An evolutionary strategy for highly efficient secretion of multidomain Staphylococcal protein A: N- to C-terminal stability gradient

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 ({approx}60 aa) antibody-binding domains on the bacterial surface with high affinity (Kd {approx}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 ({approx}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 Cterminal 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.