An alternative strained configuration was required to accommodate the peptide in the trans conformation. This was corroborated by MD analyses of the variant peptides revealing similar behaviour, consistent with the experimental data.

To explain this preference, we turned to molecular dynamics simulations. The trans-bound state of PRLR was unstable in the canonical crystal like configuration. Significant rearrangement of the trans-bound PRLR was observed after around 600 ns of simulation.

We quantified binding and found that 14-3-3 binds cis PRLR with 1000x higher affinity than trans. This isomer selectivity was conserved for PRLR and for 14-3-3 generally linked to phosphothreonine motifs.

The results were striking: Cis PRLR levels dropped instantly upon 14-3-3 addition, while trans remained largely unaffected. Over the next hour, free-state cis/trans re-equilibrated confirming the origin of the changes.

Using real-time 1D proton NMR, we can resolve distinct peaks corresponding to both cis and trans proline PRLR. Since 14-3-3 binding broadens bound signals beyond detection, we could track free cis/trans PRLR isomers concentrations over time.

Could this be a general mechanism in ID-based interactions? We had to dig deeper!

Repeating our ITC experiments confirmed: equilibration times exceeding 300 seconds! This suggested a major kinetic barrier, so we turned to NMR spectroscopy to get a direct look at the binding process.