Biology subjects

Grunewald, K.

Publications and source records attributed to Grunewald, K..

4 recordsLinked to original sources

bioRxiv2024

Molecular architecture of glideosome and nuclear F-actin in Plasmodium falciparum

Actin-based motility is required for the transmission of malaria sporozoites. While this has been shown biochemically, filamentous actin has remained elusive and has to date never been directly visualised inside the parasite. Using focused ion beam milling and electron cryo-tomography, we studied dynamic actin filaments in unperturbed Plasmodium falciparum cells for the first time. This allowed us to dissect the assembly, path and fate of actin filaments during parasite gliding and determine a complete 3D model of F-actin within sporozoites. We show that within the cell, actin assembles into micrometre long filaments, much longer than observed in in vitro studies. After their assembly at the parasites apical end, actin filaments continue to grow as they are transported down the cell as part of the glideosome machinery, and are disassembled at the basal end in a rate-limiting step. Large pores in the IMC, constrained to the basal end, may facilitate actin exchange between the pellicular space and the cytosol for its recycling and maintenance of directional actin flow for efficient gliding. The data also reveal striking and extensive actin bundles in the nucleus. Implications of these structures for motility and transmission are discussed.

microbiology↗

bioRxiv2023

Structural snapshots of phenuivirus cap-snatching and transcription

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.

biochemistry↗

bioRxiv2023

Modeling flexible protein structure with AlphaFold2 and cross-linking mass spectrometry

We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The pipeline consists of two main steps: ensemble generation using AF2, and conformer selection using XL-MS data. For conformer selection, we developed two scores - the monolink probability score (MP) and the crosslink probability score (XLP), both of which are based on residue depth. We benchmarked MP and XLP on a large dataset of decoy protein structures, and showed that our scores outperform previously developed scores. We then tested our methodology on three proteins having an open and closed conformation in the Protein Data Bank: Complement component 3 (C3), luciferase, and glutamine-binding periplasmic protein (QBP), first generating ensembles using AF2, which were then screened for the open and closed conformations using experimental XL-MS data. In five out of six cases, the most accurate model within the AF2 ensembles - or a conformation within 1 [A] of this model - was identified using crosslinks, as assessed through the XLP score. In the remaining case, only the monolinks (assessed through the MP score) successfully identified the open conformation of QBP. This serves as a compelling proof-of-concept for the effectiveness of monolinks. In contrast, the AF2 assessment score (pTM) was only able to identify the most accurate conformation in two out of six cases. Our results highlight the complementarity of AF2 with experimental methods like XL-MS, with the MP and XLP scores providing reliable metrics to assess the quality of the predicted models.

biochemistry↗

bioRxiv2022

Structural insights into viral genome replication by the Severe fever with thrombocytopenia syndrome virus L protein

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 [A]. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early- and late-stages of elongation with the RdRp core accommodating a 9-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

biochemistry↗