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Mechanism of actin N-terminal acetylation

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Science Advances  08 Apr 2020:
Vol. 6, no. 15, eaay8793
DOI: 10.1126/sciadv.aay8793
  • Fig. 1 NAA80 preferentially binds monomeric actin in vitro and in cells.

    (A) Domain diagram of NAA80, showing the two major constructs used in this study. (B) In vitro acetylation by NAA80ΔN (lacking the 77–amino acid N-terminal extension, or ΔN) of nonacetylated β/γ-actin analyzed by Western blotting, using acetylation- and isoform-specific anti–Ac-β-actin antibody. Ac-β/γ-actin purified from WT cells and actin acetylated by full-length NAA80 (NAA80FL or FL) were used as controls. Loading was controlled by anti–pan-actin staining. (C) Rescue of acetylation in NAA80 KO cells by NAA80ΔN analyzed as in (B). Lysate from WT cells was used as a control. Anti–green fluorescent protein (GFP) staining confirmed NAA80ΔN expression in cells. (D) Western blot analysis of the presence of endogenous NAA80 in the soluble (G-actin) and pellet (F-actin) fractions of HAP1 WT cells using custom anti-NAA80 antibody (1:200). Controls are profilin, which binds only G-actin, and Dia1, which binds both G- and F-actin. NAA80 KO cells are shown as a control for custom antibody specificity. Left: Representative Western blots (fig. S8). Right: Quantifications, showing data points (red circles) and means ± SD (n = 5). The statistical significance of the data was determined using an unpaired two-sided Student’s t test (***P < 0.001). (E) Representative HeLa cells expressing GFP-NAA80FL (GFP-FL) or GFP-NAA80ΔN (GFP-ΔN) and stained with Alexa Fluor 546 phalloidin to visualize F-actin. Scale bar, 10 μm. Line scans illustrate the lack of NAA80 colocalization with F-actin, as also confirmed by whole-cell correlation coefficient analysis on n = 15 cells (fig. S2, D and E). (F) Cosedimentation of NAA80FL with F-actin (Ac-α-actin) ± tropomyosin (Tpm). Data points correspond to the mean from three independent experiments ± SEM (fig. S7A). The maximum molar binding ratio (Bmax) and observed dissociation constant (Kobs) were calculated using a nonlinear least square fit (see Materials and Methods). (G and H) Isothermal titration calorimetry (ITC) titrations of NAA80FL and NAA80ΔN (CoA) into Ac-α-actin (latrunculin B, or LatB), using the indicated experimental conditions. The dissociation constant (KD) and binding stoichiometry (N) were derived from fitting to a one-site binding isotherm (red line). Errors correspond to the SD of the fits. Open symbols correspond to control titrations into buffer.

  • Fig. 2 Complex of actin-profilin is a preferred substrate of NAA80.

    (A and B) ITC titrations of NAA80FL (FL) and NAA80ΔN (ΔN) with bound CoA into Ac-α-actin–profilin (LatB) using the indicated experimental conditions. The KD and binding stoichiometry (N) were derived from fitting to a one-site binding isotherm (red line). Errors correspond to the SD of the fits. Open symbols correspond to control titrations into buffer. (C) Cosedimentation of NAA80FL with F-actin (Ac-β/γ-actin). Data points correspond to the mean from three independent experiments ± SEM (fig. S7B). The maximum molar binding ratio (Bmax) and Kobs were calculated using a nonlinear least square fit. (D and E) ITC titrations of NAA80ΔN (CoA) into acetylated and nonacetylated β/γ-actin-profilin (LatB), using the indicated experimental conditions. The KD and binding stoichiometry (N) were derived from fitting to a one-site binding isotherm (red line). Errors correspond to the SD of the fits. Open symbols correspond to control titrations into buffer. (F) ITC titrations of profilin into NAA80ΔN (CoA), using the indicated experimental conditions and analyzed as above. (G) In vitro acetylation of 10 μM nonacetylated β/γ-actin alone or in complex with profilin by NAA80FL, analyzed by Western blotting using acetylation- and isoform-specific anti–Ac-β-actin antibody. The conditions of the acetylation reaction (NAA80FL:actin or NAA80FL:actin-profilin ratio and time) are indicated in the figure. Loading was controlled by anti–pan-actin staining. Left: Representative Western blot. Right: Quantification from three independent experiments. For each blot, the band intensities were normalized to the 1:50 NAA80FL:actin-profilin band. Shown are mean relative intensities ± SD (n = 3) and individual data points (red open circles). The statistical significance was determined using an unpaired two-sided Student’s t test [not significant (n.s.), P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001].

