Excited to share our PEG model for disordered proteins in CALVADOS!

If you are interested in exploring the effects of a crowder on the global dimensions of an IDP or want to explore the phase separation behaviour of a more weakly PS-prone IDP, have a look at our preprint and give it a try.

Proline cis/trans Conformational Selection Controls 14–3–3 Binding Intrinsically disordered protein regions (IDRs) are structurally dynamic yet functional, often interacting with other proteins through short linear motifs (SLiMs). Proline residues in IDRs introduce conformational heterogeneity on a uniquely slow time scale arising from cis/trans isomerization of the Xaa-Pro peptide bond. Here, we explore the role of proline isomerization in the interaction between the prolactin receptor (PRLR) and 14–3–3. Using NMR spectroscopy, thermodynamic profiling, and molecular dynamics (MD) simulations, we uncover a unique proline isomer-dependent binding, with a cis conformation affinity 3 orders of magnitude higher than the trans. MD simulations identify structural constraints in the narrow 14–3–3 binding groove that provide an explanation for the observed isomer selectivity. The cis preference of WT PRLR introduces a slow kinetic component relevant to signal propagation and a steric component that impacts chain direction. Proline isomerization constitutes a previously unrecognized selective component relevant to the ubiquitous 14–3–3 interactome. Given the prevalence of prolines in IDRs and SLiMs, our study highlights the importance of considering the distinct properties of proline isomers in experimental design and data interpretation to fully comprehend IDR functionality.

Excited to be on my way #TGV to #BPS2025! I’ll be presenting on proline isomerization in ID-based protein interactions, covering our recent paper:
doi.org/10.1021/jacs.4c13462

Talk: Mon, Feb 17, 11:45 AM, 502B
Poster: B154

Looking forward to discussing prolines in IDPs and how to study!

If you are an undergrad or MSc student and you have good qualifications, you can apply to a @csic.es JAE-Intro fellowship. If interested in protein simulaiton and design, contact me by DM. Please retweet.

Together with DREB2A, this study highlights the multiple ways in which proline isomerization tunes IDP function #REPIN
Thanks to my amazing co-authors @bbkrage.bsky.social, Andreas Prestel and others & to the Novo Nordisk Foundation for funding this work.

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.

Experimental setup of the time dependent NMR

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.

Molecular switching in transcription through splicing and proline-isomerization regulates stress responses in plants - Nature Communications Transcription factor DREB2A interacts with Med25 to regulate stress responses. Here, the authors show that DREB2A uses splicing and proline-isomerization for this regulation and that proline cis-trans...

While studying the interaction between 14-3-3 and the disordered PRLR intracellular domain, we noticed unusual kinetics: super slow equilibration even at high temperatures, consistent with proline isomerization. Reminiscent of our recent work on DREB2A, where proline isomerization played a role.

Proline cis/trans Conformational Selection Controls 14–3–3 Binding Intrinsically disordered protein regions (IDRs) are structurally dynamic yet functional, often interacting with other proteins through short linear motifs (SLiMs). Proline residues in IDRs introduce c...

Excited for my first post on Bluesky & to share my latest work on proline isomerization in disorder-based protein interactions! A thread on what we found 🧵:
doi.org/10.1021/jacs...

The paper ”Stereochemistry in the disorder–order continuum of protein interactions” from my colleagues @estellaan.bsky.social, Johan Olsen, @bbkrage.bsky.social et al is now out:
doi.org/10.1038/s415... 🍝 🧪 🧬 🧶