Advanced

Insights into structure and dynamics of the AAA+ motor of magnesium chelatase

Lundqvist, Joakim LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Enzymet magnesium-kelatas katalyserar bildandet av magnesuim-protoporfyrin genom insättning av en magnesiumjon i protoporfyrin. Detta är det första unika steget i tillverkningen av klorofyll hos växter, alger och fotosyntetiska bakterier. Magnesium-kelatas är uppbyggt av tre olika proteiner som kallas I (vikt 40 kDa), D (70 kDa) och H (140 kDa). Tillsammans bildar dessa proteiner ett protein-komplex som är uppbyggt av ett eller flera av respektive protein som samverkar till magnesium-kelatasets enzymatiska aktivitet. Det är samverkan mellan de ingående proteinerna i komplexet som reglerar enzymets funktion. För en fullständig förståelse för den biologiska funktionen krävs det först ingående... (More)
Popular Abstract in Swedish

Enzymet magnesium-kelatas katalyserar bildandet av magnesuim-protoporfyrin genom insättning av en magnesiumjon i protoporfyrin. Detta är det första unika steget i tillverkningen av klorofyll hos växter, alger och fotosyntetiska bakterier. Magnesium-kelatas är uppbyggt av tre olika proteiner som kallas I (vikt 40 kDa), D (70 kDa) och H (140 kDa). Tillsammans bildar dessa proteiner ett protein-komplex som är uppbyggt av ett eller flera av respektive protein som samverkar till magnesium-kelatasets enzymatiska aktivitet. Det är samverkan mellan de ingående proteinerna i komplexet som reglerar enzymets funktion. För en fullständig förståelse för den biologiska funktionen krävs det först ingående karakterisering av de enskilda proteinerna som sedan kopplas till sin helhet i det funktionella komplexet. Ett viktigt steg i karakteriseringen är att bestämma proteinernas 3D-struktur, men för att få en fullständig insyn i mekanismen och funktionen behöver man även bestämma 3D-strukturen och dynamiken av det katalytiska komplexet. Exempel på experimentella metoder för strukturbestämning av individuella proteiner eller deras funktionella komplex är t.ex. röntgen-kristallografi, NMR och elektronmikroskopi (EM). För att få en bättre insyn i magnesium-kelatasets biologiska funktion och protein-komplexets strukturella dynamik har främst EM använts, som i kombination med röntgen-kristallografi, bioinformatik, mass-spektrometri och biokemiska analyser, har gett oss helt nya insikter av stor betydelse.



Vi visar att I- och D-subenheterna bildar ett 3-faldigt symmetriskt två-delat ring-komplex (s.k. ID-komplex) med en EM rekonstruktion av ID-komplexet i närvaro av substratet ADP, upplöst till 7.5 Å. ID-komplexet tillhör en stor familj av proteiner, de så kallade AAA+ proteinerna. Dessa är biologiska motorer, som behöver ATP för att utföra sin funktion. Spjälkningen (hydrolysen) av ATP inducerar en ändring av konformationen i protein-komplexet, som sedan används som energi av ett AAA+ protein kopplat unikt protein, i katalytisk syfte. I magnesium-kelataset kopplas ID-komplexet till H-subenheten som använder energin till att sätta in magnesium i protoporfyrin. Med hjälp av EM visar vi att ID-komplexet är en ATP driven AAA+ motor, som troligen är beroende av ATP hydrolys för att utföra ändringen av konformationen, vilken är nödvändig före det att H-subenheten associerar och att insättningen av magnesium kan fullföljas.



De resultat som presenteras i denna avhandling utökar vår förståelse för magnesium-kelataset och då speciellt ID-komplexet. Med tre olika protein-komponenter och tre inblandade substrat, så står det klart att magnesium-kelatas är en högt utvecklad molekylär maskin som är vida mer komplicerad än vad som någonsin kunnat förväntas. (Less)
Abstract
The insertion of Mg2+ into protoporphyrin IX represents the first committed step in the chlorophyll and bacteriochlorophyll biosynthetic pathways. The reaction is catalyzed by the multisubunit enzyme Mg-chelatase, which consists of three subunits, known as I (molecular weight ~40 kDa), D (~70 kDa), and H (~140 kDa). To fully understand this first step in chlorophyll biosynthesis each protein component of Mg-chelatase needs to be characterized and be coupled into a context of its macromolecular ensemble. Thus it is vital to study the 3D structure of individual modules, but to gain full functional insight into the system the structure and dynamics of the catalytic assemblies needs to be determined.



The results presented in... (More)
The insertion of Mg2+ into protoporphyrin IX represents the first committed step in the chlorophyll and bacteriochlorophyll biosynthetic pathways. The reaction is catalyzed by the multisubunit enzyme Mg-chelatase, which consists of three subunits, known as I (molecular weight ~40 kDa), D (~70 kDa), and H (~140 kDa). To fully understand this first step in chlorophyll biosynthesis each protein component of Mg-chelatase needs to be characterized and be coupled into a context of its macromolecular ensemble. Thus it is vital to study the 3D structure of individual modules, but to gain full functional insight into the system the structure and dynamics of the catalytic assemblies needs to be determined.



