Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK

The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK.publishedVersio

The Molecular and Regulatory Mechanism of Multi-Subunit N-Terminal Acetyltransferases

N-terminal acetylation (NTA) is one of the most widespread protein modifications, which occurs on most eukaryotic proteins, but is significantly less common on bacterial and archaea proteins. This modification is carried out by a family of enzymes called N-terminal acetyltransferases (NATs). To date, 12 NATs have been identified, harboring different composition, substrate specificity, and in some cases, modes of regulation. In the first chapter, we review the molecular features of NATs. NatA/E, NatB and NatC, are multi-subunit enzymes, responsible for the majority of eukaryotic protein NTA. Their mechanisms of action and regulation remain poorly understood before this dissertation. In the second chapter, we determined the X-ray crystal structure of yeast NatA/Naa50 as a scaffold to understand coregulation of NatA/Naa50 activity in both yeast and human. We found that Naa50 makes evolutionarily conserved contacts to both the Naa10 and Naa15 subunits of NatA. These interactions promote catalytic crosstalk within the human complex, but do so to a lesser extent in the yeast complex, where Naa50 activity is compromised. Thirdly, we reported the Cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We revealed that NAA50 and HYPK exhibit negative impacts on their binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate binding site. Fourthly, we reported the Cryo-EM structure of hNatB bound to a CoA-αSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis revealed functionally important differences with human NatA and Candida albicans NatB, resolved key hNatB protein determinants for αSyn N-terminal acetylation, and identified important residues for substrate-specific recognition and acetylation by NatB enzymes. Lastly, we report the Cryo-EM structure of S. pombe NatC with a NatE/C-type bi-substrate analogue and inositol hexaphosphate (IP6), and associated biochemistry. We find that all three subunits are prerequisite for normal NatC acetylation activity, IP6 binds tightly to NatC to stabilize the complex, and we determine the molecular basis for IP6-mediated stability of the complex and the overlapping yet distinct substrate profiles of NatC and NatE

Feasible Route for a Large Area Few-Layer MoS2 with Magnetron Sputtering

In this article, we report continuous and large-area molybdenum disulfide (MoS2) growth on a SiO2/Si substrate by radio frequency magnetron sputtering (RFMS) combined with sulfurization. The MoS2 film was synthesized using a two-step method. In the first step, a thin MoS2 film was deposited by radio frequency (RF) magnetron sputtering at 400 °C with different sputtering powers. Following, the as-sputtered MoS2 film was further subjected to the sulfurization process at 600 °C for 60 min. Sputtering combined with sulfurization is a viable route for large-area few-layer MoS2 by controlling the radio-frequency magnetron sputtering power. A relatively simple growth strategy is demonstrated here that simultaneously enhances thin film quality physically and chemically. Few-layers of MoS2 are established using Raman spectroscopy, X-ray diffractometer, high-resolution field emission transmission electron microscope, and X-ray photoelectron spectroscopy measurements. Spectroscopic and microscopic results reveal that these MoS2 layers are of low disorder and well crystallized. Moreover, high quality few-layered MoS2 on a large-area can be achieved by controlling the radio-frequency magnetron sputtering power

The NatA ribosome interaction has low μM affinity and is salt dependent.

<p>(A) Representative Western blot of NatA/ribosome co-sedimentation assay with increasing concentration of ribosome. P stands for pellet, and S stands for supernatant. The western blot targets the His tag on the Naa15 subunit (Naa10 is untagged) (B) An affinity curve of the ribosome NatA interaction quantified from the co-sedimentation assay. Assay was performed in duplicate (C) Co-sedimentation assay in increasing KCl concentrations. The first two lanes are NatA without ribosome present. S stands for supernatant and P stands for pellet. Uncropped gels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186278#pone.0186278.s001" target="_blank">S1 Fig</a>.</p

Conservation analysis and electrostatic surface of NatA show two regions important for ribosome binding.

<p>(A) A cartoon representation of the NatA complex. Naa10 is shown in cyan, Naa15 in green, and the peptide substrate in magenta. The N-terminus and internal basic helix are indicated, as is the active site where N-termini are acetylated. (B) Conservation map of the NatA complex. Magenta areas represent regions of high sequence conservation and cyan areas represent regions of low sequence conservation. (C) Electrostatic potential map of NatA. Blue areas represented regions which are electropositive, and red areas represent regions which are electronegative. Electropositive region 1 (EPR1), and electropositive region 2 (EPR2) are indicated.</p

Probing the interaction between NatA and the ribosome for co-translational protein acetylation

<div><p>N-terminal acetylation is among the most abundant protein modifications in eukaryotic cells. Over the last decade, significant progress has been made in elucidating the function of N-terminal acetylation for a number of diverse systems, involved in a wide variety of biological processes. The enzymes responsible for the modification are the N-terminal acetyltransferases (NATs). The NATs are a highly conserved group of enzymes in eukaryotes, which are responsible for acetylating over 80% of the soluble proteome in human cells. Importantly, many of these NATs act co-translationally; they interact with the ribosome near the exit tunnel and acetylate the nascent protein chain as it is being translated. While the structures of many of the NATs have been determined, the molecular basis for the interaction with ribosome is not known. Here, using purified ribosomes and NatA, a very well-studied NAT, we show that NatA forms a stable complex with the ribosome in the absence of other stabilizing factors and through two conserved regions; primarily through an N-terminal domain and an internal basic helix. These regions may orient the active site of the NatA to face the peptide emerging from the exit tunnel. This work provides a framework for understanding how NatA and potentially other NATs interact with the ribosome for co-translational protein acetylation and sets the foundation for future studies to decouple N-terminal acetyltransferase activity from ribosome association.</p></div

Pull down analysis of NatA mutants.

<p>(A) Location of NatA mutants used in this study. Mutated regions are shown in yellow. Lysines mutated in K3E and K6E constructs are shown as sticks, and the N-terminal domain deleted in the ΔN constructs is indicated in yellow. (B) Sedimentation assay with NatA mutants. Naa15 is indicated. Note for the ΔN mutants, Naa15 runs lower than WT Naa15. The faint bands above 80 kD in these lanes is not Naa15, but rather impurities from the ribosome prep. These lanes are marked with asterisks. The Naa10 band is obscured by the ribosomal proteins in the gel.</p

NatB has positively charged regions in the same configuration as NatA.

<p>(A) Cartoon representation of the NatA and NatB complex. Naa10 is shown in cyan and Naa15 in green. Naa20 is shown in yellow and Naa25 in orange. (B) Electrostatic surface representation of Naa15 and Naa25 with EPR1 and EPR2 on the Naa15 structure, and putative areas on Naa25 corresponding to these regions indicated with arrows.</p