Tamburrini, K. C. | Search bioRxiv

bioRxiv2026

Cooperativity, dynamics, and the free-energy surfaces of charge-patterned IDPs

The free-energy surfaces that underlie the conformational distributions of intrinsically disordered proteins (IDPs) are shallow and lack the deep minima characteristic of stable, folded structures. However, even in the absence of secondary or tertiary structure, sequence patterning can lead to conformational preferences and changes in chain dimensions as a function of solution conditions. While patterning effects have received extensive attention from simulation and theory, there is little corresponding data from experiment. Here we investigate the impact of charge patterning on chain dimensions and dynamics in a set of specifically designed polyampholytic IDP variants across the natural range of charge segregation with single-molecule FRET, nanosecond fluorescence correlation, circular dichroism, and NMR spectroscopy. We find that the conformational ensembles and their cooperative response to salt concentration show prominent and systematic dependencies on charge patterning, and to some extent on residue type. In contrast, the chain dynamics remain in the tens-of-nanosecond range, consistent with the absence of pronounced free-energy barriers. In close combination with molecular simulations, we show how the concept of susceptibility can be used to quantify cooperativity in the absence of barriers and relate it to the shallow free-energy surfaces of IDPs.

biophysics↗

bioRxiv2025

The C-terminal intrinsically disordered region of a fungal LPMO binds copper and displays anti-fungal properties

Lytic Polysaccharide Monooxygenases (LPMOs) are enzymes that play a crucial role in the degradation of complex polysaccharides such as cellulose and chitin. While LPMOs have attracted significant interest for industrial applications to convert biomass into biofuels, emerging evidence suggests alternative functions in fungal plant pathogenesis, microbial diseases and (micro)organism development. The AA14 LPMO family is widely distributed in filamentous fungi but remains enigmatic as its initially-suspected substrate specificity was recently challenged. In this study, we investigated the disordered C-terminal regions (dCTRs), found in more than half of AA14 family members and hypothesized to have functional relevance. Focusing on the Pycnoporus coccineus AA14A LPMO, we used small angle X-ray scattering (SAXS) and circular dichroism to show that its dCTR is highly disordered. It consists of a heavily glycosylated low complexity region followed by a charged C-terminal tail. We uncovered that the PcoAA14A dCTR binds copper ions through histidine residues located in the C-terminal tail. Using electron paramagnetic resonance (EPR), we further demonstrated that dimer formation occurs through an oxidative process involving copper and a redox-sensitive cysteine. Noting similarities between the last residues of the C-terminal tail and antimicrobial peptides, we investigated its potential antimicrobial function. We found that the positively charged region of the C-terminal tail selectively inhibits the growth of basidiomycete fungal spores. These data reveal that the dCTRs appended to AA14 LPMOs are functional regions that must be considered to unveil the role of these atypical LPMOs. Significance StatementLytic polysaccharide monooxygenases (LPMOs) degrade complex polysaccharides and are central to biomass conversion. Their biological relevance is expanding, with emerging roles in fungal development and host interactions. Despite this broad potential, research has largely focused on their catalytic domain. One area that remains poorly understood is the function of their C-terminal region, predicted to be intrinsically disordered (dCTR). The AA14 LPMO family, whose substrate specificity remains enigmatic, is enriched in dCTRs. This study provides the first experimental evidence of the disordered nature of the dCTR of an AA14 LPMO. Using multidisciplinary approaches, we demonstrate that the dCTR binds metals and exhibits antifungal activity. These findings establish dCTRs as functional regions, essential for understanding the biological roles of LPMOs.

biochemistry↗