Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants.

Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60%-80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy), some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence, and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high-resolution crystal structure suggests a model for phytate recognition that, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well conserved in PAPhys from monocot cereals, enzymes which are characterized by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta.

Oligomerization of the FliF Domains Suggests a Coordinated Assembly of the Bacterial Flagellum MS Ring.

The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and of the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 20 different proteins forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2, and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mismatch within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella enterica serovar Typhimurium FliF ortholog. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggest a mechanism for the controlled assembly of the MS ring.

Dynamic Assay for Profiling Anti-SARS-CoV-2 Antibodies and Their ACE2/Spike RBD Neutralization Capacity.

Serological assays have been widely employed during the coronavirus disease 2019 (COVID-19) pandemic to measure antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to track seroconversion in populations. However, currently available assays do not allow determination of neutralization capacity within the assay protocol. Furthermore, commercial serology assays have a high buy-in cost that is inaccessible for many research groups. We have replicated the serological enzyme-linked immunosorbent assay for the detection of SARS-CoV-2 antibody isotypes, developed at the Icahn School of Medicine at Mount Sinai, New York. Additionally, we have modified the protocol to include a neutralization assay with only a minor modification to this protocol. We used this assay to screen local COVID-19 patient sera (n = 91) and pre-COVID-19 control sera (n = 103), and obtained approximate parity with approved commercial anti-nucleoprotein-based assays with these sera. Furthermore, data from our neutralization assay closely aligns with that generated using a spike-based pseudovirus infection model when a subset of patient sera was analyzed.