Protein structure. Engineering of a superhelicase through conformational control

Abstract

Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here we show that conformational control through intramolecular cross-linking of a helicase monomer with undetectable unwinding activity converts it into a superhelicase that can unwind thousands of base pairs processively, even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into the regulation of nucleic acid unwinding activity and introduces a monomeric superhelicase without nuclease activities, which may be useful for biotechnological applications.

Fig. 1. Crosslink-mediated conformational control of helicase activity

(A) Open and closed form Rep crystal structures (PDB entry 1UAA). Domains are colored and named. Cysteine pairs that were crosslinked to lock the protein into the closed (open) conformation are shown in red (orange). Distances between the pairs are noted. Close-ups show the pairs that were crosslinked. (B) Schematics of smFRET analysis. Brightness of donor (green) and acceptor (red) changes as unwinding progresses. (C) Representative single molecule time traces for Rep-X, Rep and Rep-Y.

Fig. 2. Rep-X processivity and force generation

(A) Schematics of optical tweezers assay for Rep-X DNA unwinding. (B) 6-kbp DNA unwinding traces (colored according to overhang length, SSB and force; offset for clarity). Background colors denote two laminar flows (see inset). (C) Distribution of unwinding speed (N=38). Mean speed and standard deviation for each molecule are plotted above (colors as in B). (D) Fraction of complete DNA unwinding events. Error bars represent 95% confidence bounds. (E) Unwinding traces by Rep-X in the fixed trap assay. (F) Normalized unwinding velocities of 58 Rep-X molecules plotted vs force. Error bars = s.e.m.

Fig. 3. Conformational control of PcrA helicase

A Representative smFRET time traces for a PcrA-X monomer. (B) Representative processive unwinding traces by PcrA-X in the optical tweezers assay. (C) Fractions of enzyme-DNA binding that led to processive unwinding of 6-kbp DNA in the optical tweezers assay. (D) The effect of RepD on PcrA was measured using smFRET assay. E_FRET histograms show that the PcrA bound to RepD adduct is biased toward the closed form (high E_FRET population) compared to PcrA bound to the bare oriD DNA. In C and D, error bars represent the 95% confidence bounds.