Comparative Kd analysis of the interaction between aggregation-prone Nup153 3-2 and KapN.

Accurate quantification of binding affinities for intrinsically disordered regions (IDRs) is challenged by their flexibility and propensity for aggregation, often requiring cross-validation across distinct biophysical methods. Here, we investigated the interaction between interacting domains of karyopherin β1 (KapN) and Nup153 (Nup153 3-2), a highly aggregation-prone IDR fragment of nucleoporin 153. To circumvent experimental time constraints imposed by rapid sample aggregation, we employed a suite of short-timescale methodologies: NMR chemical shift perturbations (CSPs), lineshape analysis, 15N-edited diffusion NMR, and isothermal titration calorimetry (ITC). Remarkably, despite relying on fundamentally different physical principles, all four methods yielded apparent dissociation constants (Kd) in the low-micromolar range, with lineshape analysis, diffusion NMR, and ITC converging closely (2-3 μM) while CSP gave a modestly lower value (1.18 μM). These findings demonstrate that cross-validating short-timescale methodologies can reveal and account for method-specific biases, providing a robust strategy for characterizing aggregation-prone biomolecular systems.