Crucial residue Trp158 of lipoprotein PiaA stabilizes the ferrichrome-PiaA complex in Streptococcus pneumoniae.

The pathogenic Streptococcus pneumoniae (S. pneumoniae) has evolved a special mechanism such as pneumococcal iron acquisition ATP binding cassette (PiaABC) to take up siderophore-iron from its host. The cell-surface lipoprotein PiaA, a key component of PiaABC, is the primary receptor to bind ferrichrome (Fc). To study the structure-function relationship of PiaA, three conservative amino-acid residues, Trp63, Trp158 and Phe255, in the hydrophobic barrel of the metal binding site of PiaA, were individually and collectively mutated to alanine; and the resulted single-point mutants, W63A, W158A and F255A, and triple mutant W63A/W158A/F255A were characterized by using biochemical and biophysical methods. Experiments showed that wild-type PiaA (WT-PiaA) and the single-point mutant proteins bound Fc with a similar kinetics mode, but the reaction rate of W158A was lower than that for WT-PiaA. The binding affinity of W158A toward Fc was significantly weaker than that of the WT-PiaA-Fc (wild-type PiaA bound with Fc) interaction. Furthermore, the absence of Trp158 in the protein led to a significant impact on the secondary structure of PiaA, resulting in a labile conformational structure of W158A, with impaired resistance to thermal and chemical denaturation. Collectively, Trp158 is a crucial residue for binding Fc, playing an important role in stabilizing the PiaA-Fc complex. This study revealed the critical role of the conserved tryptophan residues in Fc-binding protein PiaA, and provided valuable information for understanding the Fc transport mechanism mediated by PiaA or its homologous proteins in bacteria.