scholarly journals Structures of human mitofusin 1 provide insight into mitochondrial tethering

2016 ◽  
Vol 215 (5) ◽  
pp. 621-629 ◽  
Author(s):  
Yuanbo Qi ◽  
Liming Yan ◽  
Caiting Yu ◽  
Xiangyang Guo ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Distal Part ◽  
Binding Pocket ◽  
Magnesium Ion ◽  
Gtpase Activity ◽  
Mitochondrial Membranes ◽  
Gtpase Domain ◽  
Gtp Hydrolysis ◽  
Mitofusin 1 ◽  
Insight Into

Mitochondria undergo fusion and fission. The merging of outer mitochondrial membranes requires mitofusin (MFN), a dynamin-like GTPase. How exactly MFN mediates membrane fusion is poorly understood. Here, we determined crystal structures of a minimal GTPase domain (MGD) of human MFN1, including the predicted GTPase and the distal part of the C-terminal tail (CT). The structures revealed that a helix bundle (HB) formed by three helices extending from the GTPase and one extending from the CT closely attaches to the GTPase domain, resembling the configuration of bacterial dynamin-like protein. We show that the nucleotide-binding pocket is shallow and narrow, rendering weak hydrolysis and less dependence on magnesium ion, and that association of HB affects GTPase activity. MFN1 forms a dimer when GTP or GDP/BeF3−, but not GDP or other analogs, is added. In addition, clustering of vesicles containing membrane-anchored MGD requires continuous GTP hydrolysis. These results suggest that MFN tethers apposing membranes, likely through nucleotide-dependent dimerization.

scholarly journals Sequences flanking the transmembrane segments facilitate mitochondrial localization and membrane fusion by mitofusin

2017 ◽  
Vol 114 (46) ◽  
pp. E9863-E9872 ◽  
Author(s):  
Xiaofang Huang ◽  
Xin Zhou ◽  
Xiaoyu Hu ◽  
Amit S. Joshi ◽  
Xiangyang Guo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Amphipathic Helix ◽  
Mitochondrial Localization ◽  
Mitochondrial Membranes ◽  
Gtpase Domain ◽  
Hydrophobic Residues ◽  
Transmembrane Segments ◽  
Mechanistic Insight ◽  
Insight Into

Mitochondria constantly divide and fuse. Homotypic fusion of the outer mitochondrial membranes requires the mitofusin (MFN) proteins, a family of dynamin-like GTPases. MFNs are anchored in the membrane by transmembrane (TM) segments, exposing both the N-terminal GTPase domain and the C-terminal tail (CT) to the cytosol. This arrangement is very similar to that of the atlastin (ATL) GTPases, which mediate fusion of endoplasmic reticulum (ER) membranes. We engineered various MFN-ATL chimeras to gain mechanistic insight into MFN-mediated fusion. When MFN1 is localized to the ER by TM swapping with ATL1, it functions in the maintenance of ER morphology and fusion. In addition, an amphipathic helix in the CT of MFN1 is exchangeable with that of ATL1 and critical for mitochondrial localization of MFN1. Furthermore, hydrophobic residues N-terminal to the TM segments of MFN1 play a role in membrane targeting but not fusion. Our findings provide important insight into MFN-mediated membrane fusion.

scholarly journals Structures of the yeast dynamin-like GTPase Sey1p provide insight into homotypic ER fusion

2015 ◽  
Vol 210 (6) ◽  
pp. 961-972 ◽  
Author(s):  
Liming Yan ◽  
Sha Sun ◽  
Wei Wang ◽  
Juanming Shi ◽  
Xiaoyu Hu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Candida Albicans ◽  
Membrane Fusion ◽  
Mammalian Cells ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Helical Bundle ◽  
The Common ◽  
Insight Into ◽  
Common Function

Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF4− but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p.

scholarly journals GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

1998 ◽  
Vol 18 (2) ◽  
pp. 827-838 ◽  
Author(s):  
Celeste J. Richardson ◽  
Sara Jones ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Membrane Fusion ◽  
Vesicular Transport ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Dominant Phenotype ◽  
Guanine Nucleotide Exchange ◽  
Membrane Morphology ◽  
Nucleotide Exchange Factor ◽  
Mutant Cells

ABSTRACT GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-freeYPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

2017 ◽  
Vol 73 (12) ◽  
pp. 970-984 ◽  
Author(s):  
Shenyuan Xu ◽  
Brian N. Long ◽  
Gabriel H. Boris ◽  
Anqi Chen ◽  
Shuisong Ni ◽  
...  
Keyword(s):  
Transition State ◽  
Binding Pocket ◽  
High Energy ◽  
Side Chain ◽  
Conformation Change ◽  
Gtp Hydrolysis ◽  
Structural Insight ◽  
Hydrolysis Of ◽  
Insight Into ◽  
Intrinsic Gtpase Activity

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

scholarly journals Structural insights into G domain dimerization and pathogenic mutation of OPA1

