Volume 25, Issue 4, Pages 641-649.e3 (April 2017) Molecular Basis of Substrate Specific Acetylation by N-Terminal Acetyltransferase NatB  Haiyan Hong, Yongfei Cai, Shijun Zhang, Hongyan Ding, Haitao Wang, Aidong Han  Structure  Volume 25, Issue 4, Pages 641-649.e3 (April 2017) DOI: 10.1016/j.str.2017.03.003 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 641-649.e3DOI: (10.1016/j.str.2017.03.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Crystal Structure of NatB in Complex with a Bisubstrate Inhibitor (A) Superposition of the NatB-sub and NatB-free complexes. Structures are presented in cartoon format with cylinder helices. NatB-sub is colored in cyan (Naa25) and olive green (Naa20). NatB-free is colored in light gray (Naa20) and dark gray (Naa25). CoA is shown in yellow sticks, and substrate peptide is shown in red. Naa25 helices are labeled with α1 to α37. Naa20 are labeled with α1-α4 and β1-β7. To the right, a 90° rotational view of the left along an x axis is shown. (B) Sequence alignment of Naa20 homologs from Candida albicans (Ca), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Homo sapiens (Hs), Arabidopsis thaliana (At), and Drosophila melanogaster (Dm). The sequence alignment was performed using Clustal Omega and generated using ESPript 3.0. Completely conserved residues are highlighted in white letters in red boxes. Highly conserved residues are labeled with red letters on white background. ▲, CoA binding residues; ★, substrate binding residues; and ▪, mutational sensitive residues. Structure 2017 25, 641-649.e3DOI: (10.1016/j.str.2017.03.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Specific Interactions between the Naa20 and Naa25 Subunits of NatB (A–D) Sub-interfaces between Naa20 and Naa25. The critical residues are shown in sticks. All hydrogen bonds are highlighted with yellow dashed lines. Secondary structural elements are labeled in orange (Naa20) and cyan (Naa25). (E and F) Mutational effects of Naa20 or Naa25 on integrity of the NatB holoenzyme. The prey Naa25 in NTA pull-down using His tag (E) or immunoprecipitation using anti-FLAG beads (F) were shown on the top gels. The associated Naa20 was detected using anti-FLAG (E) or anti-His (F) antibodies shown on the bottom gels. The inputs in lanes 1–6 (E) and 1–4 (F) using the whole-cell lysates showed that both subunits were expressed at the similar level. Structure 2017 25, 641-649.e3DOI: (10.1016/j.str.2017.03.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Substrate Recognition and Acetylation of NatB (A) Alignment of Naa20 in substrate free and bound states. Naa20 from NatB-sub is colored in olive green and that from NatB-free in gray. CoA and MES from NatB-free are shown in gray on the back. (B) Electron density and fitting of bound substrates in two complexes of one asymmetric unit. The simulated annealing omit maps are contoured at 2.5σ. The composition diagram of the bisubstrate inhibitor is shown below. (C) Detailed interactions between Naa20 and the substrate peptide in the NatB-sub complex. Naa20 is presented in an olive green cartoon with critical residues presented in sticks. The substrate peptide is shown in magenta sticks with residues labeled 1–4. Hydrogen bonds are marked with green dashed lines and CoA in gray. (D) Catalytic efficiencies of NatB wild-type and mutants. NF (not fitted) mutant has a Km > 4,000 μM, which cannot derive any catalytic parameters. ND, not detectable. Complete parameters can be found in Table 2. Structure 2017 25, 641-649.e3DOI: (10.1016/j.str.2017.03.003) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Substrate Specificity of NatB (A) Superposition of catalytic subunits from NatA–B and NatE–F presented in cartoon form. Naa20 is in olive green, Naa10 in gray, Naa50 in light green, and Naa60 in yellow orange. (B–D) Comparisons of NatB substrate binding with NatA (B), NatE (C), and NatF (D). NatB substrate peptide is colored in magenta and NatA substrate in teal blue, NatE in orange, and NatF in cyan. The key residues in substrate binding are labeled and colored accordingly. Hydrogen bonds are marked with green dashed lines. Residues involved in NatB substrate binding are shown in olive green sticks and those in other NATs in gray sticks. (E) Electrostatic surface of substrate binding pockets of NATs. Electron positive is shown in blue and electron negative in red. CoA is shown in yellow sticks and the substrate peptides in green. Only two amino acids of each substrate are shown since they are dominant in substrate specificity. The buried surface areas are 316.5 Å2 (NatB on average), 203.7 Å2 (NatA), 312.2 Å2 (NatE), and 318.2 Å2 (NatF). Structure 2017 25, 641-649.e3DOI: (10.1016/j.str.2017.03.003) Copyright © 2017 Elsevier Ltd Terms and Conditions