Adams, N. orcid.org/0000-0003-3080-3448, Hunter, C.N., Brindley, A.A. et al. (1 more author) (2016) Nanomechanical and thermophoretic analyses of the nucleotide-dependent interactions between the AAA+ subunits of magnesium chelatase. Journal of the American Chemical Society. ISSN 0002-7863
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. Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID−MgADP complexes. These experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | Copyright © 2016 American Chemical Society. This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License (http://pubs.acs.org/page/policy/authorchoice_ccbyncnd_termsofuse.html), which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
Funding Information: | Funder Grant number WASHINGTON UNIVERSITY IN ST. LOUIS WU-HT-10-12 BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL (BBSRC) BB/M000265/1 EUROPEAN RESEARCH COUNCIL SYNTHPHOTO - 338895 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 27 Jul 2016 11:48 |
Last Modified: | 06 Nov 2018 13:59 |
Published Version: | http://dx.doi.org/10.1021/jacs.6b02827 |
Status: | Published |
Publisher: | American Chemical Society |
Refereed: | Yes |
Identification Number: | 10.1021/jacs.6b02827 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:99257 |