Structure prediction of honey bee vitellogenin: a multi-domain protein important for insect immunity.

Vitellogenin (Vg) has been implicated as a central protein in the immunity of egg-laying animals. Studies on a diverse set of species suggest that Vg supports health and longevity through binding to pathogens. Specific studies of honey bees (Apis mellifera) further indicate that the vitellogenin (vg) gene undergoes selection driven by local pathogen pressures. Determining the complete 3D structure of full-length Vg (flVg) protein will provide insights regarding the structure-function relationships underlying allelic variation. Honey bee Vg has been described in terms of function, and two subdomains have been structurally described, while information about the other domains is lacking. Here, we present a structure prediction, restrained by experimental data, of flVg from honey bees. To achieve this, we performed homology modeling and used AlphaFold before using a negative-stain electron microscopy map to restrict, orient, and validate our 3D model. Our approach identified a highly conserved Ca2+ -ion-binding site in a von Willebrand factor domain that might be central to Vg function. Thereafter, we used rigid-body fitting to predict the relative position of high-resolution domains in a flVg model. This mapping represents the first experimentally validated full-length protein model of a Vg protein and is thus relevant for understanding Vg in numerous species. Our results are also specifically relevant to honey bee health, which is a topic of global concern due to rapidly declining pollinator numbers.

Cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution.

Infection of the human stomach by Helicobacter pylori remains a worldwide problem and greatly contributes to peptic ulcer disease and gastric cancer. Without active intervention approximately 50% of the world population will continue to be infected with this gastric pathogen. Current eradication, called triple therapy, entails a proton-pump inhibitor and two broadband antibiotics, however resistance to either clarithromycin or metronidazole is greater than 25% and rising. Therefore, there is an urgent need for a targeted, high-specificity eradication drug. Gastric infection by H. pylori depends on the expression of a nickel-dependent urease in the cytoplasm of the bacteria. Here, we report the 2.0 Å resolution structure of the 1.1 MDa urease in complex with an inhibitor by cryo-electron microscopy and compare it to a ß-mercaptoethanol-inhibited structure at 2.5 Å resolution. The structural information is of sufficient detail to aid in the development of inhibitors with high specificity and affinity.

Mechanism of Structural Tuning of the Hepatitis C Virus Human Cellular Receptor CD81 Large Extracellular Loop.

Hepatitis C virus (HCV) enters into human hepatocytes via tetraspanin hCD81. HCV glycoprotein E2 recognizes the "head" subdomain of the large extracellular loop (LEL) of CD81 (hCD81LEL), but the precise mechanism of virus cell attachment and entry remains elusive. Here, by combining the structural analysis of a conspicuous number of crystallized CD81LEL molecules with molecular dynamics simulations, we show that the conformational plasticity of the hCD81LEL head subdomain is a molecular property of the receptor. The observed closed, intermediate, and open conformations of the head subdomain provide distinct binding platforms. Simulations at pH 7.4 and 4.0 indicate that this dynamism is pH modulated. The crystallized double conformation of the disulfide bridge C157-C175 at the base of the head subdomain identifies this bond as the molecular zipper of the plasticity of hCD81LEL. We propose that this conformational dependence of hCD81LEL, which is finely tuned by pH and redox conditions, enables the virus-receptor interactions to diversely re-engage at endosomal conditions.

Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity.

Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043-1054. © 2016 Wiley Periodicals, Inc.

Insertion of endocellulase catalytic domains into thermostable consensus ankyrin scaffolds: effects on stability and cellulolytic activity.

Degradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8; Clostridium thermocellum) and Cel12A (C12A, GH12; Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12A catalytic domain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.