Molecular basis for N-terminal alpha-synuclein acetylation by human NatB
- Perelman School of Medicine, University of Pennsylvania, United States
- University of Pennsylvania, United States
Abstract
NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and α-synuclein (aSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in Parkinson's disease pathogenesis and as a potential therapeutic target for hepatocellular carcinoma. Here we report the cryo-EM structure of hNatB bound to a CoA-aSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for aSyn N-terminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.
Data availability
Cryo-EM data submissions to the Protein Data Bank (PDB code 6VP9), Electron Microscopy Data Bank (EMD code 21307) and Electron Microscopy Public Image Archive (EMPIAR-10477).
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cryo-EM data for NatB complexElectron Microscopy Data Bank, EMD-21307.
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cryo-EM data for NatB compleElectron Microscopy Public Image Archive, 10477.
Article and author information
Author details
Funding
National Institutes of Health (R35 GM118090)
- Ronen Marmorstein
National Institutes of Health (R01 NS103873)
- James Petersson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Deng et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Molecular basis for N-terminal alpha-synuclein acetylation by human NatB
eLife 9:e57491.
https://doi.org/10.7554/eLife.57491
Further reading
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- Structural Biology and Molecular Biophysics
In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5′ end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5′ end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.
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- Structural Biology and Molecular Biophysics
Experimental detection of residues critical for protein–protein interactions (PPI) is a time-consuming, costly, and labor-intensive process. Hence, high-throughput PPI-hot spot prediction methods have been developed, but they have been validated using relatively small datasets, which may compromise their predictive reliability. Here, we introduce PPI-hotspotID, a novel method for identifying PPI-hot spots using the free protein structure, and validated it on the largest collection of experimentally confirmed PPI-hot spots to date. We explored the possibility of detecting PPI-hot spots using (i) FTMap in the PPI mode, which identifies hot spots on protein–protein interfaces from the free protein structure, and (ii) the interface residues predicted by AlphaFold-Multimer. PPI-hotspotID yielded better performance than FTMap and SPOTONE, a webserver for predicting PPI-hot spots given the protein sequence. When combined with the AlphaFold-Multimer-predicted interface residues, PPI-hotspotID yielded better performance than either method alone. Furthermore, we experimentally verified several PPI-hotspotID-predicted PPI-hot spots of eukaryotic elongation factor 2. Notably, PPI-hotspotID can reveal PPI-hot spots not obvious from complex structures, including those in indirect contact with binding partners. PPI-hotspotID serves as a valuable tool for understanding PPI mechanisms and aiding drug design. It is available as a web server (https://ppihotspotid.limlab.dnsalias.org/) and open-source code (https://github.com/wrigjz/ppihotspotid/).
