Biology subjects

Zaccai, N. R.

Publications and source records attributed to Zaccai, N. R..

6 recordsLinked to original sources

bioRxiv2024

Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis

Clathrin mediated endocytosis (CME) is the main mechanism for swift and selective uptake of proteins into eukaryotic cells. CME is initiated by recruitment to the plasma membrane (PM) of the adaptor protein AP2, which recognizes the PM-associated lipid PtdIns(4,5)P2, as well as the protein cargo to be internalized. Nonetheless, many aspects of this process remain unclear due to their in vivo complexity. Here, a thermodynamic and time-resolved structural analysis of AP2 binding to different biomimetic PM was undertaken under physiological conditions using a combination of neutron reflectometry, interfacial tensiometry and rheology, and atomic force microscopy. The resultant in vitro data replicated previous in vivo observations, as well as yielded biophysical insights into normal and aborted CME. The presence of cargo may not be pivotal for the "activating" conformational change of AP2. However, the presence of cargo extends AP2s residence time on the membrane surface, due to slower on- and off-rates, thereby tentatively giving sufficient time for CME to proceed fully. Moreover, upon interaction with AP2, phospholipid lateral diffusion decreases markedly, inducing a gel phase attributed to creating a percolated network involving AP2 on the membrane, which could potentially serve as a mechanism for modulating subsequent clathrin binding.

biophysics↗

bioRxiv2022

Stable and ancient endocytic structures navigate the complex pellicle of apicomplexan parasites

Apicomplexan parasites have an immense impact on humanity, but their basic cellular processes are often poorly understood. The sites of endocytosis, the conservation of this process with other eukaryotes, and its functions across Apicomplexa are major unanswered questions. Yet endocytosis in Plasmodium is implicated in antimalarial drug failure. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. Moreover, conserved molecular adaptation of this structure is seen in Apicomplexa, including the kelch-domain protein K13 central to malarial drug-resistance. We determine that an essential function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata, likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

cell biology↗

bioRxiv2022

FCHO controls AP2's critical endocytic roles through a PtdIns4,5P2 membrane-dependent switch

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCPs outer edge to engage and activate further AP2s to drive CCP growth/maturation. 125 character summaryFCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s

cell biology↗

bioRxiv2021

Strikingly different roles of SARS-CoV-2 fusion peptides uncovered by neutron scattering

Coronavirus disease-2019 (COVID-19), a lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of the SARS-CoV-2 Spike protein, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium however re-orients the protein to the lipid leaflet in contact with the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed. SIGNIFICANCEWe have recreated here important elements of the critical membrane fusion mechanism of the SARS-CoV-2 coronavirus by simplifying the system down to its core elements, amenable to experimental analysis by neutron scattering. Neutrons are well suited for the study of protein - membrane interactions under physiological conditions, since they allow structural and dynamics characterization at room temperature. Our results revealed strikingly different functions encoded in the viral Spike fusion domain and thereby provide a potential calcium-dependent cell entry mechanism for SARS-CoV-2. In particular, calcium drives the proteins N-terminal to harpoon through the host membrane, while removing calcium re-orients the protein so that it is able to bridge and dehydrate lipid membranes, facilitating their fusion.

biophysics↗

bioRxiv2021

Structures of a deAMPylation complex rationalise the switch between antagonistic catalytic activities of FICD

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present long-sought crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiPs ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICDs antagonistic activities, despite a similar mode of engagement of its two substrates -- unmodified and AMPylated BiP.

biochemistry↗

bioRxiv2018

Navigating the structural landscape of de novo α-helical bundles

The association of amphipathic helices in water leads to -helical-bundle protein structures. However, the driving force for this--the hydrophobic effect--is not specific and does not define the number or the orientation of helices in the associated state. Rather, this is achieved through deeper sequence-to-structure relationships, which are increasingly being discerned. For example, for one structurally extreme but nevertheless ubiquitous class of bundle--the -helical coiled coils--relationships have been established that discriminate between all-parallel dimers, trimers and tetramers. Association states above this are known, as are antiparallel and mixed arrangements of the helices. However, these alternative states are less-well understood. Here, we describe a synthetic-peptide system that switches between parallel hexamers and various up-down-up-down tetramers in response to single-amino-acid changes and solution conditions. The main accessible states of each peptide variant are characterized fully in solution and, in most cases, to high-resolution X-ray crystal structures. Analysis and inspection of these structures helps rationalize the different states formed. This navigation of the structural landscape of -helical coiled coils above the dimers and trimers that dominate in nature has allowed us to design rationally a well-defined and hyperstable antiparallel coiled-coil tetramer (apCC-Tet). This robust de novo protein provides another scaffold for further structural and functional designs in protein engineering and synthetic biology.

biochemistry↗