Poster abstracts

Poster number 4 submitted by Tyler Billings

Intrinsic Macromolecular Flexibility in Cre-lox Recombination

Tyler Billings (Ohio State Biochemistry Program)

Abstract:
Viral integrases have found utility in gene editing for a diverse set of model organisms. The premier example is the site-specific DNA recombinase of bacteriophage P1, Cre, which exchanges genetic information between loxP sites. Two Cre monomers bind to a loxP site to form Cre2-loxP dimers. These dimers then synapse to form recombinogenic Cre4-loxP2 tetramers.1 Diagonal Cre protomers mediate single-strand exchange steps through a highly orchestrated mechanism, resulting in the formation and resolution of a Holliday junction intermediate.2,3 Despite the wealth of structural information derived from x-ray crystallography, questions regarding the role of intrinsic macromolecular flexibility and its contributions to conformational selection, protomer activation, and Holliday junction resolution remain. We have employed TROSY-based NMR techniques to record high quality spectra of the 38.5 kDa apoenzyme Cre. Aiming to simplify the spectrum of Cre and its complexes, we have designed a Cre mutant amenable to segmental isotopic labeling.4 Spectroscopic validation has revealed that this mutant is mildly structurally perturbed in the unbound state, but upon binding the loxP sequence it recovers a near wild-type fold. This strategy will enable NMR dynamics measurements of catalytically important structural motifs in tetrameric Cre-DNA complexes.

References:
1. Gibb, B.; Gupta, K.; Ghosh, K.; Sharp, R.; Chen, J.; Van Duyne, G.D. Requirements for catalysis in the Cre recombinase active site. Nucleic Acids Res., 2010, 38(17): 5817-5832.
2. Gopaul, D.N.; Guo, F.; Van Duyne, G.D. Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO, 1998, 17(14): 4175-4187.
3. Pinkey, J.N.M.; Zawadzki, P.; Mazuryk, J.; Kapanidis, A.N. Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence. Biochem., 2012, 109(51): 20871-20876.
4. Li, J.; Zhang, Y.; Soubias, O.; Khago, D.; Chao, F.; Li, Y.; Shaw, K.; Byrd, R.A. Optimization of sortase A ligation for flexible engineering of complex protein systems. J. Biol. Chem., 2020, 295(9): 2664-2675.

Keywords: NMR spectroscopy, intrinsic dynamics, Cre recombinase