Journal: bioRxiv

Article Title: Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

doi: 10.1101/2023.12.07.570580

Figure Lengend Snippet: A) Activity measurements for purified ACA8. Left: Ca 2+ dependent activity with and without CaM. Specific ATPase activity of purified WT, Δ20, Δ100 and of Δ20 with acidic phospholipids present and salipro reconstituted Δ20. WT n = 6 (Ca 2+ ) and n = 8 (Ca 2+ /CaM), Δ20 n = 4, Δ100 n = 9, Δ20 acidic phospholipids n = 6, Δ20 salipro n = 5 (Ca 2+ ) and n = 9 (Ca 2+/ CaM). Data presented as mean ± SEM. The statistical analysis is conducted as an unpaired two-tailed students t-test. Right: Fold activation of WT, Δ20, Δ100 and Δ20 activity with acidic phospholipids present and salipro reconstituted Δ20. Activity measurements are from at least two independent protein purification and expression cultures, except Δ100 which is from one expression culture. B) The 3.3Å (FSC gold standard = 0.143) density from cryo-EM in gray with the structure of ACA8 modelled. The transmembrane domain is wheat colored, the A domain is yellow, P domain is blue and N domain is red. Contour level 0.236. C) The density is colored by local resolution, from 3 Å (blue) to 5 Å (red). Contour level 0.236. D) The structure of ACA8, wheat colored, compared to E2P BeF structure of SERCA (PDB: 3B9B ) [40] in blue. Alignment on transmembrane helix 7 (TM7) to TM10.

Article Snippet: Cryo-EM data processing statistics can be found in Table S2.

Techniques: Activity Assay, Purification, Two Tailed Test, Activation Assay, Protein Purification, Expressing, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Conserved N-terminal Regulation of the ACA8 Calcium Pump with Two Calmodulin Binding Sites

doi: 10.1101/2023.12.07.570580

Figure Lengend Snippet: The core of the ACA8 E1 state predicted by AlphaFold and the low-resolution density in gray aligned with ACA8 E2P cryo-EM structure colored by domain (TM domain in wheat, A domain in yellow, P domain in blue and N domain in red), represented as cartoon with tubular helices. The autoinhibitory domain, residues 1-112, of the E1 state is excluded for clarity. Alignment on TM7-10 segment. A ) Zoom in on the Ca 2+ entry pathway of ACA8 with a side-by-side view of E1 and E2P alignment and autoinhibitory domain binding. Left: Comparison of the Ca 2+ entry pathway for the E1 and E2P state of ACA8. TM1 is shifted inward towards TM3 in the E2P structure compared to E1 thereby blocking Ca 2+ entry. Right: AlphaFold predicted E1 structure with the autoinhibitory domain (cyan) blocking Ca 2+ entry. C) Zoom in on the Ca 2+ entry pathway of ACA8 aligned with the E1 Ca 2+ AMPPCP SERCA structure (PDB: 1T5S ) D) The core of the AlphaFold predicted structure of ACA8 as a grey surface and the autoinhibitory domain in cyan. Top : Close up side view of the elongated helix with CaMBS2. The anchoring residues exposed and available for CaM interaction are shown as sticks. Bottom : CaM binding to the exposed CaMBS2. CaM in teal with Ca 2+ in magenta. CaMBS2 of the AlphaFold structure was aligned with CaMBS2 of the crystal structure of the autoinhibitory domain of ACA8 with two CaM bound (PDB: 4AQR ) (Tidow et al., 2012). E) The core of the Alphafold predicted structure represented as an electrostatic surface, red negatively charged, white neutral and blue positively charged, with the autoinhibitory domain is in cyan and the potential acidic phospholipid site is indicated by an arrow. The cutouts show a close up view of the potential acidic phospholipid binding site near F32.

Article Snippet: Cryo-EM data processing statistics can be found in Table S2.

Techniques: Cryo-EM Sample Prep, Binding Assay, Blocking Assay