Fuzzy supertertiary interactions within PSD-95 enable ligand binding.

The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by PDZ3, SH3, and Guanylate Kinase domains, regulate PSD-95 activity. Here we combined discrete molecular dynamics and single molecule Förster resonance energy transfer (FRET) to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand.

Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments.

Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Km values for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Km value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Km value in low ATP environments.