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Caiting Yu ◽  
Jinghua Zhao ◽  
Liming Yan ◽  
Yuanbo Qi ◽  
Xiangyang Guo ◽  
...  
Keyword(s):  
Optic Atrophy ◽  
Molecular Basis ◽  
Pathogenic Mutation ◽  
Mitochondrial Morphology ◽  
Membrane Association ◽  
Mitochondrial Membranes ◽  
Gtpase Domain ◽  
Gtp Hydrolysis ◽  
Catalytic Core ◽  
Structural Insights

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF3−, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.

scholarly journals Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Taylor B Updegrove ◽  
Jailynn Harke ◽  
Vivek Anantharaman ◽  
Jin Yang ◽  
Nikhil Gopalan ◽  
...  
Keyword(s):  
Active Site ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Gtpase Activity ◽  
Biological Functions ◽  
Gtp Hydrolysis ◽  
Nucleotide Base ◽  
Nucleoside Triphosphates ◽  
Nucleotide Specificity ◽  
Hydrolysis Of

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.

scholarly journals FlhF and Its GTPase Activity Are Required for Distinct Processes in Flagellar Gene Regulation and Biosynthesis in Campylobacter jejuni

2009 ◽  
Vol 191 (21) ◽  
pp. 6602-6611 ◽  
Author(s):  
Murat Balaban ◽  
Stephanie N. Joslin ◽  
David R. Hendrixson
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Campylobacter Jejuni ◽  
Regulatory System ◽  
Flagellar Gene ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Genetic Analyses ◽  
Flagellar Biosynthesis ◽  
Two Component

ABSTRACT FlhF proteins are putative GTPases that are often necessary for one or more steps in flagellar organelle development in polarly flagellated bacteria. In Campylobacter jejuni, FlhF is required for σ54-dependent flagellar gene expression and flagellar biosynthesis, but how FlhF influences these processes is unknown. Furthermore, the GTPase activity of any FlhF protein and the requirement of this speculated activity for steps in flagellar biosynthesis remain uncharacterized. We show here that C. jejuni FlhF hydrolyzes GTP, indicating that these proteins are GTPases. C. jejuni mutants producing FlhF proteins with reduced GTPase activity were not severely defective for σ54-dependent flagellar gene expression, unlike a mutant lacking FlhF. Instead, these mutants had a propensity to lack flagella or produce flagella in improper numbers or at nonpolar locations, indicating that GTP hydrolysis by FlhF is required for proper flagellar biosynthesis. Additional studies focused on elucidating a possible role for FlhF in σ54-dependent flagellar gene expression were conducted. These studies revealed that FlhF does not influence production of or signaling between the flagellar export apparatus and the FlgSR two-component regulatory system to activate σ54. Instead, our data suggest that FlhF functions in an independent pathway that converges with or works downstream of the flagellar export apparatus-FlgSR pathway to influence σ54-dependent gene expression. This study provides corroborative biochemical and genetic analyses suggesting that different activities of the C. jejuni FlhF GTPase are required for distinct steps in flagellar gene expression and biosynthesis. Our findings are likely applicable to many polarly flagellated bacteria that utilize FlhF in flagellar biosynthesis processes.

Abscisic acid induction of GTP hydrolysis in maize coleoptile plasma membranes

1998 ◽  
Vol 25 (5) ◽  
pp. 539 ◽  
Author(s):  
Helen R. Irving
Keyword(s):  
Abscisic Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Gtpase Activity ◽  
Heterotrimeric G Proteins ◽  
Gtp Hydrolysis ◽  
Maize Coleoptile

Since receptor-coupled G proteins increase GTP hydrolysis (GTPase) activity upon ligands binding to the receptor, a study was undertaken to determine if abscisic acid (ABA) induced such an effect. Plasma membranes isolated from etiolated maize (Zea mays L.) coleoptiles were enriched in GTPase activity relative to microsomal fractions. Vanadate was included in the assay to inhibit the high levels of vanadate sensitive low affinity GTPases present. Under these conditions, GTPase activity was enhanced by Mg2+, stimulated by mastoparan, and inhibited by GTPγS indicating the presence of either monomeric or heterotrimeric G proteins. The combination of NaF and AlCl3 is expected to inhibit heterotrimeric G protein activity but had little effect on GTPase activity in maize coleoptile membranes. Cholera toxin enhanced basal GTPase activity, confirming the presence of heterotrimeric G proteins in maize plasma membranes. Pertussis toxin also slightly enhanced basal GTPase activity in maize membranes. Abscisic acid enhanced GTPase activity optimally at 5 mmol/L Mg2+ in a concentration dependent manner by 1.5-fold at 10 µmol/L and up to three-fold at 100 µmol/L ABA. Abscisic acid induced GTPase activity was inhibited by GTPγS, the combination of NaF and AlCl3, and pertussis toxin. Overall, these results are typical of a receptor-coupled G protein responding to its ligand.

scholarly journals The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

2019 ◽  
Vol 116 (41) ◽  
pp. 20404-20410 ◽  
Author(s):  
Amy J. Fernandez ◽  
Earnest James Paul Daniel ◽  
Sai Pooja Mahajan ◽  
Jeffrey J. Gray ◽  
Thomas A. Gerken ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Basis ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Substrate Selectivity ◽  
Peptide Substrate ◽  
X Ray ◽  
Biochemical Studies ◽  
Cancer Mutations ◽  
Insight Into

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

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