Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity

Abstract

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Keywords: Chaetomium thermophilum; GNAT domain; N-terminal acetyltransferase; NAT; Naa50; NatE; Neurospora crassa; X-ray structure; dynein light chain protein 1; ribosome association.

Figure 1

Chaetomium thermophilum and Neurospora crassa Naa50 have N- and C-terminal extensions. Naa50 from C. thermophilum (Ct), N. crassa (Nc), Saccharomyces cerevisiae (Sc), Homo sapiens (Hs), and Arabidopsis thaliana (At) showed high conservation within the GNAT domain in a multiple sequence alignment. CtNaa50 and NcNaa50 (teal) have excessive N- and C-terminal extensions (CtNaa50 residues 1-86 and 278-445, and NcNaa50 residues 1-95 and 285-493, respectively) compared to yeast (yellow) and human/Arabidopsis (salmon) homologs. Secondary structure elements (β-strands) from a GNAT domain construct CtNaa50_82-289 are shown on top of the alignment. CtNaa50 contains a dynein light chain 1 protein binding motif (RxTQT) in the N-terminal extension. Loops β2–β3 and β3–β4 are longer in Ct and Nc. Ct/NcNaa50 feature a conserved positive patch KK(R/K)KgR at the C-terminus. Tyrosine and histidine residues (★) are catalytic residues and are absent in yeast. The sequence alignment was performed using Clustal Omega and visualized using ESPript 3.0 [56,57]. Fully conserved residues are represented as white letters in red boxes. Similarities are shown with red letters in blue frames.

Figure 2

Knockout of naa50 and NatA subunits resulted in a Neurospora crassa growth phenotype. The wild-type (wt) strain formed typical conidia under light conditions at room temperature. Knocking out hypK, naa50, naa15 (heterokaryon), and naa10 led to improper conidia formation and different growth patterns. Front growth was marked after every 24 h for 5 days.

Figure 3

CtNaa50_82-289 and NcNaa50_93-287 acetylate canonical NatC/E/F substrates. (A) Both GNAT constructs, CtNaa50_82-289 and NcNaa50_93-287, showed the highest acetylation activity towards MVNALE and MASS peptides, followed by MLGTE and insignificant activity for MDEL. SESS was not acetylated. Control reactions were performed without peptides. Acetylation by both enzymes of different peptides was compared against the peptide-free controls with a one-way ANOVA statistical analysis with Dunnett’s correction (ns = not significant, ** = p-value ≤ 0.0021, **** = p-value ≤ 0.0001). (B) Michaelis–Menten kinetics of MVNALE acetylation by CtNaa50_82-289. (C) Michaelis–Menten kinetics of MVNALE acetylation by NcNaa50_93-287. (D) CtNaa50_82-289 Y190F and H235A mutants showed reduced MVNALE acetylation. Full-length CtNaa50 also showed reduced activity. Acetylation of SESS or MVNALE by CtNaa50 variants was compared against the peptide-free controls with a one-way ANOVA with Dunnett’s correction (ns = not significant, * = p-value ≤ 0.0332, **** = p-value ≤ 0.0001). Acetylation of MVNALE by CtNaa50 variants was compared against CtNaa50_82-289 with a one-way ANOVA with Dunnett’s correction (#### = p-value ≤ 0.0001). All measurements were performed in triplicates and are presented as means ± SD.

Figure 4

CtNaa50 constructs containing the N-terminal extension have an increased thermostability. CtNaa50 and CtNaa50_1-289 feature the highest melting temperatures without the bisubstrate analog CoA-Ac-MVNAL. All constructs bind CoA-Ac-MVNAL, as highlighted by a dramatic increase in thermostability when incubated with tenfold molar excess. The melting temperatures of CtNaa50 variants without CoA-Ac-MVNAL were compared against CtNaa50 with a one-way ANOVA statistical analysis with Dunnett’s correction (* = p-value ≤ 0.0332, **** = p-value ≤ 0.0001). The melting temperatures with CoA-Ac-MVNAL were compared against melting temperatures without CoA-Ac-MVNAL with a t test (#### = p-value ≤ 0.0001). All measurements were performed in triplicates and are presented as means ± SD.

Figure 5

Structure of CtNaa50_82-289 in complex with CoA-Ac-MVNAL. (A) The structure of CtNaa50_82-289 (teal) in ribbon representation is shown in complex with CoA-Ac-MVNAL in sticks. The protein adopts a GNAT fold. The 2mF_obs-DF_calc maps are at a contour level of 1 σ. (B) CtNaa50_82-289 superimposes well with AtNaa50/CoA-Ac-MVNAL (salmon) [39] and HsNaa50/CoA/MLGPE (blue) [51]. Some loops are longer in CtNaa50, especially loop β3–β4.

