Probing inhibitory contacts between the regulatory and catalytic domains of CTP:phosphocholine cytidylyltransferase (CCT) using Transition metal ion Fluorescence Resonance Energy Transfer (tmFRET)

CCT catalyzes the rate-limiting step in phosphatidylcholine synthesis. CCT is activated when its lipid-induced amphipathic helix (domain M) binds to PC-deficient membranes and silenced when the auto-inhibitory motif (AI) within domain M binds helix αE in the catalytic domain. tmFRET was used to probe inter-domain interactions. Monobromobimane (donor) was conjugated to a cysteine engineered in αE or AI. My objective was to determine if a native di-histidine motif (89HSGH92) in the active site could function as a Cu2+ (acceptor) binding site. Fluorescence quenching occurred at Cu2+ concentrations above 10-5 M but persisted when the binding site was compromised via histidine protonation, H89S mutation, or competition with CDP-choline. Cu2+-quenching also persisted when domains M and C where dissociated by membrane binding. These results suggest that 89HSGH92 is not an effective Cu2+ binding site and quenching was likely collisional. Future work will require engineering a di-histidine motif elsewhere in the catalytic domain.