Comparative analysis of the physical properties of murine and human S100A7: Insight into why zinc piracy is mediated by human but not murine S100A7.

S100 proteins are a subfamily of EF-hand calcium-binding proteins found primarily in vertebrate animals. They are distinguished by binding of transition metals and functioning in both the intracellular and extracellular milieu. S100A7 functions in the protection of the skin and mucous membranes and is a biomarker in inflammatory skin disease. A recent study of Neisseria gonorrhoeae infection revealed that human but not murine S100A7 could be used to evade host nutritional immunity. To understand the molecular basis for this difference, we carried out a comparative analysis of the physical and structural properties of human and murine S100A7. The X-ray crystal structure of Ca2+-loaded mouse S100A7 (mS100A7) was determined to 1.69 Å resolution, and Ca2+-induced conformational changes were assessed by NMR. Unlike human S100A7 (hS100A7), which exhibits conformational changes in response to binding of Ca2+, no significant changes in mS100A7 were detected. Dynamic light scattering, circular dichroism, and a competition chelator assay were used to compare the Zn2+ affinity and the effects of ion binding on mS100A7 versus hS100A7. Alignment of their sequences revealed a substantial difference in the C-terminal region, which is an important mediator of protein-protein interactions, suggesting a rationale for the specificity of N. gonorrhoeae for hS100A7. These data, along with more detailed analysis of S100A7 sequence conservation across different species, support the proposal that, although hS100A7 is highly conserved in many mammals, the murine protein is a distinct ortholog. Our results highlight the potential limitations of using mouse models for studying bacterial infections in humans.

Characterization of the first tetrameric transcription factor of the GntR superfamily with allosteric regulation from the bacterial pathogen Agrobacterium fabrum.

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

The agar-specific hydrolase ZgAgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily.

Agars are sulfated galactans from red macroalgae and are composed of a d-galactose (G unit) and l-galactose (L unit) alternatively linked by α-1,3 and ß-1,4 glycosidic bonds. These polysaccharides display high complexity, with numerous modifications of their backbone (e.g. presence of a 3,6-anhydro-bridge (LA unit) and sulfations and methylation). Currently, bacterial polysaccharidases that hydrolyze agars (ß-agarases and ß-porphyranases) have been characterized on simple agarose and more rarely on porphyran, a polymer containing both agarobiose (G-LA) and porphyranobiose (GL6S) motifs. How bacteria can degrade complex agars remains therefore an open question. Here, we studied an enzyme from the marine bacterium Zobellia galactanivorans (ZgAgaC) that is distantly related to the glycoside hydrolase 16 (GH16) family ß-agarases and ß-porphyranases. Using a large red algae collection, we demonstrate that ZgAgaC hydrolyzes not only agarose but also complex agars from Ceramiales species. Using tandem MS analysis, we elucidated the structure of a purified hexasaccharide product, L6S-G-LA2Me-G(2Pentose)-LA2S-G, released by the activity of ZgAgaC on agar extracted from Osmundea pinnatifida By resolving the crystal structure of ZgAgaC at high resolution (1.3 Å) and comparison with the structures of ZgAgaB and ZgPorA in complex with their respective substrates, we determined that ZgAgaC recognizes agarose via a mechanism different from that of classical ß-agarases. Moreover, we identified conserved residues involved in the binding of complex oligoagars and demonstrate a probable influence of the acidic polysaccharide's pH microenvironment on hydrolase activity. Finally, a phylogenetic analysis supported the notion that ZgAgaC homologs define a new GH16 subfamily distinct from ß-porphyranases and classical ß-agarases.