(A) Gel filtration elution profile of hNatB, using a Superdex S200 column. Coomassie-stained SDS-PAGE of peak fractions is reproduced to the right of the chromatograms. (B) Comparison of hNatB activity toward different peptide substrates. All the activities are normalized to the activity of hNatB toward αSyn peptide (MDVF). (C) The dose-response curve corresponding to the titration of CoA-αSyn conjugate (CoA-MDVFMKGLSK) into hNatB acetyltransferase reactions. The calculated IC50 value is indicated. Reactions were performed in triplicate; replicates are shown in the graph as vertical dots.
(A) hNaa20 (light orange) and hNaa25 (cyan) are shown in cartoon. The CoA-αSyn conjugate inhibitor is shown in sticks and colored as magenta. The N- and C- terminal regions are indicated as ‘N-region’ and ‘C-region’, respectively. Some helices are as labeled. (B) hNaa20 (light orange) and hNaa25 (cyan) are shown overlapped with hNatA (marine blue, PDB: 6C9M). Small molecule IP6 bound to hNatA is shown as surface representation (red). (C) hNaa20 (light orange) and hNaa25 (cyan) are shown superimposed on CaNatB (slate blue, PDB:5K04).
(A) 2D and 3D classification scheme for hNatB EM map determination. (B) Local resolution map of hNatB. (C) Gold standard Fourier shell correlation (FSC) curve of hNatB EM map 3D reconstruction. (D) FSC curves of the refined model versus the overall map that it was refined against (brown); of the model refined in the first of the two independent maps used for the gold-standard FSC versus that same map (green); and of the model refined in the first of the two independent maps versus the second independent map (red).
(A) A representative micrograph of a hNatB frozen grids hole (B) Atomic model of hNatB fitted into the Cryo-EM map. (C) The fit of a helical segment from hNAA20 in the EM density. The contour level is 5 sigma. (D) The fit of a helical segment from hNAA25 in the EM density. The contour level is 5 sigma. (E) The fit of hNatB in the EM density at contour levels of 4 sigma, 3 sigma, and 2 sigma.
Species include H. sapiens (Hs), C. albicans (Ca), D. melanogaster (Dm), S. cerevisiae (Sc), and S. pombe (Sp).
(A) hNatB overlaid with hNatA-HYPK. hNatA and HYPK are colored as light salmon and red, respectively. N- and C- termini of the auxiliary subunits are indicated. (B) The extended loop connecting α31 to α32 is highlighted in green in hNatB. This loop is not present in hNatA (not shown).
(A) hNAA20 (light orange) and hNAA25 (cyan) are shown in a cartoon with major associated interface denoted. (B–G) Zoom-in views of the hydrophobic interface regions as indicated in (A).
(A) The structure of hNAA20 bound to the CoA-αSyn conjugate bound is shown in cartoon with corresponding secondary structures labeled. (B) The fit of the CoA-αSyn conjugate ligand in the EM density map. The contour level is 4.0 sigma. (C) Interaction between CoA-αSyn conjugate and hNAA20 residues is generated with LIGPLOT (Laskowski and Swindells, 2011). Hydrogen bonds are indicated by dashed green lines, and van der Waals interactions are indicated with red semicircles. (D) Highlighted polar and hydrophobic interactions between CoA-αSyn conjugate and the hNAA20 are depicted in the 3D view.
Species depicted include H. sapiens (Hs), C. albicans (Ca), D. melanogaster (Dm), S. cerevisiae (Sc), and S. pombe (Sp). Blue, black and magenta labeled indicate mutation sensitive residues, CoA-binding residues, and peptide-binding residues, respectively.
(A) Bi-substrate inhibitor-bound hNAA20 (light orange) is shown superimposed with hNAA50 (gray, PDB: 3TFY). H73, N116, and Y123 (sticks), mediate important functional roles in hNatB catalysis. (B) Residues forming the Met1 binding pocket of hNAA20 are depicted. (C) Residues forming the Met1 binding pocket of hNAA50 are depicted.
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hNaa20 (light orange) and hNaa25 (cyan) are shown in cartoon. The CoA-αSyn conjugate inhibitor is shown in sticks and colored as magenta.
Amino acid sidechains that mediate hydrogen bond and van der Waals interactions with α-synuclein are highlighted in a cartoon model of NAA20.
Sequence of primers for preparing mutants.
Both the forward and reverse primers for each mutant is indicated.