Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin

Guanine nucleotides can flip between a North and South conformation in the ribose moiety. To test the enzymatic activity of GTPases bound to nucleotides in the two conformations, we generated methanocarba guanine nucleotides in the North or South envelope conformations, i.e., (N)-GTP and (S)-GTP, re...

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Main Authors: Kiran S. Toti, John R. Jimah, Veronica Salmaso, Jenny E. Hinshaw, Kenneth A. Jacobson
Format: Article
Language:English
Published: MDPI AG 2022-04-01
Series:Biomolecules
Subjects:
Online Access:https://www.mdpi.com/2218-273X/12/4/584
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author Kiran S. Toti
John R. Jimah
Veronica Salmaso
Jenny E. Hinshaw
Kenneth A. Jacobson
author_facet Kiran S. Toti
John R. Jimah
Veronica Salmaso
Jenny E. Hinshaw
Kenneth A. Jacobson
author_sort Kiran S. Toti
collection DOAJ
description Guanine nucleotides can flip between a North and South conformation in the ribose moiety. To test the enzymatic activity of GTPases bound to nucleotides in the two conformations, we generated methanocarba guanine nucleotides in the North or South envelope conformations, i.e., (N)-GTP and (S)-GTP, respectively. With dynamin as a model system, we examined the effects of (N)-GTP and (S)-GTP on dynamin-mediated membrane constriction, an activity essential for endocytosis. Dynamin membrane constriction and fission activity are dependent on GTP binding and hydrolysis, but the effect of the conformational state of the GTP nucleotide on dynamin activity is not known. After reconstituting dynamin-mediated lipid tubulation and membrane constriction in vitro, we observed via cryo-electron microscopy (cryo-EM) that (N)-GTP, but not (S)-GTP, enables the constriction of dynamin-decorated lipid tubules. These findings suggest that the activity of dynamin is dependent on the conformational state of the GTP nucleotide. However, a survey of nucleotide ribose conformations associated with dynamin structures in nature shows almost exclusively the (S)-conformation. The explanation for this mismatch of (N) vs. (S) required for GTP analogues in a dynamin-mediated process will be addressed in future studies.
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spelling doaj.art-632a0ec8355d41b1802a038d1a5883b82023-12-01T00:57:16ZengMDPI AGBiomolecules2218-273X2022-04-0112458410.3390/biom12040584Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on DynaminKiran S. Toti0John R. Jimah1Veronica Salmaso2Jenny E. Hinshaw3Kenneth A. Jacobson4Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USALaboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USALaboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USALaboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USALaboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892, USAGuanine nucleotides can flip between a North and South conformation in the ribose moiety. To test the enzymatic activity of GTPases bound to nucleotides in the two conformations, we generated methanocarba guanine nucleotides in the North or South envelope conformations, i.e., (N)-GTP and (S)-GTP, respectively. With dynamin as a model system, we examined the effects of (N)-GTP and (S)-GTP on dynamin-mediated membrane constriction, an activity essential for endocytosis. Dynamin membrane constriction and fission activity are dependent on GTP binding and hydrolysis, but the effect of the conformational state of the GTP nucleotide on dynamin activity is not known. After reconstituting dynamin-mediated lipid tubulation and membrane constriction in vitro, we observed via cryo-electron microscopy (cryo-EM) that (N)-GTP, but not (S)-GTP, enables the constriction of dynamin-decorated lipid tubules. These findings suggest that the activity of dynamin is dependent on the conformational state of the GTP nucleotide. However, a survey of nucleotide ribose conformations associated with dynamin structures in nature shows almost exclusively the (S)-conformation. The explanation for this mismatch of (N) vs. (S) required for GTP analogues in a dynamin-mediated process will be addressed in future studies.https://www.mdpi.com/2218-273X/12/4/584conformationally lockedmethanocarbaguanine nucleotidedynaminGTPasemembrane fission
spellingShingle Kiran S. Toti
John R. Jimah
Veronica Salmaso
Jenny E. Hinshaw
Kenneth A. Jacobson
Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin
Biomolecules
conformationally locked
methanocarba
guanine nucleotide
dynamin
GTPase
membrane fission
title Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin
title_full Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin
title_fullStr Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin
title_full_unstemmed Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin
title_short Synthesis and Effect of Conformationally Locked Carbocyclic Guanine Nucleotides on Dynamin
title_sort synthesis and effect of conformationally locked carbocyclic guanine nucleotides on dynamin
topic conformationally locked
methanocarba
guanine nucleotide
dynamin
GTPase
membrane fission
url https://www.mdpi.com/2218-273X/12/4/584
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