Structure And Function Of Bacillus Subtilis Compatible Solute-binding Protein OpuCC And Streptococcus Pneumoniae Mucin-binding Protein Spr1345

(I) Structural biology of compatible solutes binding protein OpuCCThe compatible solutes ATP-binding cassette (ABC) transporters are key determinants in counteracting abrupt or remarkable alterations of osmolality in a diverse environment. The Gram-positive bacterium Bacillus subtilis has evolved a sophisticatedly stepwise osmoregulatory mechanism, in which three ABC transporters (OpuA, OpuB and OpuC) are indispensable for acquiring a variety of compatible solutes under osmotic stress. Each transporter consists of three components: taken OpuC as an example, substrate-binding protein OpuCC, which determines the substrate specificity; transmembrane proteins OpuCB/CD, providing a pathway for substrate transportation; ATPase OpuCA, driving the substrate uptake by the hydrolysis of ATP. The 34-kDa lipoprotein OpuCC is the substrate-binding protein (SBP) of OpuC system that exhibit the broadest multiple compatible solutes-binding activity as yet, especially serving as sole import pathway for carnitine, ectoine or choline-O-sulfate etc in B. subtilis. Here, fluorescence-based ligand binding measurements were used to validate substrate specificity of OpuCC compared with choline-specific OpuBC even both share 70% sequence identity. To explore structural basis of the broad spectrum for multiple compatible solutes-binding of OpuCC, we further determined crystal structures of OpuCC in complex with four selected substrates, carnitine, glycine betaine, choline and ectoine, respectively, and the structure in apo form that allows us to trace structural rearrangement upon ligand binding. These structures reveal different interaction patterns for each ligand, which help explain the multiple substrates-binding property of OpuCC. In addition, we found that a single residue mutation event is probably responsible for the remarkable functional differentiation with respect to the substrate specificity between OpuCC and OpuBC. Moreover, we also proposed a novel functional role of OpuCC as a mucin-binding protein (MucBP), which might be involved in adherence with intestinal mucins in the host. Our data will contribute to understand substrate specificity and function of compatible solutes-binding proteins. (II) Structural biology of mucin-binding protein Spr1345Streptococcus pneumoniae is a main human pathogen, whose surface proteins acting as virulence factors play an important role in pathogenesis. Surface proteins include three types: choline-binding proteins, lipoproteins and LPxTG-containing proteins. The surface protein Spr1345 from pneumococcus R6 genome is a 22 kDa mucin-binding protein (MucBP) involved in adherence and colonization of lung and respiratory tract of humans and regarded as the smallest MucBP due to the low molecular weight. It is composed of a mucin-binding domain (MucBD), a proline-rich domain (PRD) followed by an LPxTG motif sequence, which is removed by sortase to produce the mature form (MF171). The MucBD possesses comparable mucin-binding activity to MF171, and the immunofluorescence assay further proved that the MucBD adheres to the surface of human lung cell lines A549, suggesting the MucBD is indeed a functional unit for host adherence. Here, we solved the crystal structure of the MucBD at the resolution of 2.0(A|。), the first pathogenic bacteria's MucBD structure. The overall structure adopts an immunoglobulin-likeβ-sandwich fold with an elongated rod-like shape, which represents the verified minimal mucin-binding unit to date. Several conserved hydrophobic residues inβstrands, especially in the long loop region form a hydrophobic core leading to a compact architecture. Moreover, possible carbohydrate binding sites of the MucBD were predicted using the'InCa-SiteFinder'server, one of these sites lies in a PEG molecule, maybe mimicking the carbon chain of sugar interacting with the protein. These results provided structural basis for designing novel vaccine and antibiotics drugs against human diseases caused by the pathogen.(III) Characterization of yeast expression profiling under surplus ironIron is an essential micronutrient element found in almost all living organisms and participates in many fundamental metabolic processes, such as cellular respiration, iron-sulfur cluster biosynthesis and reaction of iron-containing enzymes. Iron homeostasis plays a crucial role in growth and division of cells in all kingdoms of life. Although the iron metabolism of yeast has been extensively studied, mainly through the activation of transcription factor Aft1/2 and function of iron related transporters, little is known about the molecular mechanism in response to surplus iron. In our study, the expression profiling of Saccharomyces cerevisiae upon surplus iron both in 1 h and 4 h revealed a dual effect. A cluster of stress-responsive genes are up-regulated, via the activation of the stress-resistant transcription factor Msn4, which indicates the stress effect of surplus iron on the metabolism of yeast. Meanwhile, the genes involved in aerobic metabolism and several anabolic pathways are also up-regulated at iron-surplus condition, which can significantly accelerate the growth of yeast. This dual effect suggested that surplus iron might participate in a more complex metabolic network, in addition to serving as a stress inducer. These findings will contribute to understanding the global response of yeast to fluctuating availability of iron in the environment.