BPS2026 – Clustering the ensemble of an intrinsically disordered protein by contacts between contiguous regions of hydrophobicity reveals distinguishable conformational states

Intrinsically disordered proteins (IDPs) sample a broad distribution of conformational states rather than adopting a single conformation, representing the conformational ensemble. The lack of a defined structure makes it challenging to interpret how a missense mutation in the sequence of an IDP affects its ensemble. Current methods for interpreting the effects of a mutation include clustering the ensemble by radius of gyration or structural similarity, but they can overemphasize infrequent interactions and fail to capture broader conformational trends. Here, we developed an approach that clusters an ensemble by the presence of contacts between contiguous hydrophobic regions, or h-blobs, in the long-disordered brain-derived neurotrophic factor (BDNF) prodomain sequence. Using 2 μs all-atomistic enhanced molecular dynamics simulations of seven variants of the BDNF prodomain that contain a different charge-neutral amino acid (F, M, V, L, A, Y, and I) at position 66 of the sequence, we investigated how a missense mutation affects the ensemble of the prodomain. The two naturally occurring human variants (Val/Met) have known conformational differences due to specific methionine interactions, but we observed even greater differences caused by a mutation to aromatic or other aliphatic residues at this location. To understand the underlying mechanism, we decomposed the ensemble into five clusters based on just four blob contacts identified in the Val/Met variants. We found that the frequency distribution across only five clusters is sensitive to subtle sequence changes, and the average radius of gyration across the seven sequences is strongly anticorrelated with the frequency of these key contacts. These results provide a proof of principle: a simple clustering scheme, utilizing only four contacts inspired by naturally occurring variants, offers clear insight into the mechanism underlying sequence specificity in a 91-residue IDP.