Structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin

Nitsche J, Josts I, Heidemann J, Mertens H, Maric S, Moulin M, Haertlein M, Busch S, Forsyth V, Svergun D, Uetrecht C, Tidow H, Communications Biology 1(1) (2018) DOI

SASDET4 – ACA8 complex with Calmodulin (75% deuterated) in stealth nanodisc (SANS, 100% D2O)

Membrane scaffold protein 1D1 (deuterated, 75%)
1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl)
Calcium-transporting ATPase 8, plasma membrane-type
Calmodulin-7 (deuterated 75%)
MWexperimental 136 kDa
MWexpected 168 kDa
VPorod 217 nm3
log I(s) 9.20×10-2 9.20×10-3 9.20×10-4 9.20×10-5
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) small angle scattering data  s, nm-1
ln I(s)
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) Guinier plot ln 9.20×10-2 Rg: 4.3 nm 0 (4.3 nm)-2 s2
(sRg)2I(s)/I(0)
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) Kratky plot 1.104 0 3 sRg
p(r)
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) pair distance distribution function Rg: 4.6 nm 0 Dmax: 15 nm

Data validation


Fits and models


log I(s)
 s, nm-1
ACA8 complex with Calmodulin (75% deuterated) in stealth nanodisc (SANS, 100% D2O) Rg histogram Rg, nm
Membrane scaffold protein 1D1 (deuterated, 75%) 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) Calcium-transporting ATPase 8, plasma membrane-type Calmodulin-7 (deuterated 75%) EOM/GAJOE model

SANS data from solutions of ACA8 complex with Calmodulin (75% deuterated) in stealth nanodisc (SANS, 100% D2O) in 30 mM Tris, 150 mM NaCl, 1mM MgCl2, 1 mM CaCl2, pH 7.5 were collected on the SANS-1 camera at the Heinz Maier-Leibnitz Zentrum storage ring (Garching, Germany) using a Array of 128 3-He position sensitive tubes MLZ detector at a sample-detector distance of 5.5 m and at a wavelength of λ = 5 nm , the range of momentum transfer 0.120 < s < 2.400 nm-1 was covered (l(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 3.2 and 3.2 mg/ml were measured at 10°C. One 7200 second frame was collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

ACA8 protein complex with Calmodulin (75% deuterated) encapsulated in a partially deuterated "stealth-nanodisc": (sND). The Calmodulin and disc components (MSP1D1 scaffold protein - 75% deuterated, and POPC lipid - heads/tails 78% and 92% deuterated) are effectively matched out at 100% D2O. Thus the expected molecular weight should correspond to that of the ACA8 protein alone (~118 kDa). Data measured using a single instrument configuration: 5.5 m (sample-detector distances) with a fixed neutron wavelength (λ) of 5 Å.

Membrane scaffold protein 1D1 (deuterated, 75%) (d75-msp1d1)
Mol. type   Protein
Olig. state   Dimer
Mon. MW   24.7 kDa
Sequence   FASTA
 
1-palmitoyl-2-palmitoleoyl-sn-glycero-3-phosphocholine (deuteration: 78% head, 92% acyl) (d(78,92)-POPC)
Mol. type   Other
Organism   Escherichia coli. (AL95 strain)
Olig. state   Unknown
Mon. MW   0.8 kDa
Sequence   FASTA
 
Calcium-transporting ATPase 8, plasma membrane-type (ACA8)
Mol. type   Protein
Organism   Arabidopsis thaliana
Olig. state   Monomer
Mon. MW   118.2 kDa
 
UniProt   Q9LF79
Sequence   FASTA
 
Calmodulin-7 (deuterated 75%) (d75-Cam7)
Mol. type   Protein
Organism   Arabidopsis thaliana
Olig. state   Monomers
 
UniProt   P59220