Structure of a Ternary Naa50p (NAT5/SAN) N-terminal Acetyltransferase Complex Reveals the Molecular Basis for Substrate-specific Acetylation

Liszczak, Glen; Arnesen, Thomas; Marmorstein, Ronen

doi:10.1074/jbc.m111.282863

Cited by 89 publications

(188 citation statements)

References 37 publications

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“…These include the transcription factor Runt-related transcription factor 2 (Runx2) (11), the enzyme methionine sulfoxide reductase A (MSRA) (12), and myosin light chain kinase (MLCK) (13). These findings were surprising to us because the structures of all NATs determined to date (9, 14 -17), including Naa10 (9), contain an extended loop that seems to occlude lysine side chains within a polypeptide from lying across the active site as they do in KATs (15)(16)(17)(18). In addition, other reports demonstrating that Naa10 acetylates a lysine residue on Hif-1␣ have failed to be replicated (19 -21), making the question of whether Naa10 is able to acetylate lysine residues controversial.…”

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confidence: 67%

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

Magin

March

Marmorstein

2016

Journal of Biological Chemistry

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The N-terminal acetyltransferase NatA is a heterodimeric complex consisting of a catalytic subunit (Naa10/ARD1) and an auxiliary subunit (Naa15). NatA co-translationally acetylates the N termini of a wide variety of nascent polypeptides. In addition, Naa10 can act independently to posttranslationally acetylate a distinct set of substrates, notably actin. Recent structural studies of Naa10 have also revealed the molecular basis for N-terminal acetylation specificity. Surprisingly, recent reports claim that Naa10 may also acetylate lysine residues of diverse targets, including methionine sulfoxide reductase A, myosin light chain kinase, and Runt-related transcription factor 2. Here we used recombinant proteins to reconstitute and assess lysine acetylation events catalyzed by Naa10 in vitro. We show that there is no difference in lysine acetylation of substrate proteins with or without Naa10, suggesting that the substrates may be acetylated chemically rather than enzymatically. Together, our data argue against a role for Naa10 in lysine acetylation.

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confidence: 67%

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

Magin

March

Marmorstein

2016

Journal of Biological Chemistry

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“…NAA50 uses a two-base mechanism to carry out substrate α-amino group deprotonation and facilitate catalysis (16). These two bases, NAA50-Y73 and NAA50-H112, are on the β3 and β4 strands, respectively, and are representative of the traditional general base positions in GNAT enzymes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Procedures detailing X-ray diffraction data collection and structure determination also can be found in SI Materials and Methods. Acetyltransferase assays were performed as previously described (16). Protocols describing acetyltransferase assays for both substrates and calculation of catalytic parameters can be found in the SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

“…The only two eukaryotic NATs that have been structurally characterized are Schizosaccharomyces pombe NatA, consisting of the Naa10p catalytic subunit and Naa15p regulatory subunit, and human NatE, which is the independently active NAA50 enzyme (15,16). NatA, which harbors the most diversity for substrate selection, is responsible for modifying the majority of all amino-terminally acetylated proteins, which it accomplishes by recognizing proteins with amino-terminal Ala-, Cys-, Gly-, Ser-, Thr-, or Val-residues (5).…”

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confidence: 99%

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Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

Liszczak

Marmorstein

2013

Proc. Natl. Acad. Sci. U.S.A.

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Amino-terminal acetylation is a ubiquitous modification in eukaryotes that is involved in a growing number of biological processes. There are six known eukaryotic amino-terminal acetyltransferases (NATs), which are differentiated from one another on the basis of substrate specificity. To date, two eukaryotic NATs, NatA and NatE, have been structurally characterized, of which NatA will acetylate the α-amino group of a number of nonmethionine amino-terminal residue substrates such as serine; NatE requires a substrate amino-terminal methionine residue for activity. Interestingly, these two NATs use different catalytic strategies to accomplish substrate-specific acetylation. In archaea, where this modification is less prevalent, only one NAT enzyme has been identified. Surprisingly, this enzyme is able to acetylate NatA and NatE substrates and is believed to represent an ancestral NAT variant from which the eukaryotic NAT machinery evolved. To gain insight into the evolution of NAT enzymes, we determined the X-ray crystal structure of an archaeal NAT from Sulfolobus solfataricus (ssNAT). Through the use of mutagenesis and kinetic analysis, we show that the active site of ssNAT represents a hybrid of the NatA and NatE active sites, and we highlight features of this protein that allow it to facilitate catalysis of distinct substrates through different catalytic strategies, which is a unique characteristic of this enzyme. Taken together, the structural and biochemical data presented here have implications for the evolution of eukaryotic NAT enzymes and the substrate specificities therein.structural biology | evolutionary biology | enzymology | X-ray crystallography T he cotranslational process of amino-terminal acetylation occurs on a majority of eukaryotic proteins and mediates many biological processes, including cellular apoptosis, enzymatic regulation, protein localization, and protein degradation (1-4). In humans, three major amino-terminal acetyltransferase (NAT) complexes, called NatA, NatB, and NatC, are responsible for modifying ∼85% of all proteins that undergo amino-terminal acetylation (5). These complexes are conserved in yeast, exist as obligate heterodimers, and are differentiated from one another on the basis of substrate specificity, which is dictated by the amino-terminal sequence of the substrate protein (5, 6). Each complex consists of a single unique catalytic subunit and an additional unique auxiliary subunit that has been shown to potentiate activity and alter substrate specificity of the enzymatic component, as well as anchor the complex to the ribosome during translation (6-11). Three additional human NAT enzymes, NatD-NatF, have also been identified, but they have a much more limited set of physiological substrates, appear to be independently active, and are not well characterized across eukaryotes (12)(13)(14).The only two eukaryotic NATs that have been structurally characterized are Schizosaccharomyces pombe NatA, consisting of the Naa10p catalytic subunit and Naa15p regulatory subunit, and ...

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“…In addition, human Ard1 was found to translocate into the nucleus. 45 However, the reports on the ability of N-terminal acetyltransferases to modify internal lysines [46][47][48][49] remain controversial, 50,51 and our attempts to acetylate Smc proteins in vitro using recombinant Nat3/Mdm20 and Ard1/Nat1 N-terminal acetyltransferases were unsuccessful. It is conceivable that it would be necessary to couple an acetylation assay to an in vitro translation system.…”

Section: Role Of Post-translational Modificationsmentioning

confidence: 99%

The torments of the cohesin ring

Chavda

Ang

Ivanov

2017

Nucleus

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Cohesin is a ring-shaped protein complex which comprises the Smc1, Smc3 and Scc1 subunits. It topologically embraces chromosomal DNA to connect sister chromatids and stabilize chromatin loops. It is required for proper chromosomal segregation, DNA repair and transcriptional regulation. We have recently reported that cohesin rings can adopt a "collapsed" rod-like conformation which is driven by the interaction between the Smc1 and Smc3 coiled coil arms and is regulated by post-translational modifications. The "collapsed" conformation plays a role in cohesin ring assembly and its loading on the DNA. Here we speculate about the mechanism of cohesin's conformational transitions in relation to its loading on the DNA and draw parallels with other Smc-like complexes.

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Structure of a Ternary Naa50p (NAT5/SAN) N-terminal Acetyltransferase Complex Reveals the Molecular Basis for Substrate-specific Acetylation

Cited by 89 publications

References 37 publications

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

The torments of the cohesin ring

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