and NCOCX (red) are illustrated. Correlations with matching Cα and CO chemical shifts are connected with solid lines.

**Figure 7**
¹³C zfr-INADEQUATE 2D spectrum of U-gVp–NA-DNA complex. The spectrum depicts one-bond connectivities (dashed lines) at their sum frequencies in the double-quantum indirect dimension. Many of the residues involved were not observed in other dipolar-based spectra. A Lorentz-to-Gauss transformation was applied in both dimensions.

**Figure 8**
Comparison of several typical regions from¹³C-¹³C DARR correlation spectra of free gVp (green, mixing time 100 ms) and ssDNA-bound gVp (orange, mixing time 15 ms). Fourteen contours were drawn at multiples of 1.4 with the lowest contour at a signal-to-noise value of 10. Clear spectral changes occur upon complexation across the entire protein.

**Figure 9**
Weighted RMSD values extracted from chemical shift perturbations of free and bound gVp. (A) RMSD plot calculated by giving the¹⁵N shifts one-quarter the weight of carbons. The different regions of the protein are colored as follows: blue, DNA binding loop (residues 13–31); yellow, complex loop (residues 36–43); green, dyad loop (residues 68–78); purple, remainder of the protein. A five-point moving average was plotted as a dashed line. There were no data for residues 1, 41, 42, and 87. (B) RMSD values plotted on the structure of the dimer structure from X-ray crystallography (pdb id 1vqb).

**Figure 10**
Quadratic average of the backbone torsion-angle differences $Δ ϕ = ϕ_{f r e e} - ϕ_{b o u n d}$ and $Δ ψ = ψ_{f r e e} - ψ_{b o u n d}$ , given by $\sqrt{0.5 (Δ ϕ^{2} + Δ ψ^{2})}$ , between the free and ssDNA-bound gVp. Predictions with low reliability in TALOS are marked with ‘x’. The dashed line represents the average of all entries having values < 30 degrees and equals 11.6 degrees. The dotted line is the 5-point moving average.

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This research was funded by the Israel Science Foundation (ISF), grant number 847/17, and by the ISF-NFSC, grant number 2423/18.

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Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR

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Conformational Changes in Ff Phage Protein gVp upon Complexation with Its Viral Single-Stranded DNA Revealed Using Magic-Angle Spinning Solid-State NMR

Authors

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Conflict of interest statement

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