Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Abstract

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterisation is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a YSD saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used SPR with purified proteins to validate the kinetics measured using surface display. Positions where CcdB mutations lead to slower rejuvenation rates are largely involved in CcdA-binding, though there were several notable exceptions. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring kinetics of ternary complex formation for individual CcdB mutants in solution by FRET studies.

Mechanistic insights into global suppressors of protein folding defects

“…S60E, though not involved directly in CcdA interaction, alters the conformation and rigidity of the 39–46 loop that contacts CcdA and might therefore affect CcdB function ( S7 Fig ). V46L also similarly affects the rigidity of the 8–14 and 39–46 loops important for CcdA binding and GyrA14 rejuvenation [ 49 , 50 ]. In CcdB, the E11R and M32T suppressor mutations had lower stability than WT and conversely, the L42E mutant which is more stable than WT, failed to act as a suppressor.…”

“…These data demonstrate that while most suppressor mutations show small stabilization effects in the WT background, increased stability is neither necessary nor sufficient for global suppressor mutations. In separate studies from our laboratory, it was observed that though the individual suppressor mutations do not greatly alter affinity towards CcdA, mutations at all these positions significantly affected the rejuvenation process [ 50 ]. The functional importance of these CcdB residues, explains why the experimentally identified global suppressor mutations are not found in naturally occurring ccdB genes.…”

“…The CcdB positions 10, 12 and 46 are involved in CcdA binding and mutations at these positions as well as the nearby E11 residue will affect CcdA interaction and conformation of the CcdA interacting loop from residues 8 to 15 ( 8 YKRESRYR 15 ) (S7 Fig) . S60E, though not involved directly in CcdA interaction, alters the conformation and rigidity of the 39-46 loop that contacts CcdA and might therefore affect CcdB function (S7 Fig) . V46L also similarly affects the rigidity of the 8-14 and 39-46 loops important for CcdA binding and GyrA14 rejuvenation [49,50]. In CcdB, the E11R and M32T suppressor mutations had lower stability than WT and conversely, the L42E mutant which is more stable than WT, failed to act as a suppressor.…”