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

Abstract 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. Competing Interest Statement The authors have declared no competing interest.