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.
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Liang
L
Zhang
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Nan
N
Li
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Kun
K
Cao
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Xiao-Yan
XY
Yang
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
Guandi
G
Zeng
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. Electronic address: tsunxs@jnu.edu.cn.
Xuesong
X
Sun
Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. Electronic address: tqyhe@jnu.edu.cn.
Qing-Yu
QY
He
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005291:Ferrichrome / Q000737:chemistry* / Q000378:metabolism; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D008074:Lipoproteins; D046912:Multiprotein Complexes; D005291:Ferrichrome; D014364:Tryptophan
Ferrichrome; Iron transport; Iron-binding protein; PiaA; Streptococcus pneumoniae