Water-organizing motif continuity is critical for potent ice nucleation protein activity

Jordan Forbes, Akalabya Bissoyi, Lukas Eickhoff, Naama Reicher, Thomas Hansen, Christopher G. Bon, Virginia K. Walker, Thomas Koop, Yinon Rudich, Ido Braslavsky, Peter L. Davies*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


Bacterial ice nucleation proteins (INPs) can cause frost damage to plants by nucleating ice formation at high sub-zero temperatures. Modeling of Pseudomonas borealis INP by AlphaFold suggests that the central domain of 65 tandem sixteen-residue repeats forms a beta-solenoid with arrays of outward-pointing threonines and tyrosines, which may organize water molecules into an ice-like pattern. Here we report that mutating some of these residues in a central segment of P. borealis INP, expressed in Escherichia coli, decreases ice nucleation activity more than the section’s deletion. Insertion of a bulky domain has the same effect, indicating that the continuity of the water-organizing repeats is critical for optimal activity. The ~10 C-terminal coils differ from the other 55 coils in being more basic and lacking water-organizing motifs; deletion of this region eliminates INP activity. We show through sequence modifications how arrays of conserved motifs form the large ice-nucleating surface required for potency.

Original languageAmerican English
Article number5019
JournalNature Communications
Issue number1
StatePublished - Dec 2022

Bibliographical note

Funding Information:
This work was in part funded by the CIHR Foundation award FRN 148422 to P.L.D., who holds the Canada Research Chair in Protein Engineering. A.B. acknowledges funding by the Planning and Budgeting Committee of the CHE in Israel, I.B. is grateful to the Israel Science Foundation (ISF 930/16), and V.K.W. acknowledges funding from NSERC Discovery Grant RGPIN-13289. We are thankful for the scientific contributions and technical expertise of Heather Tomalty and Rob Eves.

Funding Information:
Project Title: “Estudi de la presència i la representació de Catalunya a la premsa internacional (2019)”, funded by Diplocat, Generalitat de Catalunya, Reference number 7230-2019-40.

Publisher Copyright:
© 2022, The Author(s).


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