Martinez-Yamout, M. A. | Search bioRxiv

bioRxiv2026

From Hummingbird to Elephant: Amyloid Formation in Natural Transthyretin Variants

Transthyretin (TTR) is a secreted protein associated with cardiac and other amyloid diseases via misfolding. We have previously shown that agitation of human TTR solutions at neutral pH results in aggregation and fibril formation. Here we report that agitation-induced aggregation of TTR from species with very different heart rates (Annas hummingbird, hbTTR, and African elephant, aeTTR) differs from that of human TTR (huTTR). Aggregation of hbTTR is slow and favors formation of smaller, fibrillar aggregates, while aeTTR aggregation is rapid and favors larger, more amorphous particles. Spherical, early-stage oligomeric intermediates were found for all variants by mass photometry and electron microscopy. The slow aggregation of hbTTR matches its resistance to denaturation by 8 M urea. The widely different aggregation behavior exhibited by these naturally occurring TTR variants in response to mechanical agitation under close to physiological conditions provides insight into how small sequence differences can contribute to the evolutionary fitness of different animals.

biophysics↗

bioRxiv2025

Linker Length and Composition within Disordered Binding Motifs modulates the Avidity and Reversibility of a Multivalent Protein Interaction Switch

Intrinsically disordered proteins that mediate the cellular transcriptional response to hypoxia play important roles in turning on and turning off oxygen stress genes. In particular, the feedback inhibitor CITED2 operates a unidirectional switch that efficiently terminates the hypoxic response by displacing the C-terminal activation domain of the hypoxia-inducible factor HIF-1 from its complex with the TAZ1 domain of the transcriptional coactivators CBP and p300. Unidirectionality of the switch arises from subtle allosteric conformational changes in TAZ1 and from differences in the strength of thermodynamic coupling between the TAZ1-binding motifs in the multivalent HIF-1 and CITED2 activation domains. To investigate the role of binding cooperativity, we mutated a linker sequence in the HIF-1 activation domain to enhance or reduce the thermodynamic coupling between its TAZ1-binding motifs. An efficient native-gel assay shows that certain linker mutations enhance the affinity of HIF-1 for TAZ1, and fluorescence anisotropy competition and NMR measurements show that these mutants can compete with CITED2 for TAZ1 more effectively than wild-type HIF-1. The wide range of mutants, which include insertion, deletion, replacement and scrambling of residues in the linker, provide insights into the molecular basis for the exquisite tuning of the hypoxic switch: the TAZ1 affinity and consequent CITED2 competition enhancement depends both on the flexibility of the linker sequence (particularly the presence of glycine residues) and the unfavorable electrostatic interactions of a highly conserved arginine side chain in the center of the linker with an electropositive surface of TAZ1.

biophysics↗