Biology subjects

Vercellino, I.

Publications and source records attributed to Vercellino, I..

5 recordsLinked to original sources

bioRxiv2026

Cryo-EM reveals the central steps of mitochondrial complex III assembly and the cooperative assembly of supercomplex CIII2CIV

Mitochondrial complex III is the central component of the respiratory chain and is conserved across eukaryotes. Complex III is an obligate dimer which assembles through a stepwise mechanism, involving 20 subunits and other assembly factors. Defects in the assembly of complex III are associated with metabolic diseases. The assembly mechanism of complex III has long been investigated using biochemical approaches, which suggested a process where folded subunits are added sequentially and in parallel for the two protomers after dimerization. Our structural investigation of CIII2 assembly challenges these assumptions: we observe using cryo-EM that incorporation and folding of its subunits can be uncoupled (as we show for cytochrome c1) and that after dimerization the assembly of the complex does not proceed in parallel for the two protomers (as we show for the folding of the intermembrane space domain). Our structures also reveal the mechanism of formation of supercomplex CIII2CIV for non-vertebrates, intertwined with the assembly of CIII2. This work reshapes our knowledge of complex III assembly and proposes a generalizable model for the maturation of the complex, alongside a model for supercomplex formation that supports the cooperative assembly model. Furthermore, as the observed assembly steps cannot be predicted by AlphaFold, our work also showcases the central role of cryo-EM in the study of assembly mechanism of protein complexes.

molecular biology↗

bioRxiv2025

Cryo-EM structures of naturally occurring dimeric photosystem II complexes lacking the Mn4CaO5 cluster

Robust oxygenic photosynthesis requires the efficient assembly and repair of the multi-subunit oxygen-evolving photosystem II (PSII) complex. Previous electron cryo-microscopy (cryo-EM) structures of PSII assembly/disassembly intermediates have relied on the analysis of deletion mutants or removal of PSII subunits in vitro. Here we have determined the cryo-EM structures of naturally occurring dimeric PSII intermediates from the cyanobacterium Thermosynechococcus vestitus at a resolution of 2.2 [A]. These intermediates contain inactive dimers lacking the oxygen-evolving complex (OEC) and semi-active dimers with the OEC present in one of the two monomers. Our structural data provide a mechanism for how assembly and disassembly of the Mn4CaO5 cluster is coordinated with the binding and release of the extrinsic proteins: restructuring of the C-terminal tail of D1 subunit during assembly or disassembly of the Mn cluster triggers conformational changes in D2, CP47 and CP43 to drive the binding/release of the extrinsic proteins. A combination of structural and mass spectrometry data indicates that the inactive PSII complexes are disassembly complexes and that oxidation of D1-His332, a monodentate ligand to one of the Mn ions of the OEC, is an early event in the photoinactivation of PSII.

biochemistry↗

bioRxiv2022

The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we have used cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the Mn4CaO5 cluster that catalyzes water oxidation, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides new insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.

biochemistry↗

bioRxiv2021

Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c / Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signaling is well established, the function of their transmembrane (TM) regions remains unknown. Here we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 [A] resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands. One-Sentence SummaryStructure of M. tuberculosis membrane adenylyl cyclase Cya

biochemistry↗

bioRxiv2018

Structural basis of sterol recognition by human hedgehog receptor PTCH1

Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4 [A] cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by ten sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol sensing domain, SSD, and the SSD-like domain, SSDL, which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.

biochemistry↗