N-terminal acetyltransferases (NATs) belong to the superfamily of acetyltransferases. They are enzymes catalysing the transfer of an acetyl group from acetyl coenzyme A to the N-terminus of polypeptide chains. N-terminal acetylation is one of the most common protein modifications. To date, not much is known on the molecular basis for the exclusive substrate specificity of NATs. All NATs share a common fold called GNAT. A characteristic of NATs is the β6β7 hairpin loop covering the active site and forming with the α1α2 loop a narrow tunnel surrounding the catalytic site in which cofactor and polypeptide meet and exchange an acetyl group. We investigated the dynamics-function relationships of all available structures of NATs covering the three domains of Life. Using an elastic network model and normal mode analysis, we found a common dynamics pattern conserved through the GNAT fold; a rigid V-shaped groove formed by the β4 and β5 strands and splitting the fold in two dynamical subdomains. Loops α1α2, β3β4 and β6β7 all show clear displacements in the low frequency normal modes. We characterized the mobility of the loops and show that even limited conformational changes of the loops along the low-frequency modes are able to significantly change the size and shape of the ligand binding sites. Based on the fact that these movements are present in most low-frequency modes, and common to all NATs, we suggest that the α1α2 and β6β7 loops may regulate ligand uptake and the release of the acetylated polypeptide.

Computational Biology Unit Department of Informatics University of Bergen Bergen Norway

Department of Biological Sciences University of Bergen Bergen Norway

Department of Biomedicine University of Bergen Bergen Norway

Department of Chemistry University of Bergen Bergen Norway

Department of Informatics University of Bergen Bergen Norway

Department of Surgery Haukeland University Hospital Bergen Norway

Faculty of Informatics Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Arnesen T., Van Damme P., Polevoda B., Helsens K., Evjenth R., Colaert N. Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans. Proc Natl Acad Sci U S A. 2009;106:8157–8162. PubMed PMC

Drazic A., Myklebust L.M., Ree R., Arnesen T. The world of protein acetylation. Biochim Biophys Acta - Proteins Proteomics. 2016;1864:1372–1401. PubMed

Kalvik T.V., Arnesen T. Protein N-terminal acetyltransferases in cancer. Oncogene. 2013;32:269–276. PubMed

Myklebust L.M., Van Damme P., Støve S.I., Dörfel M.J., Abboud A., Kalvik T.V. Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects. Hum Mol Genet. 2014;24:1956–1976. PubMed PMC

Dörfel M.J., Lyon G.J. The biological functions of Naa10 - From amino-terminal acetylation to human disease. Gene. 2015;567:103–131. PubMed PMC

Foyn H., Jones J.E., Lewallen D., Narawane R., Varhaug J.E., Thompson P.R. Design, synthesis, and kinetic characterization of protein N-terminal acetyltransferase inhibitors. ACS Chem Biol. 2013;8:1121–1127. PubMed

Vetting M.W., Luiz L.P., Yu M., Hegde S.S., Magnet S., Roderick S.L. Structure and functions of the GNAT superfamily of acetyltransferases. Arch Biochem Biophys. 2005;433:212–226. PubMed

Lu L., Berkey K.A., Casero R.A. RGFGIGS is an amino acid sequence required for acetyl coenzyme A binding and activity of human spermidine/spermine N1 acetyltransferase*. J Biol Chem. 1996;271:18920–18924. PubMed

Marmorstein R. Structure of histone acetyltransferases. J Mol Biol. 2001;311:433–444. PubMed

Rathore O.S., Faustino A., Prudêncio P., Van Damme P., Cox C.J., Martinho R.G. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms. Sci Rep. 2016;6:1–13. PubMed PMC

Dinh T.V., Bienvenut W.V., Linster E., Feldman-Salit A., Jung V.A., Meinnel T. Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling. Proteomics. 2015;15:2426–2435. PubMed PMC

Drazic A., Aksnes H., Marie M., Boczkowska M., Varland S., Timmerman E. NAA80 is actin’s N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility. Proc Natl Acad Sci. 2018 PubMed PMC

Yoshikawa A., Isono S., Sheback A., Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12. MGG Mol Gen Genet. 1987;209:481–488. PubMed

