ORCID Profile
0000-0002-6198-1194
Current Organisation
The University of Texas at Austin
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Publisher: American Society for Microbiology
Date: 04-2013
DOI: 10.1128/JVI.06868-11
Abstract: Human noroviruses are genetically and antigenically highly ergent. Monoclonal antibodies raised in mice against one kind of norovirus virus-like particle (VLP), however, were found to have broad recognition. In this study, we present the crystal structure of the antigen-binding fragment (Fab) for one of these broadly reactive monoclonal antibodies, 5B18, in complex with the capsid-protruding domain from a genogroup II genotype 10 (GII.10) norovirus at 3.3-Å resolution and, also, the cryo-electron microscopy structure of the GII.10 VLP at ∼10-Å resolution. The GII.10 VLP structure was more similar in overall architecture to the GV.1 murine norovirus virion than to the prototype GI.1 human norovirus VLP, with the GII.10 protruding domain raised ∼15 Å off the shell domain and rotated ∼40° relative to the GI.1 protruding domain. In the crystal structure, the 5B18 Fab bound to a highly conserved region of the protruding domain. Based on the VLP structure, this region is involved in interactions with other regions of the capsid and is buried in the virus particle. Despite the occluded nature of the recognized epitope in the VLP structure, enzyme-linked immunosorbent assay (ELISA) binding suggested that the 5B18 antibody was able to capture intact VLPs. Together, the results provide evidence that the norovirus particle is capable of extreme conformational flexibility, which may allow for antibody recognition of conserved surfaces that would otherwise be buried on intact particles.
Publisher: Research Square Platform LLC
Date: 27-04-2023
DOI: 10.21203/RS.3.RS-2837968/V1
Abstract: CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction. Target RNA cleavage at regular intervals is characteristic of type III effector complexes however, the mechanism has remained enigmatic. Here, we determine the structures of the Synechocystis type III-Dv complex, an evolutionary intermediate in type III effectors, in pre- and post-cleavage states, which show metal ion coordination in the active sites. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we reveal the structure and dynamics of the three catalytic sites, where a 2’-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Thus, type III CRISPR-Cas complexes function as protein-assisted ribozymes, and their programmable nature has important implications for how these complexes could be repurposed for applications.
Publisher: American Society for Microbiology
Date: 03-2016
DOI: 10.1128/JVI.02916-15
Abstract: The capsid protein (VP1) of all caliciviruses forms an icosahedral particle with two principal domains, shell (S) and protruding (P) domains, which are connected via a flexible hinge region. The S domain forms a scaffold surrounding the nucleic acid, while the P domains form a homodimer that interacts with receptors. The P domain is further sub ided into two subdomains, termed P1 and P2. The P2 subdomain is likely an insertion in the P1 subdomain consequently, the P domain is ided into the P1-1, P2, and P1-2 subdomains. In order to investigate capsid antigenicity, N-terminal (N-term)/S/P1-1 and P2/P1-2 were switched between two sapovirus genotypes GI.1 and GI.5. The chimeric VP1 constructs were expressed in insect cells and were shown to self-assemble into virus-like particles (VLPs) morphologically similar to the parental VLPs. Interestingly, the chimeric VLPs had higher levels of cross-reactivities to heterogeneous antisera than the parental VLPs. In order to better understand the antigenicity from a structural perspective, we determined an intermediate-resolution (8.5-Å) cryo-electron microscopy (cryo-EM) structure of a chimeric VLP and developed a VP1 homology model. The cryo-EM structure revealed that the P domain dimers were raised slightly (∼5 Å) above the S domain. The VP1 homology model allowed us predict the S domain (67–229) and P1-1 (229–280), P2 (281–447), and P1-2 (448–567) subdomains. Our results suggested that the raised P dimers might expose immunoreactive S/P1-1 subdomain epitopes. Consequently, the higher levels of cross-reactivities with the chimeric VLPs resulted from a combination of GI.1 and GI.5 epitopes. IMPORTANCE We developed sapovirus chimeric VP1 constructs and produced the chimeric VLPs in insect cells. We found that both chimeric VLPs had a higher level of cross-reactivity against heterogeneous VLP antisera than the parental VLPs. The cryo-EM structure of one chimeric VLP (Yokote/Mc114) was solved to 8.5-Å resolution. A homology model of the VP1 indicated for the first time the putative S and P (P1-1, P2, and P1-2) domains. The overall structure of Yokote/Mc114 contained features common among other caliciviruses. We showed that the P2 subdomain was mainly involved in the homodimeric interface, whereas a large gap between the P1 subdomains had fewer interactions.
Publisher: Springer Science and Business Media LLC
Date: 20-05-2022
DOI: 10.1038/S41467-022-30402-8
Abstract: CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3′ binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.
Publisher: Cold Spring Harbor Laboratory
Date: 14-06-2022
DOI: 10.1101/2022.06.13.496011
Abstract: CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are a type of adaptive immune response in bacteria and archaea that utilize crRNA (CRISPR RNA)-guided effector complexes to target complementary RNA or DNA for destruction. The prototypical type III-A and III-B CRISPR-Cas systems utilize multi-subunit effector complexes composed of in idual proteins to cleave ssRNA targets at 6-nt intervals, as well as non-specifically degrading ssDNA and activating cyclic oligoadenylate (cOA) synthesis. Recent studies have shown that type III systems can contain subunit fusions yet maintain canonical type III RNA-targeting capabilities. To understand how a multi-subunit fusion effector functions, we determine structures of a variant type III-D effector and biochemically characterize how it cleaves RNA targets. These findings provide insights into how multi-subunit fusion proteins are tethered together and assemble into an active and programmable RNA endonuclease, how the effector utilizes a novel mechanism for target RNA seeding, and the structural basis for the evolution of type III effector complexes. Furthermore, our results provide a blueprint for fusing subunits in class 1 effectors for design of user-defined effector complexes with disparate activities. While this manuscript was in preparation, a manuscript describing the structure of the type III-E effector was published 1 . We reference these important findings however, a careful comparison of the structures will follow once the coordinates have been released by the PDB.
Publisher: Elsevier BV
Date: 12-2020
Publisher: Cold Spring Harbor Laboratory
Date: 18-04-2020
DOI: 10.1101/2020.04.18.045682
Abstract: CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. We investigated the previously uncharacterized type I-D interference complex from Synechocystis and revealed it is a genetic and structural hybrid with similarity to both type I and III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across erse type I-D, I-B and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution. Internal translation of large subunit transcripts drives small subunit synthesis in erse type I CRISPR-Cas interference complexes
Location: United States of America
No related grants have been discovered for David Taylor.