Biology subjects

Lyons, J. A.

Publications and source records attributed to Lyons, J. A..

8 recordsLinked to original sources

bioRxiv2025

Revisiting flippase specificity: Drs2-Cdc50 transports multiple anionic lipid substrates

P4-ATPase lipid flippases maintain transbilayer lipid asymmetry in eukaryotic membranes, which is essential for many cellular processes. In yeast, the Drs2-Cdc50 flippase complex was previously shown to specifically transport phosphatidylserine (PS) from the exoplasmic to the cytosolic leaflet of the trans-Golgi network (TGN), thereby controlling vesicular trafficking in the secretory and endocytic pathways. Using an improved proteoliposome-based lipid flippase assay, we now show that the Drs2-Cdc50 complex transports multiple anionic glycerophospholipids, including PS, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. In vivo cell-based lipid uptake assays further support the transport of these lipids. To understand the basis of this substrate promiscuity, we analyzed cryo-EM structures of the complex with occluded lipids. These structures revealed that the water network surrounding the lipid headgroup plays a critical role in enabling Drs2-Cdc50 to recognize different lipids. These data unveil an unexpected broad specificity of the Drs2-Cdc50 complex for anionic lipids, which may significantly impact their transbilayer distribution in the yeast TGN.

biochemistry↗

bioRxiv2024

Cryo-EM analysis of complement C3 reveals a reversible major opening of the macroglobulin ring

The C3 protein is the central molecule within the complement system and undergoes pattern-recognition-dependent proteolytic activation to C3b in the presence of pathogens and damage-associated patterns. Spontaneous pattern-independent activation of C3 occurs via hydrolysis, resulting in C3(H2O). However, the structural details of C3 hydrolysis remain elusive. Here, we show that the conformation of the C3(H2O) analog, C3MA, in which the C3 thioester is broken by aminolysis is indistinguishable from C3b except for the 77-residue anaphylatoxin (ANA) domain. In contrast, the reaction intermediate C3* formed during C3 adopts a dynamic conformation dramatically different from both C3 and C3MA/C3b. In C3*, unlocking of the macroglobulin (MG) 3 domain creates a large opening in the MG-ring through which the ANA domain translocates. In support of this mechanism, C3MA formation is inhibited by an MG3/MG4-interface-specific nanobody and prevented by linking the ANA domain to the C3 {beta}-chain. Our study reveals an unexpected dynamic behavior of C3 where an exceptional conformational change allows the translocation of an entire domain through a large dynamic opening. These results form the basis for elucidation of the in vivo contribution of C3 hydrolysis to complement activation and offer a rational approach for modulation of C3(H2O) with the potential for preventing complement activation caused by intravascular hemolysis and surface contacts.

biochemistry↗

bioRxiv2023

Functional characterization of ATP13A2 variants associated with distinct neurodegenerative disorders

ATP13A2 is a late endolysosomal transporter that exports the polyamines spermine and spermidine from the organellar lumen to the cytosol. Loss-of-function variants in ATP13A2 are causative for Kufor-Rakeb syndrome (KRS, a recessive juvenile-onset parkinsonism with dementia) and have also been identified in early-onset PD (EOPD) and hereditary spastic paraplegia (HSP). Furthermore, candidate pathogenic ATP13A2 variants have been identified in neuronal ceroid lipofuscinosis (NCL; M854R), multiple system atrophy (MSA; Y1020C) and amyotrophic lateral sclerosis (ALS; I411M) suggesting that ATP13A2 may be implicated in a broader range of neurodegenerative disorders. Since the functional consequences of the NCL, MSA, and ALS variants have not yet been examined, we here characterized these ATP13A2 variants in terms of subcellular localization, cellular polyamine uptake, and transport activity. We found that the homozygous NCL-associated M854R variant results in an instable protein with low expression levels, leading to complete loss of ATPase and cellular polyamine uptake activity. The heterozygous MSA-linked Y1020C variant is properly localized and presents only partially decreased ATPase activity without affecting cellular polyamine uptake. The ALS-associated I411M variant is also correctly localized and exhibits a minor effect on cellular polyamine uptake, however, without a significant impact on ATPase activity. Taken together, only the homozygous NCL variant of ATP13A2 causes a complete loss-of-function, validating that ATP13A2 dysfunction is implicated in NCL. The ALS and MSA variants only presented a subtle functional defect, questioning whether these heterozygous variants are pathogenic and whether ATP13A2 dysfunction may cause MSA or ALS.

molecular biology↗

bioRxiv2023

Activation and substrate specificity of the human P4-ATPase ATP8B1

Asymmetric distribution of phospholipids in eukaryotic membranes is essential for maintaining cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. In this study, we present a total of nine cryo-electron microscopy structures at a resolution ranging from 2.4 to 3.1 [A], along with functional and computational studies of the human flippase ATP8B1-CDC50A complex, describing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as its regulation by phosphoinositides. Our findings show that the P4-ATPase transport site is filled with water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identified and characterized the PI(3,4,5)P3 binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of ATP8B1 broad specificity for lipids and identifies a new transport substrate for P4-ATPases, phosphatidylinositol (PI). We report the critical role of the sn-2 ester bound of glycerophospholipids in substrate recognition by ATP8B1. These findings provide fundamental insights into ATP8B1 regulation, the catalytic cycle, and substrate recognition in P4-ATPases.

biochemistry↗

bioRxiv2022

Variations of a theme - crystal forms of the amino acid transporter MhsT

The bacterial amino acid transporter MhsT of the SLC6A family was crystallized in complex with different substrates in order to understand the determinants of substrate specificity of the transporter. Surprisingly, crystals of the different MhsT-substrate complexes showed interrelated, but different crystal packing arrangements. Space group assignment and structure determination of these different crystal forms presented challenging combinations of pseudosymmetry, twinning and tNCS. SynopsisAn unusual case of protein-substrate complexes obtained in similar conditions, but containing different packing arrangements. The crystals exhibit a combination of various crystal imperfections (pseudosymmetry, twinning and tNCS) masking the true crystal symmetries and challenging data processing and structure determination.

biochemistry↗

bioRxiv2021

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, e.g. in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 [A] resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C- termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

biochemistry↗

bioRxiv2020

Computational Studies of Substrate Transport and Specificity in a Phospholipid Flippase

Type 4 P-type ATPases are lipid flippases which help maintain asymmetric phospholipid distribution in eukaryotic membranes by driving unidirectional translocation of phospholipid substrates. Recent cryo-EM and crystal structures have provided a detailed view of flippases, and we here use molecular dynamics simulations to study the first steps of phospholipid transport and lipid substrate specificity. Our simulations and new cryo-EM structure shows phospholipid binding to a groove and subsequent movement towards the centre of the membrane, and reveal a preference for phosphatidylserine lipids. We find that only the lipid head group stays in the groove while the lipid tails remain in the membrane, thus visualizing how flippases have evolved to transport large substrates. The flippase also induces deformation and thinning of the outer leaflet facilitating lipid recruitment. Our simulations provide insight into substrate binding to flippases and suggest that multiple sites and steps in the functional cycle contribute to substrate selectivity.

biophysics↗

bioRxiv2019

Structure and autoregulation of a P4-ATPase lipid flippase

P4-ATPases are lipid flippases that drive active transport of phospholipids from the exoplasmic or lumenal to the cytosolic leaflets of eukaryotic membranes to maintain their asymmetric lipid composition. The molecular architecture of P4-ATPases and how they work in lipid recognition and transport has remained elusive. Using cryo-electron microscopy we have determined the structures of a P4-ATPase, specifically of the Saccharomyces cerevisiae Drs2p-Cdc50p, which is a phosphatidylserine and phosphatidylethanolamine specific lipid flippase. Drs2p-Cdc50p is autoinhibited by the Drs2p C-terminal tail and activated by phosphatidylinositol-4 phosphate (PI4P). We present three structures representing an autoinhibited, an intermediate, and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of auto-inhibition and PI4P-dependent activation. We observe the opening of a putative flippase pathway engaging conserved residues Ile508 of transmembrane segment 4 and Lys1018 and polar residues of transmembrane segment 5 in the centre of the lipid bilayer.

molecular biology↗