Tanaka S., Matsushita Y., Yoshikawa A., Isono K. Cloning and molecular characterization of the gene rimL which encodes an enzyme acetylating ribosomal protein L12 of Escherichia coli K12. Mol Gen Genet. 1989;217:289–293. PubMed

Mackay D.T., Botting C.H., Taylor G.L., White M.F. An acetylase with relaxed specificity catalyses protein N-terminal acetylation in Sulfolobus solfataricus. Mol Microbiol. 2007;64:1540–1548. PubMed

Aksnes H., Ree R., Arnesen T. Co-translational, post-translational, and non-catalytic roles of N-terminal acetyltransferases. Mol Cell. 2019 PubMed PMC

Liszczak G., Arnesen T., Marmorsteins R. Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation. J Biol Chem. 2011;286:37002–37010. PubMed PMC

Liszczak G., Goldberg J.M., Foyn H., Petersson E.J., Arnesen T., Marmorstein R. Molecular basis for N-terminal acetylation by the heterodimeric NatA complex. Nat Struct Mol Biol. 2013;20:1098–1105. PubMed PMC

Chen J.Y., Liu L., Cao C.L., Li M.J., Tan K., Yang X. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase. Sci Rep. 2016;6:1–12. PubMed PMC

Magin R.S., Liszczak G.P., Marmorstein R. The molecular basis for histone H4- and H2A-specific amino-terminal acetylation by NatD. Structure. 2015;23:332–341. PubMed PMC

Grauffel C., Abboud A., Liszczak G., Marmorstein R., Arnesen T., Reuter N. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases. PLoS One. 2012:7. PubMed PMC

Myklebust L.M., Van Damme P., Støve S.I., Dörfel M.J., Abboud A., Kalvik T.V. Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects. Hum Mol Genet. 2014:24. PubMed PMC

Micheletti C. Comparing proteins by their internal dynamics: exploring structure-function relationships beyond static structural alignments. Phys Life Rev. 2013;10:1–26. PubMed

Carnevale V., Raugei S., Micheletti C., Carloni P. Convergent dynamics in the protease enzymatic superfamily. J Am Chem Soc. 2006;128:9766–9772. PubMed

Tiwari S.P., Reuter N. Similarity in shape dictates signature intrinsic dynamics despite no functional conservation in TIM barrel enzymes. PLoS Comput Biol. 2016;12:1–26. PubMed PMC

Fuglebakk E., Tiwari S.P., Reuter N. Comparing the intrinsic dynamics of multiple protein structures using elastic network models. Biochim Biophys Acta. 2015;1850:911–922. PubMed

Rueda M., Chacón P., Orozco M. Thorough validation of protein normal mode analysis: a comparative study with essential dynamics. Structure. 2007;15:565–575. PubMed

Berendsen H.J., Hayward S. Collective protein dynamics in relation to function. Curr Opin Struct Biol. 2000;10:165–169. PubMed

Sathiyamoorthy K., Vijayalakshmi J., Tirupati B., Fan L., Saper M.A. Structural analyses of the Haemophilus influenzae peptidoglycan synthase activator LpoA suggest multiple conformations in solution. J Biol Chem. 2017;292:17626–17642. PubMed PMC

Chirasani V.R., Revanasiddappa P.D., Senapati S. Structural plasticity of cholesteryl ester transfer protein assists the lipid transfer activity. J Biol Chem. 2016;291:19462–19473. PubMed PMC

Schushan M., Rimon A., Haliloglu T., Forrest L.R., Padan E., Ben-Tal N. A model-structure of a periplasm-facing state of the NhaA antiporter suggests the molecular underpinnings of pH-induced conformational changes. J Biol Chem. 2012;287:18249–18261. PubMed PMC

Valadié H., Lacapčre J.J., Sanejouand Y.H., Etchebest C. Dynamical properties of the MscL of Escherichia coli: a normal mode analysis. J Mol Biol. 2003;332:657–674. PubMed

Konagurthu A.S., Whisstock J.C., Stuckey P.J., Lesk A.M. MUSTANG: a multiple structural alignment algorithm. Proteins. 2006;64:559–574. PubMed

Chang Y.-Y., Hsu C.-H. Structural basis for substrate-specific acetylation of Nα-acetyltransferase Ard1 from Sulfolobus solfataricus. Sci Rep. 2015;5:8673. PubMed PMC

