Dynein light chain 8 (LC8) is a dimeric hub protein that symmetrically and sequentially binds a pair of intrinsically disordered regions of proteins (IDPs) to two identical binding grooves. LC8 has >100 varied interaction partners, regulating critical cellular processes like apoptosis and viral interactions. While all known physiological roles for LC8 involve homotypic binding of the same client at both sites, it has remained unknown whether structural properties of LC8 alone can favor symmetric binding. We performed isothermal titration calorimetry (ITC) experiments with uncertainty quantified by Bayesian inference, which reveal that LC8 exhibits sequence-dependent cooperativity: the identity of the first-bound client differentially affects the affinities of the second site depending on client sequence. To our knowledge, this phenomenon has not been reported before and its mechanisms are unknown. To understand the structural behavior underpinning the thermodynamic measurements, we use multi-μs molecular dynamics (MD) simulations of half-bound LC8 to different client peptides and quantify fluctuations at the unbound site, i.e., the “site of cooperativity.” The use of replicate simulations and an alignment-free analysis, based on inter-residue distances, enable a stringent assessment of cooperativity differences among peptides. Our work establishes an apparently novel biophysical phenomenon and attempts to build a bridge between structural mechanisms and binding thermodynamics.
Case et al. (Sun,) studied this question.