An evolutionary strategy for highly efficient secretion of multidomain Staphylococcal protein A: N- to C-terminal stability gradient
Year: 2026
Type: preprint
Abstract: Surface factors of Staphylococcus aureus have become therapeutic targets, including staphylococcal protein A (SpA), a key contributor to virulence. SpA displays five nearly identical helical ( ≈ 60 aa) antibody-binding domains on the bacterial surface with high affinity ( K d ≈ 0.4 − 4 nM). Folding free energies have been determined for only domains E and B. Here we used intrinsic fluorescence-detected urea denaturation to measure the folding thermodynamics of each domain in isolation and in the native multidomain context (SpA-N, the N-terminal half of the mature protein). Domains D, A, B, and C have identical stabilities in isolation and in SpA-N, confirming independent folding; E domain is stabilized ≈ 1 kcal/mol by its multidomain context. To determine role of the conserved linker between domains D through C we used fluorescence-detected urea denaturation of 1–5 B-domain repeat constructs. Analysis of these data using a nearest-neighbor matrix model demonstrated unambiguously that adjacent domains are thermodynamically uncoupled. We also find a steep N-to C-terminal gradient of increasing domain stability (0.97 ± 0.05 to 5.69 ± 0.28 kcal/mol), which has not been reported for any repeat protein. We propose this gradient facilitates efficient co-translational secretion of SpA through the SecYEG translocon: the least stable E domain enters the channel first, lowering the energetic barrier to translocation of this highly abundant surface protein. Significance Staphylococcal protein A (SpA) is a highly expressed virulence factor of Staphylococcus aureus that evades host immunity by binding antibodies with high affinity. SpA displays on the bacterial surface five nearly identical small helical domains that are connected by highly flexible linkers. We measured the folding thermodynamics of each domain in isolation and in their native multidomain context. We unambiguously demonstrated that adjacent domains are thermodynamically uncoupled. We also discovered a steep N-to C-terminal gradient of increasing domain stability. We propose this gradient facilitates efficient post-translational secretion of SpA through the SecYEG translocon: the least stable domains enter first, lowering the energetic barrier to translocation for the most highly expressed protein in S. aureus .
Institution: Duke University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7163172997