Aksnes H., Goris M., Strømland Ø., Drazic A., Waheed Q., Reuter N. Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane. J Biol Chem. 2017;292:6821–6837. PubMed PMC

Liszczak G., Marmorstein R. Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog. Proc Natl Acad Sci U S A. 2013;110:14652–14657. PubMed PMC

Goris M., Magin R.S., Foyn H., Myklebust L.M., Varland S., Ree R. Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80. Proc Natl Acad Sci. 2018 PubMed PMC

Fuglebakk E., Echave J., Reuter N. Measuring and comparing structural fluctuation patterns in large protein datasets. Bioinformatics. 2012;28:2431–2440. PubMed

Van Damme P., Evjenth R., Foyn H., Demeyer K., De Bock P.-J., Lillehaug J.R. Proteome-derived peptide libraries allow detailed analysis of the substrate specificities of Nα-acetyltransferases and point to hNaa10p as the post-translational actin Nα-acetyltransferase. Mol Cell Proteomics. 2011;10 M110.004580. PubMed PMC

Tiwari S.P., Reuter N. Conservation of intrinsic dynamics in proteins—what have computational models taught us? Curr Opin Struct Biol. 2018;50:75–81. PubMed

Tiwari S.P., Reuter N. Similarity in shape dictates signature intrinsic dynamics despite no functional conservation in TIM barrel enzymes. PLOS Comput Biol. 2016;12 PubMed PMC

Myklebust L.M., Van Damme P., Stove S.I., Dorfel M.J., Abboud A., Kalvik T.V. Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects. Hum Mol Genet. 2014:24. PubMed PMC

Zen A., Carnevale V., Lesk A.M., Micheletti C. Correspondences between low-energy modes in enzymes: dynamics-based alignment of enzymatic functional families. Protein Sci. 2008;17:918–929. PubMed PMC

Yang L.W., Bahar I. Coupling between catalytic site and collective dynamics: a requirement for mechanochemical activity of enzymes. Structure. 2005;13:893–904. PubMed PMC

Zheng W., Tekpinar M. Large-scale evaluation of dynamically important residues in proteins predicted by the perturbation analysis of a coarse-grained elastic model. BMC Struct Biol. 2009;9:45. PubMed PMC

Reuter N., Hinsen K., Lacapère J.J. Transconformations of the SERCA1 Ca-ATPase: a normal mode study. Biophys J. 2003;85:2186–2197. PubMed PMC

Fenwick R.B., Orellana L., Esteban-Martín S., Orozco M., Salvatella X. Correlated motions are a fundamental property of β-sheets. Nat Commun. 2014:5. PubMed

Zhang S., Li H., Krieger J.M., Bahar I., Ozkan B. Shared signature dynamics tempered by local fluctuations enables fold adaptability and specificity. Mol Biol Evol. 2019 PubMed PMC

Popp B., Støve S.I., Endele S., Myklebust L.M., Hoyer J., Sticht H. De novo missense mutations in the NAA10 gene cause severe non-syndromic developmental delay in males and females. Eur J Hum Genet. 2015;23:602–609. PubMed PMC

Shahmoradi A., Sydykova D.K., Spielman S.J., Jackson E.L., Dawson E.T., Meyer A.G. Predicting evolutionary site variability from structure in viral proteins: buriedness, packing, flexibility, and design. J Mol Evol. 2014;79:130–142. PubMed PMC

Bahar I., Cheng M.H., Lee J.Y., Kaya C., Zhang S. Structure-encoded global motions and their role in mediating protein-substrate interactions. Biophys J. 2015;109:1101–1109. PubMed PMC

Lovera S., Morando M., Pucheta-Martinez E., Martinez-Torrecuadrada J.L., Saladino G., Gervasio F.L. Towards a molecular understanding of the link between imatinib resistance and kinase conformational dynamics. PLoS Comput Biol. 2015:11. PubMed PMC

Hong H., Cai Y., Zhang S., Ding H., Wang H., Han A. Molecular basis of substrate specific acetylation by N-terminal acetyltransferase NatB. Structure. 2017;25 641–649.e3. PubMed

