Tardigrades are microscopic aquatic animals that exhibit remarkable resistance to environmental stress, including radiation, cryogenic temperatures and desiccation, that would be fatal for the vast majority of living organisms. Although the origins of this extremotolerance are poorly understood, tardigrade-specific intrinsically disordered proteins are known to play a crucial role, allowing the organism to survive in a dormant state for long periods of time (years), and to function normally upon return to ambient conditions. We previously used NMR spectroscopy to demonstrate [1] that one such protein, CAHS-8 from Hypsibius exemplaris, is largely disordered in solution, with long (94 and 33 amino acids) dynamic tails at the N- and C-termini, flanking a central helical domain comprising two helices of 56 and 28 amino acids. Remarkably, this monomeric protein undergoes a reversible conformational transition in response to environmental change, self-assembling to form a fibrous hydrogel in a transition thought to be integral to tardigrade stress resistance.
The extraordinary properties of CAHS proteins to preserve biological material under extreme stress conditions without impacting the viability of the organism has made them the subject of curiosity and considerable research interest, not only because of their functional role, but also because of their exciting potential in terms of cryoprotection and biological preservation. Despite intense scrutiny, the molecular origin of the protection conferred by this conformational transition remains elusive.
Here we have used an integrated approach [2] , combining X-ray crystallography (ESRF) with NMR, atomic force and electron microscopy (IBS), to determine the atomic structure of the fibrils. The crystal structure of the fibril was resolved to 2.6Å resolution, while EM and AFM were combined to describe the supramolecular assembly, and NMR and molecular modelling to probe the behaviour of the disordered tails in the fibrillar assembly. Individual fibrils are formed from a single extended helix that dimerises via two distinct interfaces situated on alternate faces of the same helix. The combination of these two interfaces mediates a unique mode of assembly resulting in a continuous fibril. Fibrils interact in a pairwise manner, apparently via their disordered domains, to form straight fibres.
This study will inform further investigation of the molecular basis of stress resistance in tardigrades, while providing new avenues for peptide pharmaceuticals, drug delivery, preservation of biomass or design of stress-tolerant proteins.
A. Malki (IBS), J.-M. Toulon (IBS), E. Mikkola (IBS), D. Maurin (IBS), J.-L. Pellequer (IBS), M. Nanao (ESRF) and M. Blackledge (IBS)
[1] Malki A, Teulon JM, Camacho-Zarco AR, Chen SW et al. (2022) Angew Chem Int Ed Engl, 61, e202109961
[2] Malki A, Teulon JM, Mikkola EA, Maurin D et al. (2026) Angew Chem Int Ed Engl, 65, e19912
Figure 1: We present the atomic structure of the fibril formed by an intrinsically disordered protein that has been shown to be responsible for remarkable environmental stress in tardigrades. X-ray crystallography reveals a unique mechanism of fibril assembly whereby a single helix forms a stable, antiparallel coiled-coil dimer, which then assembles into a single continuous fibril via a second coiled-coil interface on the opposing face of the helix. (background negative staining EM, top left AFM, atomic structures (yellow and blue/magenta – X-ray crystallography).
Figure 2: NMR reveals that the long, disordered tails (red and blue) remain flexible in the fibril. Interactions between these domains appear to stabilise the formation of the straight fibre formed by adjacent fibrils.