Adaptive disorder as the hallmark of nanobodies antigen-binding loops

Nanobodies are antigen-binding proteins of great interest as diagnostics and therapeutics. Accurate and fast characterization of their complementarity-determining regions (CDRs) is crucial to uncover the principles guiding their design. Yet, this task remains challenging, as random recombination and somatic mutations generate highly diverse CDR sequences that escape motif-based or structure-prediction approaches currently used to identify them. To overcome this hurdle, we employed two independent strategies that converged on the same conclusion. At the sequence level, we developed a deep learning model to identify nanobody CDRs directly from the primary sequence. At the structural level, we applied an energy decomposition method, revealing CDRs as residues highly uncoupled to the rest of the fold. Explainability analyses showed the network captured intrinsic CDR properties, which notably aligned with these energy values. CDRs emerge as fuzzy regions capable of adopting diverse conformational ensembles, from which a preferred state is selected upon antigen binding. This finding supports a model where chaos in both sequence and structure appears adaptive and disorder emerges as the hallmark of nanobody CDRs. This work aims to advance the definition of rules for the design of antigen binding regions, paving the way for the next-generation immune diagnostics and therapeutics.