Abstract
Multistep protein-protein interactions underlie most biological processes, but their characterization through methods such as isothermal titration calorimetry (ITC) is largely confined to simple models that provide little information on the intermediate, individual steps. We examine the hub protein LC8, which binds to disordered regions of 100+ client proteins in a wide range of stoichiometries. Despite evidence that LC8 binds clients cooperatively, prior ITC thermodynamic analyses have relied on models that do not accommodate allostery, and furthermore do not account for critical uncertainties in analyte concentrations. To characterize allostery in a more rigorous fashion, we build on existing Bayesian approaches to ITC to quantify thermodynamic parameters for multi-step binding interactions impacted by significant uncertainty in protein concentration. Notably, we account for a previously unrecognized intrinsic ambiguity in concentrations in standard binding models and clarify how this ambiguity impacts the extent to which binding parameters can be determined in cases of highly uncertain analyte concentrations. Our approach is applicable to a host of multi-step binding interactions, and we use it to investigate two systems. First, we deeply examine 2:2 LC8 binding and find it to be significantly positively cooperative with high confidence for multiple clients. Building on observations in the LC8 system, we develop a system-agnostic ‘phase diagram’ calculated from synthetic data demonstrating that certain binding parameters intrinsically inflate parameter uncertainty in ITC analysis, independent of experimental uncertainties. Second, we study 2:2 binding between the dynein intermediate chain and binding protein NudE, where in contrast, we find little evidence of allostery.
Competing Interest Statement
The authors have declared no competing interest.