Characterization of the protrimer intermediate in the folding pathway of the interdigitated β-helix tailspike protein

P22 tailspike is a homotrimeric, thermostable adhesin that recognizes the O-antigen lipopolysaccharide of Salmonella typhimurium. The 70 kDa subunits include long beta-helix domains. After residue 540, the polypeptide chains change their path and wrap around one another, with extensive interchain contacts. Formation of this interdigitated domain intimately couples the chain folding and assembly mechanisms. The earliest detectable trimeric intermediate in the tailspike folding and assembly pathway is the protrimer, suspected to be a precursor of the native trimer structure. We have directly analyzed the kinetics of in vitro protrimer formation and disappearance for wild type and mutant tailspike proteins. The results confirm that the protrimer intermediate is an on-pathway intermediate for tailspike folding. Protrimer was originally resolved during tailspike folding because its migration through nondenaturing polyacrylamide gels was significantly retarded with respect to the migration of the native tailspike trimer. By comparing protein mobility versus acrylamide concentration, we find that the retarded mobility of the protrimer is due exclusively to a larger overall size than the native trimer, rather than an altered net surface charge. Experiments with mutant tailspike proteins indicate that the conformation difference between protrimer and native tailspike trimer is localized toward the C-termini of the tailspike polypeptide chains. These results suggest that the transformation of the protrimer to the native tailspike trimer represents the C-terminal interdigitation of the three polypeptide chains. This late step may confer the detergent-resistance, protease-resistance, and thermostability of the native trimer.