Full-Length Context Disrupts Folding of IgG-Binding Domains of Protein A.

Multidomain proteins are often thought to fold as collections of independently stable domains, a modularity that underpins many assumptions in structural biology and design. Here, we challenge this view by examining the folding behavior of full-length Staphylococcal protein A (SpA), a 516-residue multidomain protein containing five immunoglobulin (Ig)-binding domains. Although each of the five Ig-binding domains of SpA folds stably in isolation (as it is already known experimentally and also confidently predicted by AI models), here, we show that the full-length construct and the individual Ig-binding domains in the full-length construct fail to adopt a stable three-dimensional structure in solution (despite being predicted to be folded by AI models). Instead, full-length SpA populates a compact yet predominantly disordered ensemble with residual secondary structure, where the folded state of each Ig-binding domain is thermodynamically unfavorable. These findings not only challenge long-held assumptions about the modular architecture and stability of SpA but also underscore the limitations of AI-based predictors when decoupled from the thermodynamic context. This work has implications for validating structure predictions, understanding multidomain architecture, and designing modular proteins for biotechnology and medicine.