  • Fig. 3 Structural basis of NAA80 binding to actin-profilin.

    (A) Structure of the ternary complex of NAA80ΔN (ΔN) with CoA bound (magenta), Ac-α-actin (LatB; blue), and profilin (green). Gray broken lines illustrate two flexible regions of NAA80 that were not visualized in the electron density map. A zoom in the middle highlights the catalytic cleft of NAA80 (indicated by a rectangle), showing CoA and the acetylated N terminus of α-actin. Key amino acids are numbered. (B and C) Analysis of actin-NAA80 contact interface according to sequence conservation and charge complementarity. Conservation scores (fig. S1) were plotted onto the surfaces of Ac-α-actin and NAA80 derived from the structure of their complex. Amino acid conservation decreases from green to magenta (as indicated), using a conservation range of 100 to 50% for actins (which are highly conserved) and 100 to 0% for the NATs (which are poorly conserved). The contact surfaces on actin (blue) and NAA80 (magenta) are highlighted yellow, and the profilin contact surface is highlighted green. Actin subdomains are numbered 1 to 4 (bold, italic characters). NAA80 interacts almost exclusively with actin subdomain 1, whose surface is predominantly negatively charged, whereas the counterpart contact surface on NAA80 is mostly positively charged, as revealed by their respective electrostatic-surface representations. See also movie S1.

  • Fig. 4 NAA80 specifically recognizes actin-profilin through extensive surface contacts.

    (A) Alignment of the N-terminal sequences of the six human actin isoforms as they exist in cells after processing and Nt-acetylation. (B) E. coli– and mammalian cell–expressed NAA80 can acetylate N-terminal peptides derived from all six human actin isoforms but not a prototypical NatA/NAA10-substrate peptide, shown as a control (see fig. S9 for full peptide sequences and characterization). Acetylation is reported as the amount of radioactive product measured in disintegrations per minute (DPM). Error bars are ±SD (n = 3; red open circles). The statistical significance of the measurements was determined using an unpaired two-sided Student’s t test, comparing each of the actin peptides to the control (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Surface representation of the structure of NAA80, showing the actin-binding (yellow) and profilin-binding (green) interfaces and groups of residues replaced in mutants M1 (V220D, F221D, R225D, and L226D; blue), M2 (L230D and L231D; cyan), and M3 (P266D and P267D; orange). (D) In vitro activity of NAA80FL mutants M1, M2, M3, and M4 (M1 + M3) immunoprecipitated from HeLa cells toward N-terminal actin peptides (fig. S9). The activity was normalized against the expression level and immunoprecipitation efficiency of each construct. Error bars are ±SD (n = 3; red open circles). The statistical significance of the measurements was determined using an unpaired two-sided Student’s t test (n.s., P ≥ 0.05, **P < 0.01, ***P < 0.001). (E) ITC titrations of NAA80FL mutant M4 (CoA) into Ac-α-actin–profilin (LatB) using the indicated experimental conditions. The data could not be fit to a binding isotherm. Open symbols correspond to a control titration into buffer. (F) NAA80 mutant M4 cannot acetylate nonacetylated β/γ-actin, as monitored by Western blot analysis using acetylation- and isoform-specific anti–Ac-β-actin antibody. Shown as a control is Ac-β/γ-actin purified from WT cells, and loading is controlled by anti–pan-actin staining. (G) Rescue of β/γ-actin acetylation in NAA80 KO cells transfected with V5-NAA80FL WT and mutants M1 to M4 analyzed by Western blot using acetylation- and isoform-specific anti-actin antibodies. Anti-V5 staining shows levels of NAA80 expression, whereas anti–pan-actin and anti-vinculin staining provide loading controls (see also fig. S2C). For each mutant, the intensities of the Ac-β-actin and Ac-γ-actin bands were first normalized to the expression level (V5 signal) and then to the level of WT Ac-β/γ-actin. Error bars are ±SD for n = 6 (WT, M2, and M3) or n = 3 (M1 and M4) independent experiments shown as red open circles. The statistical significance of the measurements was determined using a two-way analysis of variance (ANOVA) test between WT and each mutant (n.s., P ≥ 0.05, *P < 0.05, **P < 0.01).