The results presented in this thesis are mainly derived from electron microscopy (EM) single-particle techniques. In combination with several other established methods in protein characterization, e.g. X-ray crystallography, bioinformatics, biochemical assays and mass-spectrometry it has provided a poweful tool to obtain quasi-atomic information of the structure and dynamics of R. capsulatus Mg-chelatase. It is presented here that the I- and D-subunits form bipartite chaperone-like complex (ID-complex) with a C3 point-group symmetry and the single-particle cryo-EM 3D reconstruction of the ID-complex in presence of ADP is solved to 7.5 Å resolution.



The largest subunit H, carries the protoporphyrin IX, and it is known to associate with a preformed ID-complex. The results presented here give the first insights into the structure of an H-subunit. Single-particle EM 3D-reconstruction was used to solve the structure in apo and substrate bound conformations at resolution of 25 Å, and revealed a conformational change upon substrate binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model.



The presented quasi-atomic model of the ADP-induced ID-complex reveals the complex structure of the D-ring, which belongs to a unique clade of ATPase inactive AAA+ proteins that has not yet been characterized structurally, and how it assembles with the ATPase active I-ring. Furthermore, the ID-complex is an ATP-fuelled AAA+ motor that is most likely dependent upon ATP-hydrolysis for the conformational rearrangement that is required before H-subunit association may take place and the metal insertion can be completed. An autoinhibitory mechanism for the ATP-fuelled motions of the ID-complex is presented, that may be of general importance for the mechanistic understanding of all bipartite AAA+ family members. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr Jensen, Poul Erik, University of copenhagen
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Bioinformatik, biomathematics biometrics, Bioinformatics, medical informatics, Rhodobacter capsulatus, Tetrapyrrole, Mutants, Magnesium chelatase, Cryo-Electron microscopy single-particle 3D reconstruction, Chlorophyll biosynthesis, AAA+ proteins, ATPase, medicinsk informatik, biomatematik, Molecular biophysics, Molekylär biofysik, Natural science, Naturvetenskap
pages
133 pages
publisher
Molecular Biophysics, Lund University
defense location
Lecture hall B Chemical centre getingev 60 Lund
defense date
2007-11-10 10:00
ISBN
978-91-7422-171-8
language
English
LU publication?
yes
id
e9208d98-2d45-4198-b0b5-ea2807c87842 (old id 599105)
date added to LUP
2007-11-13 08:25:46
date last changed
2016-09-19 08:45:12
@misc{e9208d98-2d45-4198-b0b5-ea2807c87842,
  abstract     = {The insertion of Mg2+ into protoporphyrin IX represents the first committed step in the chlorophyll and bacteriochlorophyll biosynthetic pathways. The reaction is catalyzed by the multisubunit enzyme Mg-chelatase, which consists of three subunits, known as I (molecular weight ~40 kDa), D (~70 kDa), and H (~140 kDa). To fully understand this first step in chlorophyll biosynthesis each protein component of Mg-chelatase needs to be characterized and be coupled into a context of its macromolecular ensemble. Thus it is vital to study the 3D structure of individual modules, but to gain full functional insight into the system the structure and dynamics of the catalytic assemblies needs to be determined.<br/><br>
<br/><br>
The results presented in this thesis are mainly derived from electron microscopy (EM) single-particle techniques. In combination with several other established methods in protein characterization, e.g. X-ray crystallography, bioinformatics, biochemical assays and mass-spectrometry it has provided a poweful tool to obtain quasi-atomic information of the structure and dynamics of R. capsulatus Mg-chelatase. It is presented here that the I- and D-subunits form bipartite chaperone-like complex (ID-complex) with a C3 point-group symmetry and the single-particle cryo-EM 3D reconstruction of the ID-complex in presence of ADP is solved to 7.5 Å resolution.<br/><br>
<br/><br>
The largest subunit H, carries the protoporphyrin IX, and it is known to associate with a preformed ID-complex. The results presented here give the first insights into the structure of an H-subunit. Single-particle EM 3D-reconstruction was used to solve the structure in apo and substrate bound conformations at resolution of 25 Å, and revealed a conformational change upon substrate binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model.<br/><br>
<br/><br>
The presented quasi-atomic model of the ADP-induced ID-complex reveals the complex structure of the D-ring, which belongs to a unique clade of ATPase inactive AAA+ proteins that has not yet been characterized structurally, and how it assembles with the ATPase active I-ring. Furthermore, the ID-complex is an ATP-fuelled AAA+ motor that is most likely dependent upon ATP-hydrolysis for the conformational rearrangement that is required before H-subunit association may take place and the metal insertion can be completed. An autoinhibitory mechanism for the ATP-fuelled motions of the ID-complex is presented, that may be of general importance for the mechanistic understanding of all bipartite AAA+ family members.},
  author       = {Lundqvist, Joakim},
  isbn         = {978-91-7422-171-8},
  keyword      = {Bioinformatik,biomathematics biometrics,Bioinformatics,medical informatics,Rhodobacter capsulatus,Tetrapyrrole,Mutants,Magnesium chelatase,Cryo-Electron microscopy single-particle 3D reconstruction,Chlorophyll biosynthesis,AAA+ proteins,ATPase,medicinsk informatik,biomatematik,Molecular biophysics,Molekylär biofysik,Natural science,Naturvetenskap},
  language     = {eng},
  pages        = {133},
  publisher    = {ARRAY(0xa3bbdc0)},
  title        = {Insights into structure and dynamics of the AAA+ motor of magnesium chelatase},
  year         = {2007},
}