Kurkcuoglu Z., Bakan A., Kocaman D., Bahar I., Doruker P. Coupling between catalytic loop motions and enzyme global dynamics. PLoS Comput Biol. 2012;8:1–11. PubMed PMC

Støve S.I.I., Magin R.S.S., Foyn H., Haug B.E.E., Marmorstein R., Arnesen T. Crystal structure of the Golgi-associated human Nα-acetyltransferase 60 reveals the molecular determinants for substrate-specific acetylation. Structure. 2016;24:1044–1056. PubMed PMC

Vetting M.W., Bareich D.C., Yu M., Blanchard J.S. Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18. Protein Sci. 2008;17:1781–1790. PubMed PMC

Montgomery D.C., Sorum A.W., Meier J.L. Chemoproteomic profiling of lysine acetyltransferases highlights an expanded landscape of catalytic acetylation. J Am Chem Soc. 2014;136:8669–8676. PubMed PMC

Liu Z., Liu Y., Wang H., Ge X., Jin Q., Ding G. Patt1, a novel protein acetyltransferase that is highly expressed in liver and downregulated in hepatocellular carcinoma, enhances apoptosis of hepatoma cells. Int J Biochem Cell Biol. 2009;41:2528–2537. PubMed

Yang X., Yu W., Shi L., Sun L., Liang J., Yi X. HAT4, a Golgi apparatus-anchored B-type histone acetyltransferase, acetylates free histone H4 and facilitates chromatin assembly. Mol Cell. 2011;44:39–50. PubMed

Yoon H., Kim H.L., Chun Y.S., Shin D.H., Lee K.H., Shin C.S. NAA10 controls osteoblast differentiation and bone formation as a feedback regulator of Runx2. Nat Commun. 2014;5:1–14. PubMed

Ohkawa N., Sugisaki S., Tokunaga E., Fujitani K., Hayasaka T., Setou M. N-acetyltransferase ARD1-NAT1 regulates neuronal dendritic development. Genes Cells. 2008;13:1171–1183. PubMed

Wang Z.L.W. Inactivation of androgen-induced regulator ARD1 inhibits androgen receptor acetylation and prostate tumorigenesis. Proc Natl Acad Sci U S A. 2012;188:2015–2016. PubMed PMC

Seo J.H., Park J.H., Lee E.J., Vo T.T.L., Choi H., Kim J.Y. ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation. Nat Commun. 2016;7:1–14. PubMed PMC

Lim J.H., Park J.W., Chun Y.S. Human arrest defective 1 acetylates and activates beta-catenin, promoting lung cancer cell proliferation. Cancer Res. 2006;66:10677–10682. PubMed

Seo J.H., Cha J.H., Park J.H., Jeong C.H., Park Z.Y., Lee H.S. Arrest defective 1 autoacetylation is a critical step in its ability to stimulate cancer cell proliferation. Cancer Res. 2010;70:4422–4432. PubMed

Muller Reinke T., Travers Timothy, Cha Hi-Jea, Phillips Joshua L., Gnanakaran S., Pos K.M. Switch loop flexibility affects substrate transport of the AcrB efflux pump. J Mol Biol. 2017:22. PubMed

Jager M., Deechongkit S., Koepf E.K., Nguyen H., Gao J., Powers E.T. Understanding the mechanism of beta-sheet folding from a chemical and biological perspective. Biopolymers. 2008;90:751–758. PubMed

Dawson N.L., Lewis T.E., Das S., Lees J.G., Lee D., Ashford P. CATH: an expanded resource to predict protein function through structure and sequence. Nucleic Acids Res. 2017 PubMed PMC

Andreeva A., Howorth D., Chothia C., Kulesha E., Murzin A.G. SCOP2 prototype: a new approach to protein structure mining. Nucleic Acids Res. 2014;42:D310–D314. PubMed PMC

Andreeva A., Kulesha E., Gough J., Murzin A.G. The SCOP database in 2020: expanded classification of representative family and superfamily domains of known protein structures. Nucleic Acids Res. 2020 PubMed PMC

Velankar S., Van Ginkel G., Alhroub Y., Battle G.M., Berrisford J.M., Conroy M.J. PDBe: Improved accessibility of macromolecular structure data from PDB and EMDB. Nucleic Acids Res. 2016;44:D385–D395. PubMed PMC

Sigrist C.J.A., De Castro E., Cerutti L., Cuche B.A., Hulo N., Bridge A. New and continuing developments at PROSITE. Nucleic Acids Res. 2013;41:344–347. PubMed PMC

Kolde R. Package ‘pheatmap’. Bioconductor. 2012:1–6.

