Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

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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Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713026461 ◽

2014 ◽

Vol 70 (2) ◽

pp. 242-252 ◽

Cited By ~ 4

Author(s):

Sonia Fieulaine ◽

Michel Desmadril ◽

Thierry Meinnel ◽

Carmela Giglione

Keyword(s):

Sequence Similarity ◽

Three Dimensional ◽

Binding Pocket ◽

Dimensional Structure ◽

Structural Basis ◽

Human Counterpart ◽

Low Activity

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of theN-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeablein vivoand display similar propertiesin vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

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Structural basis of dynamic P5CS filaments

10.1101/2021.12.02.470899 ◽

2021 ◽

Author(s):

Jiale Zhong ◽

Chen-Jun Guo ◽

Xian Zhou ◽

Chia-Chun Chang ◽

Boqi Yin ◽

...

Keyword(s):

Conformational Changes ◽

Essential Role ◽

Point Mutations ◽

Underlying Mechanism ◽

Structural Basis ◽

Multiple Interfaces ◽

Cryo Electron Microscopy ◽

Neurocutaneous Syndrome

AbstractThe bifunctional enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS) is central to the synthesis of proline and ornithine. Pathogenic mutations in P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants, which has an essential role in agriculture and human health. Recently, P5CS has been demonstrated forming the cytoophidium in vivo and filaments in vitro. However, the underlying mechanism for the function of P5CS filamentation and catalyze the synthesis of P5C is hardly accessible without structural basis. Here, we have succeeded in determining the full-length structures of Drosophila P5CS filament in three states at resolution from 3.1 to 4.3 Å under cryo-electron microscopy, we observed the distinct ligand-binding states and conformational changes for GK and GPR domain separately. These structures show the distinctive spiral filament is assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations that deplete such interactions disturb P5CS filamentation and greatly reduce the activity. Our findings reveal a previously undescribed mechanism that filamentation is crucial for the coordination between GK and GPR domains, and provide insights into structural basis for catalysis function of P5CS filament.

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Structural basis for a complex I mutation that blocks pathological ROS production

Nature Communications ◽

10.1038/s41467-021-20942-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhan Yin ◽

Nils Burger ◽

Duvaraka Kula-Alwar ◽

Dunja Aksentijević ◽

Hannah R. Bridges ◽

...

Keyword(s):

Point Mutation ◽

Complex I ◽

Structural Changes ◽

Ischemia Reperfusion ◽

Single Point ◽

Structural Basis ◽

Single Point Mutation ◽

Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

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Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs

10.1101/442459 ◽

2018 ◽

Cited By ~ 2

Author(s):

David M. Kern ◽

SeCheol Oh ◽

Richard K. Hite ◽

Stephen G. Brohawn

Keyword(s):

Lipid Bilayers ◽

Critical Role ◽

Anion Channel ◽

Lipid Binding ◽

Channel Structure ◽

Anion Channels ◽

Cryo Electron Microscopy ◽

Lipid Nanodiscs ◽

Insight Into

AbstractHypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

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Analysis of the structural requirements of sugar binding to the liver, brain and insulin-responsive glucose transporters expressed in oocytes

Biochemical Journal ◽

10.1042/bj2940753 ◽

1993 ◽

Vol 294 (3) ◽

pp. 753-760 ◽

Cited By ~ 22

Author(s):

C A Colville ◽

M J Seatter ◽

G W Gould

Keyword(s):

