Domain-level rocking motion within a polymerase that translocates on single-stranded nucleic acid

Area of Science:

• Structural biology
• Virology
• Biochemistry

Background:

• Vaccinia virus poly(A) polymerase (VP55) is a unique enzyme capable of independent single-stranded nucleic acid (ssNA) translocation.
• Previous structural studies of VP55 were limited to its complex with the VP39 processivity factor.

Purpose of the Study:

• To determine the crystal structure of unliganded monomeric VP55.
• To elucidate the structural basis for VP55's independent translocation mechanism.
• To compare the structural dynamics of VP55 with and without its processivity factor, VP39.

Main Methods:

• X-ray crystallography of unliganded VP55 at 2.86 Å resolution.
• Comparative structural analysis of VP55 monomer, VP39-VP55 heterodimer, and VP39 monomer.

Main Results:

• The crystal structure of unliganded monomeric VP55 was solved, revealing backbone structural isoforms for the first time.
• VP55 monomers exhibit a `rocking' motion of the N-terminal domain, suggesting conformational flexibility absent in the VP39-bound state.
• Conformational changes were observed at the primer contact site and catalytic center in the absence of ligand.

Conclusions:

• The unliganded VP55 structure provides insights into its independent translocation mechanism.
• VP55's conformational flexibility may be modulated by VP39 binding, leading to different translocation strategies.
• This study completes the structural characterization of VP55 and VP39 in their monomeric and heterodimeric forms.