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The accessory sec system consisting of seven conserved components is commonly distributed among pathogenic Gram-positive bacteria for the secretion of serine-rich-repeat proteins (SRRPs). Asp1/2/3 protein complex in the system is responsible for both the O-acetylation of GlcNAc and delivering SRRPs to SecA2. However, the molecular mechanism of how Asp1/2/3 transport SRRPs remains unknown. Here, we report the complex structure of Asp1/2/3 from Streptococcus pneumoniae at 2.9 A. Further functional assays indicated that Asp1/2/3 can stimulate the ATPase activity of SecA2. In addition, the deletion of asp1/2/3 gene resulted in the accumulation of a secreted version of PsrP with an altered glycoform in protoplast fraction of the mutant cell, which suggested the modification/transport coupling of the substrate. Altogether, these findings not only provide structural basis for further investigations on the transport process of SRRPs, but also uncover the indispensable role of Asp1/2/3 in the accessory sec system.

L-amino acids represent the most common amino acid form, most notably as protein residues, whereas D-amino acids, despite their rare occurrence, play significant roles in many biological processes. Amino acid racemases are enzymes that catalyze the interconversion of L- and/or D-amino acids. McyF is a pyridoxal 5′-phosphate (PLP) independent amino acid racemase that produces the substrate D-aspartate for the biosynthesis of microcystin in the cyanobacterium Microcystis aeruginosa PCC7806. Here we report the crystal structures of McyF in complex with citrate, L-Asp and D-Asp at 2.35, 2.63 and 2.80 A, respectively. Structural analyses indicate that McyF and homologs possess highly conserved residues involved in substrate binding and catalysis. In addition, residues Cys87 and Cys195 were clearly assigned to the key catalytic residues of “two bases” that deprotonate D-Asp and L-Asp in a reaction independent of PLP. Further site-directed mutagenesis combined with enzymatic assays revealed that Glu197 also participates in the catalytic reaction. In addition, activity assays proved that McyF could also catalyze the interconversion of L-MeAsp between D-MeAsp, the precursor of another microcystin isoform. These findings provide structural insights into the catalytic mechanism of aspartate racemase and microcystin biosynthesis.

The choline-binding proteins play essential roles in pneumococcal colonization and virulence. It has been suggested that the choline-binding protein J (termed CbpJ; encoded by the gene sp_0378) from Streptococcus pneumoniae TIGR4 involves in the colonization in host and contributes to evasion of neutrophil killing. Here we report the 2.0 A crystal structure of CbpJ in complex with choline. CbpJ consists of an N-terminal putative functional domain (N-domain) followed by a C-terminal choline-binding domain (CBD). The N-domain harbors four degenerated choline-binding repeats (CBRs) that lose the capacity of binding to choline, whereas the CBD is composed of seven typical CBRs. Further functional assays showed that the CBD contributes to the pneumococcal adhesion to human lung epithelial cell A549. These findings provide insights into the pneumococcal pathogenesis and broaden our understanding on the functions of choline-binding proteins.

XPA (xeroderma pigmentosum complementation group A), a key scaffold protein in nucleotide excision repair (NER) pathway, is important in DNA damage verification and repair proteins recruitment. Earlier studies had mapped the minimal DNA-binding domain (MBD) of XPA to a region corresponding to residues 98–219. However, recent studies indicated that the region involving residues 98–239 is the redefined DNA-binding domain (DBD), which binds to DNA substrates with a much higher binding affinity than MBD and possesses a nearly identical binding affinity to the full-length XPA protein. However, the structure of the redefined DBD domain of XPA (XPA-DBD) remains to be investigated. Here, we present the crystal structure of XPA-DBD at 2.06 A resolution. Structure of the C-terminal region of XPA has been extended by 21 residues (Arg211–Arg231) as compared with previously reported MBD structures. The structure reveals that the C-terminal extension (Arg211–Arg231) is folded as an α-helix with multiple basic residues. The positively charged surface formed in the last C-terminal helix suggests its critical role in DNA binding. Further structural analysis demonstrates that the last C-terminal region (Asp217–Thr239) of XPA-DBD might undergo a conformational change to directly bind to the DNA substrates. This study provides a structural basis for understanding the possible mechanism of enhanced DNA-binding affinity of XPA-DBD.

Carboxysome, encapsulating an enzymatic core within an icosahedral-shaped semipermeable protein shell, could enhance CO2 fixation under low CO2 conditions in the environment. The shell of Halothiobacillus neapolitanus α-carboxysome possesses two 38% sequence-identical pentameric proteins, namely CsoS4A and CsoS4B. However, the functions of two paralogous pentameric proteins in α-carboxysome assembly remain unknown. Here we report the crystal structure of CsoS4B at 2.15 A resolution. It displays as a stable pentamer, each subunit of which consists of a β-barrel core domain, in addition to an insertion of helix α1 that forms the central pore. Structural comparisons and multiple-sequence alignment strongly indicate that CsoS4A and CsoS4B differ from each other in interacting with various components of α-carboxysome, despite they share a similar overall structure. These findings provide the structural basis for further investigations on the self-assembly process of carboxysome.

S100A9 is a member of the S100 family of proteins that contain two EF-hand calcium-binding motifs. We previously reported that S100A9 was differentially expressed during the early airway response phase of asthma and can be regulated by acupuncture. To understand the possible role of S100A9 in asthma, the effects of the S100A9 were investigated in a rat model of asthma and in isolated tracheal spirals. The pulmonary function and isometric tension were measured after the administration of purified recombinant S100A9. The results of in vivo experiments showed that S100A9 (0.1 μg/kg) significantly decreased the pulmonary resistance and increased the dynamic compliance. The in vitro experimental results showed that S100A9 (100, 200, 400, or 800 ng/ml, final concentrations) significantly reduced the isometric tension of isolated tracheal spirals. These results suggest that S100A9 elicits dose-dependent anti-asthmatic effects and may provide further insight into the treatment of asthma.