Figure 6

CtNaa50_82-289 active site and substrate peptide binding. (A) M1_p resides in a hydrophobic pocket formed by residues in loops α1–α2 (L107, P108, and V109), β6–β7 (Y264 and L267), and β5 (W237). (B) The second peptide residue, V2_p, sits in an amphiphilic pocket formed by Y111, F115, R151, Y190, Q192, and Y263. (C) The active site contains a catalytically important water molecule, which is bound by the amide group of M1_p, Y190 hydroxyl, I191 main-chain carbonyl, and H235 main-chain amide groups. Structural details of the active site are shown in the ribbon representation in light blue with residues shown in stick representation.

Figure 7

C. thermophilum pull-outs indicate that CtNaa50 does not bind to CtNatA. (A) CtNaa50 does not copurify with NatA subunits in a pull-out, while Naa15 copurifies with Naa10 and HypK. SDS-PAGE gel was stained with Coomassie. (B) Mass spectrometry experiments confirm that CtNaa50 and CtNaa15 pull-outs do not lead to enrichment (red data points) of the respective other protein, highlighting that they do not interact. Putative dynein light chain 1 protein (DLC1) and two hypothetical 14-3-3 proteins were copurified with Naa50. A ribonuclease E-like protein, a ubiquitin carboxyl-terminal hydrolase-like protein (UCTH), and the NatA subunits Naa10 and HypK were enriched the most during Naa15 pull-out. TMT LC-MS/MS experiments were performed in triplicates.

Figure 8

Analysis of CtNatA/CtNaa50 interaction using yeast two-hybrid assays and in vitro interaction studies. (A) Only CtNaa15 and CtNaa10 subunits interacted with each other in yeast two-hybrid assays. CtNaa15 and CtNaa50 constructs did not interact. Proteins were fused to Gal4 activation (AD) or binding domains (BD). Growth on SDC -Leu/-Trp indicates successful transformation. (B) CtNaa50_82-289 incubated with CtNatA did not coelute on an analytical size-exclusion column (Superdex 200 10/300GL). (C) Peak fractions in (B) are visualized by SDS-PAGE stained with Coomassie.

Figure 9

CtNaa50 binds to CtDLC1 with its N-terminal RATQT motif. (A) CtNAA50 full-length (FL) interacted with CtDLC1 in a yeast two-hybrid assay. Naa50 truncation variants lacking the N-terminus (ΔN or ΔN/C) and a poly-Ala mutant (DLC1 binding motif ₃₄RATQT₃₈ mutated to ₃₄AAAAA₃₈) did not interact with CtDLC1. Coding sequences were fused to Gal4 activation (AD) or binding domains (BD). Growth on SDC -Leu/-Trp indicated successful transformation. (B) CtNaa50_1-289 incubated with CtDLC1 led to an elution peak shift on an analytical size-exclusion column. Peak fractions are visualized by SDS-PAGE with Coomassie stain. (C) CtNaa50_1-289 and CtDLC1 did not significantly differ in melting temperatures T_m when tested alone or in complex.

Figure 10

CtNaa50 coelutes with Ct80S ribosomes via its C-terminus. In a Flag-tag pull-down experiment, Flag-tagged proteins were eluted (E) from Flag beads after incubation with Ct80S ribosomes (R; ribosome flow-through). The negative controls YFP and empty beads do not bind ribosomes. CtNatA and CtNaa50_82-445 (ΔN) coelute with ribosomes, while CtNaa50_82-289 (ΔN/C) does not bind to the ribosome. M = Protein size marker; * highlights a pure ribosomal fraction.

Figure 11

Diversity of Naa50 in different organisms. (A) Naa50 (teal) from filamentous fungi contains significant N- and C-terminal extensions and shows N-terminal acetylation activity towards typical NatC/E/F-type substrates (1). The N-terminus increases its thermostability (2). It does not interact with NatA, and therefore a NatE complex is not formed in filamentous fungi (3). In C. thermophilum, a binding motif within the N-terminus allows for interaction with DLC1 (4). Positive patches within the C-terminus enable ribosome binding without NatA (5). (B) Naa50 in animals and plants (blue) is active and can form a NatE complex with NatA. (C) Yeast Naa50 (grey) is inactive due to missing catalytic residues. It forms a NatE complex and can bind to the ribosome as NatE.