Tiwari S.P., Fuglebakk E., Hollup S.M., Skjærven L., Cragnolini T., Grindhaug S.H. WEBnm@ v2. 0: Web server and services for comparing protein flexibility. BMC Bioinf. 2014;15:427. PubMed PMC

Hinsen K. Analysis of domain motions by approximate normal mode calculations. Proteins Struct Funct Genet. 1998;33:417–429. 10.1002/(SICI)1097-0134(19981115)33:3<417::AID-PROT10>3.0.CO;2-8. PubMed

Hinsen K., Petrescu A.J., Dellerue S., Bellissent-Funel M.C., Kneller G.R. Harmonicity in slow protein dynamics. Chem Phys. 2000;261:25–37.

Hinsen K. The molecular modeling toolkit: a new approach to molecular simulations. J Comput Chem. 2000;21:79–85. 10.1002/(SICI)1096-987X(20000130)21:2<79::AID-JCC1>3.0.CO;2-B.

Ichiye T., Karplus M. Collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations. Proteins Struct Funct Bioinforma. 1991;11:205–217. PubMed

Miloshevsky G.V., Jordan P.C. The open state gating mechanism of gramicidin A requires relative opposed monomer rotation and simultaneous lateral displacement. Structure. 2006;14:1241–1249. PubMed

Mahajan S., Sanejouand Y.-H. Jumping between protein conformers using normal modes. J Comput Chem. 2017 PubMed

Chaudhury S., Lyskov S., Gray J.J. PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta. Bioinformatics. 2010;26:689–691. PubMed PMC

Rohl C.A., Strauss C.E.M., Misura K.M.S., Baker D. Protein structure prediction using Rosetta. Methods Enzymol. 2004;383:66–93. PubMed

Alford R.F., Leaver-Fay A., Jeliazkov J.R., O’Meara M.J., DiMaio F.P., Park H. The Rosetta all-atom energy function for macromolecular modeling and design. J Chem Theory Comput. 2017;13:3031–3048. PubMed PMC

Nivón L.G., Moretti R., Baker D. A pareto-optimal refinement method for protein design scaffolds. PLoS ONE. 2013 PubMed PMC

Jurcik A., Bednar D., Byska J., Marques S.M., Furmanova K., Daniel L. Analyst 2.0: analysis and visualization of channels and tunnels in protein structures and molecular dynamics trajectories. Bioinformatics. 2018:9–10. PubMed PMC

Jurčík A, Byška J, Sochor J, Kozlíková B. Visibility-based approach to surface detection of tunnels in proteins. Proc 31st Spring Conf Comput Graph – SCCG’15 2015:65–72. https://doi.org/10.1145/2788539.2788548.

Robert X., Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014:42. PubMed PMC

Oke M., Carter L.G., Johnson K.A., Liu H., McMahon S.A., Yan X. The Scottish structural proteomics facility: targets, methods and outputs. J Struct Funct Genomics. 2010;11:167–180. PubMed PMC

Chang Y.Y., Hsu C.H. Multiple conformations of the loop region confers heat-resistance on SsArd1, a Thermophilic NatA. ChemBioChem. 2016;17:214–217. PubMed

Ma C., Pathak C., Jang S., Lee S.J., Nam M., Kim S.J. Structure of Thermoplasma volcanium Ard1 belongs to N-acetyltransferase family member suggesting multiple ligand binding modes with acetyl coenzyme A and coenzyme A. Biochim Biophys Acta - Proteins Proteomics. 2014;1844:1790–1797. PubMed

Vetting M.W., De Carvalho L.P.S., Roderick S.L., Blanchard J.S. A novel dimeric structure of the RimL Na-acetyltransferase from Salmonella typhimurium. J Biol Chem. 2005;280:22108–22114. PubMed

Sakamoto K., Murayama K., Oki K., Iraha F., Kato-Murayama M., Takahashi M. Genetic encoding of 3-iodo-l-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography. Structure. 2009;17:335–344. PubMed