  • Fig. 5 There is one NAA80 molecule for every 3000 actin molecules in cells.

    (A) Western blot analysis of the amount of endogenous NAA80 in HAP1 WT cell lysates (left) compared to specific amounts of purified NAA80 added to NAA80 KO HAP1 cell lysates (right) and the resulting standard curve. The arrow points to the NAA80 band detected by a custom-made antibody. (B) Western blot analysis of the amount of endogenous actin in HAP1 WT cell lysates (left) compared to specific amounts of purified actin (right) and the resulting standard curve. (C) Quantification of the absolute amounts of NAA80 and actin in HAP1 WT cells (n = 4). Errors correspond to the SD.

  • Table 1 Crystallographic data and refinement statistics.

    StructureAc-α-actin–
    profilin–
    NAA80ΔN
    (CoA)
    Ac-α-actin–
    profilin–
    NAA80ΔN
    (AcCoA)
    β/γ-actin–
    profilin–
    NAA80ΔN
    (AnCoA)
    Data collection
    BeamlineMacCHESS F1MacCHESS F1MacCHESS F1
    Date data
    collection
    11/27/201711/27/201711/27/2017
    Wavelength (Å)0.9770.9770.977
    Space groupC2221C2221C2221
    Cell a, b, c (Å)104.5, 115.9,
    132.5
    103.8, 114.9,
    132.3
    104.4, 115.6,
    132.2
    Cell α, β, γ (°)90, 90, 9090, 90, 9090, 90, 90
    Resolution (Å)50.0–2.0
    (2.07–2.0)
    50.0–2.9
    (3.0–2.9)
    50.0–2.5
    (2.59–2.5)a
    Rmerge (%)9.3 (57.6)17.3 (55.8)11.6 (56.3)
    I/σI30.2 (4.5)24.3 (6.3)21.0 (4.0)
    No. of unique
    reflections
    54,090 (5321)17,781 (1744)28,132 (2785)
    Multiplicity26.3 (25.6)39.4 (40.3)13.1 (13.3)
    Completeness
    (%)
    100.0 (100.0)100.0 (100.0)100.0 (100.0)
    CC1/2b0.997 (0.947)0.994 (0.969)0.996 (0.974)
    Wilson B-factor
    2)
    26.441.633
    Resolution (Å)33.4–2.0 (2.08–2.0)28.7–2.9 (3.0–2.9)26.1–2.5 (2.59–2.5)
    No. of unique
    reflections
    54,009 (5172)17,714 (1633)27,972 (2736)
    Completeness
    (%)
    99.6 (97.0)99.1 (92.1)99.4 (98.2)
    Rwork (%)15.9 (18.7)18.4 (22.1)17.8 (22.7)
    Rfree (%)18.4 (22.8)23.9 (29.1)20.9 (28.02)
    No. of atoms604655295598
    Protein544953835259
    Ligands137140135
    Solvent (H2O)4606204
    RMS deviations
    Bond lengths (Å)0.0050.0030.003
    Bond angles (°)0.860.690.72
    B-factors (Å2)
    Protein33.340.339.9
    Solvent and
    ligands
    33.831.334.9
    Ramachandran
    (%)
    Favored97.295.297.1
    Outliers0.20.60.5
    PDBc code6NBE6NAS6NBW

    a, Numbers in parenthesis correspond to highest resolution shell; b, Pearson’s correlation coefficient; c, Protein Data Bank.

    Supplementary Materials

    • Supplementary Materials

      Mechanism of actin N-terminal acetylation

      Grzegorz Rebowski, Malgorzata Boczkowska, Adrian Drazic, Rasmus Ree, Marianne Goris, Thomas Arnesen, Roberto Dominguez

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      The PDF file includes:

      • Figs. S1 to S9
      • Legend for movie S1
      • References

      Other Supplementary Material for this manuscript includes the following:

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