Journal: Cell / Year: 2020 Title: Dual Functions of a Rubisco Activase in Metabolic Repair and Recruitment to Carboxysomes. Authors: Mirkko Flecken / Huping Wang / Leonhard Popilka / F Ulrich Hartl / Andreas Bracher / Manajit Hayer-Hartl / Abstract: Rubisco, the key enzyme of CO fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone ...Rubisco, the key enzyme of CO fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.
History
Deposition
Aug 3, 2020
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Header (metadata) release
Sep 23, 2020
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Map release
Sep 23, 2020
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Update
Apr 7, 2021
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Current status
Apr 7, 2021
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Organism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 sTAR
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
2.2 mg/mL
Buffer
pH: 8.4 Component:
Concentration
Formula
Name
10.0 mM
C4H6O5
Malic acid
20.0 mM
C6H13NO4S
2-ethanesulfonic acid
20.0 mM
C4H11NO3
2-Amino-2-hydroxymethyl-propane-1,3-diol
50.0 mM
KCl
Potassium chloride
10.0 mM
MgCl2
Magnesium chloride
5.0 mM
NaHCO3
Sodium bicarbonate
0.004 mM
C6H14O13P2
2-Carboxyarabinitol-1,5-diphosphate
2.0 mM
C10H16N5O13P3
Adenosine triphosphate sodium salt
2.0 mM
C10H12Li4N5O12P3S
Adenosine 5'-(3-thiotriphosphate) tetralithium salt
Grid
Model: Quantifoil / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details
NosRubisco (1 uM) was incubated with NaHCO3 (10 mM) at 298 K for 10 min followed by addition of CABP (8 uM). CABP-inhibited NosRubisco (0.5 uM) was then incubated with NosRcaDC (10 uM) in the presence of ATP (2 mM) for 10 s, followed by the addition of ATP-gammaS (2 mM), and incubated at 298 K for another 10 min before preparing the cryo-grids.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 9042 / Average exposure time: 2.8 sec. / Average electron dose: 60.0 e/Å2 / Details: 31 frames per image
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
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