Poster abstracts

Poster number 4 submitted by Tyler Billings

Cre recombinase adopts an auto-inhibited state enabled by an α-β secondary structure transition

Tyler Billings (Ohio State Biochemistry Program), Aparna Unnikrishnan, Deepak Yadav Kumar, Mark P. Foster (Department of Chemisty and Biochemistry)

Abstract:
Cre is a DNA recombinase capable of conservative, site-specific recombination between its target loxP sites. As a gene editing tool, Cre has been used extensively to study the genetic basis of human disease. Important aspects of its mechanism have been illuminated by crystallographic and recent cryo-EM structures of Cre-lox complexes.1-3 These structures indicate that the C-terminal helix N is the most persistent inter-protomer contact, which remains docked in trans so long as there is an adjacent protomer. Unexpectedly, NMR data on free Cre suggest helix N is rigid, whereas missing density in the map of a Cre-lox monomer complex suggests this region is flexible when not docked onto a neighboring protomer.4 This finding suggests that the protein must undergo a large conformational change to bind DNA, with potentially important functional consequences.

Characterizing the structures of Cre responsible for initial binding of loxP will reveal the structural transitions between the free enzyme and the numerous Cre-lox complexes. We are working to refine the NMR-based solution structure for the autoinhibited conformation of the Cre catalytic domain.4 Secondary NMR chemical shifts and NOEs suggest that residues from the inter-domain linker and helix N exhibit unexpected β-sheet propensity. We posit that this autoinhibited conformation plays an important role in target site discrimination by sequestering a motif that might otherwise promote complex assembly at off-target sites.

References:
1. Ghosh, Kaushik, et al. “Synapsis of LoxP Sites by Cre Recombinase*.” JBC, vol. 282, no. 33, Aug. 2007, pp. 24004–16. https://doi.org/10.1074/jbc.M703283200.
2. Gopaul, Deshmukh N., et al. “Structure of the Holliday Junction Intermediate in Cre–LoxP Site-Specific Recombination.” EMBO, vol. 17, no. 14, July 1998, pp. 4175–87. https://doi.org/10.1093/emboj/17.14.4175.
3. Stachowski K., et al. “Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM.” NAR, vol. 50, no. 3, Feb. 2022, pp. 1753-1769. https://doi.org/10.1093/nar/gkac032.
4. Unnikrishnan, Aparna, et al. “DNA Binding Induces a Cis-to-Trans Switch in Cre Recombinase to Enable Intasome Assembly.” PNAS, vol. 117, no. 40, Oct. 2020, pp. 24849–58. PubMed. https://doi.org/10.1073/pnas.2011448117.

Keywords: DNA recombination, NMR, protein dynamics