Hydrogen Bond ◽

Binding Sites ◽

Glucose Transporters ◽

Binding Pocket ◽

Structural Basis ◽

Sugar Binding ◽

Glucose Analogues ◽

Sugar Recognition

We have expressed the liver (GLUT 2), brain (GLUT 3) and insulin-responsive (GLUT 4) glucose transporters in oocytes from Xenopus laevis by microinjection of in vitro-transcribed mRNA. Using a range of halogeno- and deoxy-glucose analogues, and other hexoses, we have studied the structural basis of sugar binding to these different isoforms. We show that a hydrogen bond to the C-3 position is involved in sugar binding for all three isoforms, but that the direction of this hydrogen bond is different in GLUT 2 from either GLUT 1, 3 or 4. Hydrogen-bonding at the C-4 position is also involved in sugar recognition by all three isoforms, but we propose that in GLUT 3 this hydrogen bond plays a less significant role than in GLUT 2 and 4. In all transporters we propose that the C-4 position is directed out of the sugar-binding pocket. The role of the C-6 position is also discussed. In addition, we have analysed the ability of fructopyranose and fructofuranose analogues to inhibit the transport mediated by GLUT2. We show that fructofuranose analogues, but not fructopyranose analogues, are efficient inhibitors of transport mediated by GLUT 2, and therefore suggest that GLUT 2 accommodates D-glucose as a pyranose ring, but D-fructose as a furanose ring. Models for the binding sites of GLUT 2, 3 and 4 are presented.

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Sterol trafficking between the endoplasmic reticulum and plasma membrane in yeast

Biochemical Society Transactions ◽

10.1042/bst0340356 ◽

2006 ◽

Vol 34 (3) ◽

pp. 356-358 ◽

Cited By ~ 63

Author(s):

D.P. Sullivan ◽

H. Ohvo-Rekilä ◽

N.A. Baumann ◽

C.T. Beh ◽

A.K. Menon

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Wild Type Strain ◽

Binding Pocket ◽

Oxysterol Binding Protein ◽

Sterol Transport ◽

Transport Cycle ◽

Independent Mechanism

We recently showed that transport of ergosterol from the ER (endoplasmic reticulum) to the sterol-enriched PM (plasma membrane) in yeast occurs by a non-vesicular (Sec18p-independent) mechanism that results in the equilibration of sterol pools in the two organelles [Baumann, Sullivan, Ohvo-Rekilä, Simonot, Pottekat, Klaassen, Beh and Menon (2005) Biochemistry 44, 5816–5826]. To explore how this occurs, we tested the role of proteins that might act as sterol transporters. We chose to study oxysterol-binding protein homologues (Osh proteins), a family of seven proteins in yeast, all of which contain a putative sterol-binding pocket. Recent structural analyses of one of the Osh proteins [Im, Raychaudhuri, Prinz and Hurley (2005) Nature (London) 437, 154–158] suggested a possible transport cycle in which Osh proteins could act to equilibrate ER and PM pools of sterol. Our results indicate that the transport of newly synthesized ergosterol from the ER to the PM in an OSH deletion mutant lacking all seven Osh proteins is slowed only 5-fold relative to the isogenic wild-type strain. Our results suggest that the Osh proteins are not sterol transporters themselves, but affect sterol transport in vivo indirectly by affecting the ability of the PM to sequester sterols.

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Structural basis for spectrin recognition by ankyrin

Blood ◽

10.1182/blood-2009-11-255604 ◽

2010 ◽

Vol 115 (20) ◽

pp. 4093-4101 ◽

Cited By ~ 62

Author(s):

Jonathan J. Ipsaro ◽

Alfonso Mondragón

Keyword(s):

Membrane Proteins ◽

Structural Model ◽

Membrane Integrity ◽

Adaptor Protein ◽

Principal Component ◽

Structural Basis ◽

Protein Protein Interaction ◽

The Individual ◽

Spectrin Repeats

Maintenance of membrane integrity and organization in the metazoan cell is accomplished through intracellular tethering of membrane proteins to an extensive, flexible protein network. Spectrin, the principal component of this network, is anchored to membrane proteins through the adaptor protein ankyrin. To elucidate the atomic basis for this interaction, we determined a crystal structure of human βI-spectrin repeats 13 to 15 in complex with the ZU5-ANK domain of human ankyrin R. The structure reveals the role of repeats 14 to 15 in binding, the electrostatic and hydrophobic contributions along the interface, and the necessity for a particular orientation of the spectrin repeats. Using structural and biochemical data as a guide, we characterized the individual proteins and their interactions by binding and thermal stability analyses. In addition to validating the structural model, these data provide insight into the nature of some mutations associated with cell morphology defects, including those found in human diseases such as hereditary spherocytosis and elliptocytosis. Finally, analysis of the ZU5 domain suggests it is a versatile protein-protein interaction module with distinct interaction surfaces. The structure represents not only the first of a spectrin fragment in complex with its binding partner, but also that of an intermolecular complex involving a ZU5 domain.

