Crystal structures of YeiE from Cronobacter sakazakii and the role of sulfite tolerance in gram-negative bacteria.

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

High-resolution structures of annexin A5 in a two-dimensional array.

Annexins are soluble cytosolic proteins that bind to cell membranes. Annexin A5 self-assembles into a two-dimensional (2D) array and prevents cell rupture by attaching to damaged membranes. However, this process is not fully understood at the molecular level. In this study, we determined the crystal structures of annexin A5 with and without calcium (Ca2+) and confirmed the Ca2+-dependent outward motion of a tryptophan residue. Strikingly, the two structures exhibited the same crystal packing and 2D arrangement into a p3 lattice, which agrees well with the results of low-resolution structural imaging. High-resolution structures indicated that a three-fold interaction near the tryptophan residue is important for mediating the formation of the p3 lattice. A hypothesis on the promotion of p3 lattice formation by phosphatidyl serine (PS) is also suggested. This study provides molecular insight into how annexins modulate the physical properties of cell membranes as a function of Ca2+ concentration and the phospholipid composition of the membrane.

Structural basis for the transglycosylase activity of a GH57-type glycogen branching enzyme from Pyrococcus horikoshii.

Glycogen branching enzyme (GBE) catalyzes the formation of α-1,6-branching points during glycogenesis by cleaving α-1,4 bonds and making new α-1,6 bonds. Most GBEs belong to the glycoside hydrolase 13 family (GH13), but new GBEs in the GH57 family have been isolated from Archaea. Here, we determined the crystal structure of a GH57 GBE from the hyperthermophilic archaeon Pyrococcus horikoshii (PhGBE) at a resolution of 2.3 Å. PhGBE exhibits both α-1,6-branching activity and endo-α-1,4 hydrolytic activity. PhGBE has a central (ß/α)7-barrel domain that contains an embedded helix domain and an α-helix-rich C-terminal domain. The active-site cleft is located at the interface of the central and C-terminal domains. Amino acid substitution at Trp22, which is separate from the catalytic nucleophilic residue, abolished both enzymatic activities, indicating that Trp22 might be responsible for substrate recognition. We also observed that shortening of the flexible loop near the catalytic residue changed branched chain lengths of the reaction products with increased hydrolytic activity. Taken together, our findings propose a molecular mechanism for how GH57 GBEs exhibit the two activities and where the substrate binds the enzyme.