Biology subjects

Steimle, S.

Publications and source records attributed to Steimle, S..

4 recordsLinked to original sources

bioRxiv2026

A nucleotide-triggered molecular switch orchestrating septin polymerization

Septins are conserved cytoskeletal GTP-binding proteins that form higher-order structures critical for cytokinesis and polarized growth across opisthokonts. In budding yeast and humans, four homologous septins assemble into hetero-octameric protofilaments through alternating interactions between adjacent G-domains (G-interface) or N- and C-termini (NC-interface), which then polymerize end-to-end into filaments. How nucleotide binding and hydrolysis control filament assembly has remained elusive. We uncover a septin-specific mechanism in which nucleotide binding stabilizes the monomeric G-interface and primes it for protofilament formation. Cryo-EM and DEER spectroscopy reveal nucleotide-induced conformational changes that engage the G-interface and enable an NC-compatible conformation absent in the nucleotide-free state. In vitro binding and reconstitution assays confirm that G-interface formation is strictly nucleotide-dependent and required for subsequent NC-interface assembly. These findings establish unidirectional allosteric signaling from the G-to the NC-interface, revealing the molecular basis for controlled septin protofilament assembly.

molecular biology↗

bioRxiv2025

Horizontal gene transfer of the functional archaellum machinery to Bacteria

Motility in Archaea is driven by a nanomachinery called the archaellum. So far, archaella have been exclusively described for the archaeal domain; however, a recent study reported the presence of archaellum gene clusters in bacterial strains of the SAR202 clade (Chloroflexota). Here, we show that bona fide archaellum gene clusters are widespread in several members of the Chloroflexota, which in turn lack any bacterial flagellar components. Analysis of archaellum encoding loci and predicted structures show remarkable similarity to the archaellum machinery. Moreover, using cryoEM single particle analysis, we solved the structure of the bacterial archaellum from Litorilinea aerophila, demonstrating the successful expression and assembly of this machinery in Bacteria and its function in swimming motility. Finally, a phylogenomic analysis revealed two horizontal gene transfer events from euryarchaeal members to Chloroflexota. In summary, our study demonstrates that a functional and assembled archaellum machinery can be successfully exchanged between the two prokaryotic domains.

microbiology↗

bioRxiv2021

Structural visualization of de novo initiation of RNA polymerase II transcription

Structural studies of the initiation-elongation transition of RNA polymerase II (pol II) transcription were previously facilitated by the use of synthetic oligonucleotides. Here we report structures of initiation complexes de novo converted from pre-initiation complex (PIC) through catalytic activities and stalled at different template positions. Contrary to previous models, the closed-to-open promoter transition was accompanied by a large positional change of the general transcription factor TFIIH which became in closer proximity to TFIIE for the active delivery of the downstream DNA to the pol II active center. The initially-transcribing complex (ITC) reeled over 80 base pairs of the downstream DNA by scrunching, while retaining the fixed upstream contact, and underwent the transition to elongation when it encountered promoter-proximal pol II from a preceding round of transcription. TFIIH is therefore conducive to promoter melting, TSS scanning, and promoter escape, extending far beyond synthesis of a short transcript.

biophysics↗

bioRxiv2020

Cryo-EM Structures of Respiratory bc1-cbb3 type CIII2CIV Super-complex and Electronic Communication Between the Complexes

The respiratory electron transport complexes convey electrons from nutrients to oxygen and generate a proton-motive force used for energy (ATP) production in cells. These enzymes are conserved among organisms, and organized as individual complexes or combined forming large super-complexes (SC). Bacterial electron transport pathways are more branched than those of mitochondria and contain multiple variants of such complexes depending on their growth modes. The Gram-negative species deploy a mitochondrial-like cytochrome bc1 (Complex III, CIII2), and may have bacteria-specific cbb3-type cytochrome c oxidases (Complex IV, CIV) in addition to, or instead of, the canonical aa3-type CIV. Electron transfer between these complexes is mediated by two different carriers: the soluble cytochrome c2 which is similar to mitochondrial cytochrome c and the membrane-anchored cytochrome cy which is unique to bacteria. Here, we report the first cryo-EM structure of a respiratory bc1-cbb3 type SC (CIII2CIV, 5.2Å resolution) and several conformers of native CIII2 (3.3Å resolution) from the Gram-negative bacterium Rhodobacter capsulatus. The SC contains all catalytic subunits and cofactors of CIII2 and CIV, as well as two extra transmembrane helices attributed to cytochrome cy and the assembly factor CcoH. Remarkably, some of the native CIII2 are structural heterodimers with different conformations of their [2Fe-2S] cluster-bearing domains. The unresolved cytochrome c domain of cy suggests that it is mobile, and it interacts with CIII2CIV differently than cytochrome c2. Distance requirements for electron transfer suggest that cytochrome cy and cytochrome c2 donate electrons to heme cp1 and heme cp2 of CIV, respectively. For the first time, the CIII2CIV architecture and its electronic connections establish the structural features of two separate respiratory electron transport pathways (membrane-confined and membrane-external) between its partners in Gram-negative bacteria.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsQquinoneQH2Quinol or hydroquinoneComplex IIICIII2 or cytochromebc1ubiquinolcytochrome c oxidoreductaseComplex IV or CIVcbb3-type cytochrome c oxidasecytcytochromecyt c2cytochrome c2, soluble cytochrome ccyt cycytochrome cy, membrane-anchored cytochrome ccyt S-cysoluble part of cytochrome cy without its membrane anchorSCsuper-complexMSmass spectrometryXL-MScross-linking mass spectrometryXLcross-linksTMBZ3,3’,5,5’-tetramethyl-benzidineDBH22,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinoneFeSRieske iron-sulfur proteinFeS-EDmembrane-extrinsic domain of FeS proteinb positionlocation of the [2Fe-2S] cluster near heme bLc positionlocation of the [2Fe-2S] cluster near heme c1cryo-EMcryogenic electron microscopyBN-PAGEblue native polyacrylamide gel electrophoresisSDS-PAGEsodium dodecylsulfate polyacrylamide gel electrophoresisC-terC-terminusN-terN-terminusHis-tag8-histidine tagFLAG-tagDYKDDDDK-tagSECsize exclusion chromatographyTMHtransmembrane helixDSSdisuccinimidyl suberateDSBUdisuccinimidyl dibutyric urea; DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chlorideheme-Feheme-ironEmredox midpoint potentialheme cp1N-ter located c-type heme 1 of CcoPheme cp2C-ter located c-type heme 2 of CcoPheme coc-type heme of CcoOSO32-sulfiteSO42-sulfateRMSDroot-mean-square deviationDDMn-dodecyl β-D-maltoside.View Full Text

biochemistry↗