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The Structural Basis for Glycerol Permeation by human AQP7

10.1101/858332 ◽

2019 ◽

Cited By ~ 1

Author(s):

Li Zhang ◽

Deqiang Yao ◽

Fu Zhou ◽

Qing Zhang ◽

Ying Xia ◽

...

Keyword(s):

Physiological Condition ◽

Critical Role ◽

Binding Pocket ◽

Structural Basis ◽

Glycerol Metabolism ◽

Functional Assay ◽

Substrate Binding Pocket ◽

Glycerol Molecule ◽

Cytoplasmic Side

AbstractHuman glycerol channel AQP7 conducts glycerol release from adipocyte and entry into the cells in pancreatic islets, muscles and kidney tubule, and thus regulate glycerol metabolism in those tissues. Compared with other human aquaglyceroporins, AQP7 shows a less conserved “NPA” motif in the center cavity, and a pair of aromatic residues at Ar/R selectivity filter. To understand the structural basis for the glycerol conductance, we crystallized the human AQP7 and determined the structure at 3.7 Å. A substrate binding pocket was found near to the Ar/R filter and the bound glycerol molecule stabilized by R229. In vivo functional assay on human AQP7 as well as AQP3 and AQP10 demonstrated strong glycerol transportation activities at physiological condition. The human AQP7 structure reveals a fully closed conformation with its permeation pathway strictly confined by Ar/R filter at the exoplasmic side and the gate at the cytoplasmic side, and the dislocation of the residues at narrowest parts of glycerol pathway in AQP7 play a critical role in controlling the glycerol flux.

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Mutations in efflux pump Rv1258c (Tap) cause resistance to pyrazinamide and other drugs inM. tuberculosis

10.1101/249102 ◽

2018 ◽

Author(s):

Jiayun Liu ◽

Wanliang Shi ◽

Shuo Zhang ◽

Gail Cassell ◽

Dmitry A. Maslov ◽

...

Keyword(s):

Drug Resistance ◽

Drug Targets ◽

Drug Exposure ◽

Efflux Pump ◽

Active Form ◽

Point Mutations ◽

Drug Susceptibility ◽

Clinical Isolates

AbstractAlthough drug resistance inM. tuberculosisis mainly caused by mutations in drug activating enzymes or drug targets, there is increasing interest in possible role of efflux in causing drug resistance. Previously, efflux genes are shown upregulated upon drug exposure or implicated in drug resistance in overexpression studies, but the role of mutations in efflux pumps identified in clinical isolates in causing drug resistance is unknown. Here we investigated the role of mutations in efflux pump Rv1258c (Tap) from clinical isolates in causing drug resistance inM. tuberculosisby constructing point mutations V219A, S292L in Rv1258c in the chromosome ofM. tuberculosisand assessed drug susceptibility of the constructed mutants. Interestingly, V219A, S292L point mutations caused clinically relevant drug resistance to pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM), but not to other drugs inM. tuberculosis. While V219A point mutation conferred a low level resistance, the S292L mutation caused a higher level of resistance. Efflux inhibitor piperine inhibited INH and PZA resistance in the S292L mutant but not in the V219A mutant. S292L mutant had higher efflux activity for pyrazinoic acid (the active form of PZA) than the parent strain. We conclude that point mutations in the efflux pump Rv1258c in clinical isolates can confer clinically relevant drug resistance including PZA and could explain some previously unaccounted drug resistance in clinical strains. Future studies need to take efflux mutations into consideration for improved detection of drug resistance inM. tuberculosisand address their role in affecting treatment outcome in vivo.

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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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