Structural And Functional Studies On β-galactosidase Bgac And Siderophore Binding Protein PiaA From Streptococcus Pneumoniae

1. Structural and functional research on β-galactosidase BgaC from Streptococcus pneumoniaeThe surface-exposed P-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity towards the p(1,3)-linked galactose and N-acetylglucosamine [Ga1β(1,3)NAG] moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at1.8A and its complex with galactose at1.95A. At pH5.5to8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains:a catalytic domain of a classic (α/β)8TIM barrel, followed by two all-β domains(ABDs) of unknown function. The side-walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp243and Tyr455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Ga1p(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp240and Tyr455contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp243is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain. 2. Structural and functional research on the siderophore binding protein PiaA from Streptococcus pneumoniaeIron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at2.7and2.1A resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an a-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues:Glu119and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome.