Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. Here, we show that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. Our results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR and uncover new roles for an EHD protein at synapses.

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The protein-binding pocket of Botulinum neurotoxin B accommodates a preassembled synaptotagmin / ganglioside complex

10.1101/2021.10.18.464377 ◽

2021 ◽

Author(s):

Jorge Ramirez-Franco ◽

Fodil Azzaz ◽

Marion Sangiardi ◽

G&eacuteraldine Ferracci ◽

Fahamoe Youssouf ◽

...

Keyword(s):

Botulinum Neurotoxin ◽

Transmembrane Domain ◽

Binding Pocket ◽

Lipid Binding ◽

Membrane Topology ◽

Neuronal Membranes ◽

Natural Variant ◽

Synaptotagmin 1 ◽

Binding Pockets ◽

Serotype B

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of this therapeutic tool bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside. This interaction allows a lipid-binding loop of BoNT/B to engage in a series of concomitant interactions with the glycone part of GT1b and the transmembrane domain of synaptotagmin. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

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Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

10.1101/2020.08.11.243204 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tilak Kumar Gupta ◽

Sven Klumpe ◽

Karin Gries ◽

Steffen Heinz ◽

Wojciech Wietrzynski ◽

...

Keyword(s):

Membrane Integrity ◽

Point Mutations ◽

Binding Pocket ◽

Thylakoid Membranes ◽

Lipid Binding ◽

Structural Basis ◽

Amphipathic Helices ◽

Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

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Structural basis for p50RhoGAP BCH domain–mediated regulation of Rho inactivation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014242118 ◽

2021 ◽

Vol 118 (21) ◽

pp. e2014242118

Author(s):

Vishnu Priyanka Reddy Chichili ◽

Ti Weng Chew ◽

Srihari Shankar ◽

Shi Yin Er ◽

Cheen Fei Chin ◽

...

Keyword(s):

Binding Pocket ◽

Lipid Binding ◽

Small Gtpases ◽

Regulatory Function ◽

Structural Basis ◽

Myoblast Differentiation ◽

Scaffolding Proteins ◽

Spatiotemporal Regulation ◽

Self Association

Spatiotemporal regulation of signaling cascades is crucial for various biological pathways, under the control of a range of scaffolding proteins. The BNIP-2 and Cdc42GAP Homology (BCH) domain is a highly conserved module that targets small GTPases and their regulators. Proteins bearing BCH domains are key for driving cell elongation, retraction, membrane protrusion, and other aspects of active morphogenesis during cell migration, myoblast differentiation, and neuritogenesis. We previously showed that the BCH domain of p50RhoGAP (ARHGAP1) sequesters RhoA from inactivation by its adjacent GAP domain; however, the underlying molecular mechanism for RhoA inactivation by p50RhoGAP remains unknown. Here, we report the crystal structure of the BCH domain of p50RhoGAP Schizosaccharomyces pombe and model the human p50RhoGAP BCH domain to understand its regulatory function using in vitro and cell line studies. We show that the BCH domain adopts an intertwined dimeric structure with asymmetric monomers and harbors a unique RhoA-binding loop and a lipid-binding pocket that anchors prenylated RhoA. Interestingly, the β5-strand of the BCH domain is involved in an intermolecular β-sheet, which is crucial for inhibition of the adjacent GAP domain. A destabilizing mutation in the β5-strand triggers the release of the GAP domain from autoinhibition. This renders p50RhoGAP active, thereby leading to RhoA inactivation and increased self-association of p50RhoGAP molecules via their BCH domains. Our results offer key insight into the concerted spatiotemporal regulation of Rho activity by BCH domain–containing proteins.

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A Novel Variant in the TBC1D24 Lipid-Binding Pocket Causes Autosomal Dominant Hearing Loss: Evidence for a Genotype-Phenotype Correlation

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2020.585669 ◽

2020 ◽

Vol 14 ◽

Author(s):

Thomas Parzefall ◽

Alexandra Frohne ◽

Martin Koenighofer ◽

Juergen Neesen ◽

Franco Laccone ◽

...

Keyword(s):

Hearing Loss ◽

Autosomal Dominant ◽

Binding Pocket ◽

Lipid Binding ◽

Phenotype Correlation ◽

Genotype Phenotype Correlation ◽

Novel Variant

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Skywalker to the Rescue – Sky/TBC1D24 epilepsy mutations in a novel lipid-binding pocket cause presynaptic dysfunction

10.26226/morressier.5b31ec422afeeb001345a064 ◽

2018 ◽

Author(s):

Kevin Luethy

Keyword(s):

Binding Pocket ◽

Lipid Binding

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Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

The Journal of Cell Biology ◽

10.1083/jcb.200905007 ◽

2009 ◽

Vol 187 (6) ◽

pp. 889-903 ◽

Cited By ~ 150

Author(s):

Timothy A. Schulz ◽

Mal-Gi Choi ◽

Sumana Raychaudhuri ◽

Jason A. Mears ◽

Rodolfo Ghirlando ◽

...

Keyword(s):

Binding Protein ◽

Binding Pocket ◽

Lipid Binding ◽

Dependent Manner ◽

Oxysterol Binding Protein ◽

The Core ◽

Contact Sites ◽

Core Lipid ◽

Related Proteins ◽

Distal Site

Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

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Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

Biochemical Society Symposium ◽

10.1042/bss2007c05 ◽

2007 ◽

Vol 74 ◽

pp. 47-57 ◽

Cited By ~ 6

Author(s):

David J. Gill ◽

Hsiangling Teo ◽

Ji Sun ◽

Olga Perisic ◽

Dmitry B. Veprintsev ◽

...

Keyword(s):

Protein Complexes ◽

Multivesicular Body ◽

Binding Pocket ◽

Lipid Binding ◽

Pleckstrin Homology Domain ◽

Class Iii ◽

Membrane Recruitment ◽

Early Endosomes ◽

Phosphoinositide 3 Kinase ◽

Vacuolar Protein

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.

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Unique structural features of the AIPL1–FKBP domain that support prenyl lipid binding and underlie protein malfunction in blindness

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704782114 ◽

2017 ◽

Vol 114 (32) ◽

pp. E6536-E6545 ◽

Cited By ~ 7

Author(s):

Ravi P. Yadav ◽

Lokesh Gakhar ◽

Liping Yu ◽

Nikolai O. Artemyev

Keyword(s):

Binding Pocket ◽

Structural Features ◽

Biochemical Properties ◽

Lipid Binding ◽

Severe Form ◽

Nmr Studies ◽

Interacting Protein ◽

Aryl Hydrocarbon ◽

Dynamics Simulations ◽

Fkbp Domain

FKBP-domain proteins (FKBPs) are pivotal modulators of cellular signaling, protein folding, and gene transcription. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a distinctive member of the FKBP superfamily in terms of its biochemical properties, and it plays an important biological role as a chaperone of phosphodiesterase 6 (PDE6), an effector enzyme of the visual transduction cascade. Malfunction of mutant AIPL1 proteins triggers a severe form of Leber congenital amaurosis and leads to blindness. The mechanism underlying the chaperone activity of AIPL1 is largely unknown, but involves the binding of isoprenyl groups on PDE6 to the FKBP domain of AIPL1. We solved the crystal structures of the AIPL1–FKBP domain and its pathogenic mutant V71F, both in the apo form and in complex with isoprenyl moieties. These structures reveal a module for lipid binding that is unparalleled within the FKBP superfamily. The prenyl binding is enabled by a unique “loop-out” conformation of the β4-α1 loop and a conformational “flip-out” switch of the key W72 residue. A second major conformation of apo AIPL1–FKBP was identified by NMR studies. This conformation, wherein W72 flips into the ligand-binding pocket and renders the protein incapable of prenyl binding, is supported by molecular dynamics simulations and appears to underlie the pathogenicity of the V71F mutant. Our findings offer critical insights into the mechanisms that underlie AIPL1 function in health and disease, and highlight the structural and functional diversity of the FKBPs.

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