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Title: Nanomechanical and thermophoretic analyses of the nucleotide-dependent interactions between the AAA+ subunits of magnesium chelatase

Abstract

In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg2+ ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA+ proteins ChlI and ChlD, form a ChlID–MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID–MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID–MgATP complex. The N-terminal AAA+ domain of ChlD is essential for complex formation, but some stability is preserved in the absence of the C-terminal integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy (SMFS) was used to determine the factors that stabilize formation of the ChlID–MgADP complex at the single molecule level; ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show that the N-terminal AAA+ domain of ChlD mediates this process, in agreement with the microscale thermophoresis data.more » Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID–MgADP complexes. Finally, these experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex.« less

Authors:
 [1];  [1];  [1];  [1]
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  1. Univ. of Sheffield, Sheffield (United Kingdom)
Publication Date:
Research Org.:
Univ. of Sheffield, Sheffield (United Kingdom)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1346002
Grant/Contract Number:  
SC0001035
Resource Type:
Accepted Manuscript
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Volume: 138; Journal Issue: 20; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Adams, Nathan B. P., Vasilev, Cvetelin, Brindley, Amanda A., and Hunter, C. Neil. Nanomechanical and thermophoretic analyses of the nucleotide-dependent interactions between the AAA+ subunits of magnesium chelatase. United States: N. p., 2016. Web. doi:10.1021/jacs.6b02827.
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Adams, Nathan B. P., Vasilev, Cvetelin, Brindley, Amanda A., & Hunter, C. Neil. Nanomechanical and thermophoretic analyses of the nucleotide-dependent interactions between the AAA+ subunits of magnesium chelatase. United States. https://doi.org/10.1021/jacs.6b02827
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Adams, Nathan B. P., Vasilev, Cvetelin, Brindley, Amanda A., and Hunter, C. Neil. Sat . "Nanomechanical and thermophoretic analyses of the nucleotide-dependent interactions between the AAA+ subunits of magnesium chelatase". United States. https://doi.org/10.1021/jacs.6b02827. https://www.osti.gov/servlets/purl/1346002.
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@article{osti_1346002,
title = {Nanomechanical and thermophoretic analyses of the nucleotide-dependent interactions between the AAA+ subunits of magnesium chelatase},
author = {Adams, Nathan B. P. and Vasilev, Cvetelin and Brindley, Amanda A. and Hunter, C. Neil},
abstractNote = {In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg2+ ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA+ proteins ChlI and ChlD, form a ChlID–MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID–MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID–MgATP complex. The N-terminal AAA+ domain of ChlD is essential for complex formation, but some stability is preserved in the absence of the C-terminal integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy (SMFS) was used to determine the factors that stabilize formation of the ChlID–MgADP complex at the single molecule level; ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show that the N-terminal AAA+ domain of ChlD mediates this process, in agreement with the microscale thermophoresis data. Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID–MgADP complexes. Finally, these experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex.},
doi = {10.1021/jacs.6b02827},
journal = {Journal of the American Chemical Society},
number = 20,
volume = 138,
place = {United States},
year = {Sat Apr 30 00:00:00 EDT 2016},
month = {Sat Apr 30 00:00:00 EDT 2016}
}
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https://doi.org/10.1021/jacs.6b02827
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Works referenced in this record:

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The NTA–His6 bond is strong enough for AFM single-molecular recognition studies
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Interplay between an AAA module and an integrin I domain may regulate the function of magnesium chelatase
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Structure and function of the AAA+ nucleotide binding pocket
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Crystal structure of the catalytic subunit of magnesium chelatase
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Determinants of catalytic activity with the use of purified I, D and H subunits of the magnesium protoporphyrin IX chelatase from Synechocystis PCC68031
journal, September 1998

  • Jensen, Poul E.; Gibson, Lucien C. D.; Hunter, C. Neil
  • Biochemical Journal, Vol. 334, Issue 2
  • DOI: 10.1042/bj3340335

Single-molecule dissection of the high-affinity cohesin-dockerin complex
journal, November 2012

  • Stahl, S. W.; Nash, M. A.; Fried, D. B.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 50
  • DOI: 10.1073/pnas.1211929109

Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX
journal, March 1999

  • Grafe, S.; Saluz, H. -P.; Grimm, B.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 5
  • DOI: 10.1073/pnas.96.5.1941

Structure of the Cyanobacterial Magnesium Chelatase H Subunit Determined by Single Particle Reconstruction and Small-angle X-ray Scattering
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Evolutionary Relationships and Structural Mechanisms of aaa+ Proteins
journal, June 2006

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    Works referencing / citing this record:

    The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase
    journal, July 2019

    • Farmer, David A.; Brindley, Amanda A.; Hitchcock, Andrew
    • Biochemical Journal, Vol. 476, Issue 13
    • DOI: 10.1042/bcj20190095

    Phenotype of ribonuclease 1 deficiency in mice
    journal, May 2019

    Hexameric structure of the ATPase motor subunit of magnesium chelatase in chlorophyll biosynthesis
    journal, January 2020

    • Gao, Yong‐Shan; Wang, Yan‐Li; Wang, Xiao
    • Protein Science, Vol. 29, Issue 4
    • DOI: 10.1002/pro.3816
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