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

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 host Escherichia 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.

Atomic-Resolution Structure of the Protein Encoded by Gene V of fd Bacteriophage in Complex with Viral ssDNA Determined by Magic-Angle Spinning Solid-State NMR

“…There are several significant structural changes detected throughout the sequence, reflected overall by a backbone (N, Co, C α ) RMSD of 6.4 Å between the ensemble-averaged structure of bound gVp and the X-ray structure of free gVp. Such significant conformational changes accompanying the nucleoprotein complex assembly process are expected given the CSP analysis previously reported by our lab and agree well with the results reported here, as will be shown in detail.…”

“…Some examples of unambiguous long-range contacts restricting this region (connecting carbon sites at the C-terminus region to carbon sites belonging to the hydrophobic core) are provided in the SI (Figure S7 and S8, Tables S11 and S12), corroborating this aspect of the calculated structure. Also, this result agrees well with the findings from the previous CSP results . The motional pathway for the transition between the free and bound gVp molecular conformations remains puzzling in terms of the change to the location of the C-terminus with respect to the core, and further research is required in order to decipher the trajectory of such a structural change.…”

“…(2) We assumed that any pair of carbon sites further apart than 16 Å in the X-ray structure of free gVp will not be close enough in the ssDNA-bound form of the protein to result in a spectrally detectable cross-peak. While evidence previously reported by our lab has demonstrated that the structure of the gVp protein undergoes significant changes upon binding, we can safely assume that the complex assembly process does not entail a complete rearrangement of the overall fold . (3) We removed contacts with a high probability of association with the homodimer interface.…”

“…34 In work previously reported by our lab, the isotropic chemical shifts of both free gVp and ssDNAbound gVp have been assigned. 35,36 Quantification and analysis of chemical shift perturbations (CSPs) led to the conclusion that gVp undergoes significant structural changes upon binding, and regions expected to undergo the most extensive structural modifications�including the DNA-binding loop, the core loop, and the C-terminus�were identified. 36 MAS ssNMR is also useful for protein structure elucidation based on acquisition of internuclear distance-dependent interactions and prediction of backbone torsion angles based on chemical shifts, 37 as well as acquisition of additional structural restraints.…”

“…35,36 Quantification and analysis of chemical shift perturbations (CSPs) led to the conclusion that gVp undergoes significant structural changes upon binding, and regions expected to undergo the most extensive structural modifications�including the DNA-binding loop, the core loop, and the C-terminus�were identified. 36 MAS ssNMR is also useful for protein structure elucidation based on acquisition of internuclear distance-dependent interactions and prediction of backbone torsion angles based on chemical shifts, 37 as well as acquisition of additional structural restraints. 38,39 Given a sufficient amount of restraints distributed along different regions of the sequence, a carefully designed calculation process can lead to a converged, viable structural model.…”