Expression, Crystallization and Substrate Binding Studies of Human Histone N-terminal Acetyltransferase Nat11

Abstract

Abstract: The alpha-amino groups of histones H4 and H2A can be acetylated by histone N-terminal acetyltransferase 11（Nat11）, which plays an important role in epigenetic regulation. The cDNA of human Nat11 was amplified and cloned into pSUMO vector. The resultant construct was transformed into E.coli strain BL21（DE3）for recombinant protein expression. Homogenous Nat11 was highly purified through a series of purification procedures including nickel column affinity chromatography. Using isothermal titration calorimetry（ITC）, we measured micromolar binding constants between Nat11 and histone H4 peptides. MALDI-TOF mass spectrometry analysis revealed that purified Nat11 was pre-bound with acetyl coenzyme A or coenzyme A that was co-purified from E.coli. After ITC titration using unmodified peptide as ligand, N-acetylated product was detected by mass spectrometry, suggesting that the purified Nat11 is active. We performed crystallization screening and successfully obtained single crystal of a truncate form of Nat11 and substrate-enzyme recombinant protein after optimization.

Key words: Alpha-amino acetyltransferase 11 , Recombinant protein expression , Protein aggregation , Enzyme-substrate binding , Crystallization

Huang Jiaxin ,Li Haitao. Expression, Crystallization and Substrate Binding Studies of Human Histone N-terminal Acetyltransferase Nat11[J]. Biotechnology Bulletin, 2014, 0(11): 193-200.

References

[1]Starheim KK, Gevaert K, Arnesen T. Protein N-terminal acetyltransf-erases：when the start matters[J]. Trends in Biochemical Sciences, 2012, 37（4）：152-161.
[2]Arnesen T, Van Damme P, Polevoda B, et al. Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast and humans[J]. Proceedings of the National Academy of Sciences, 2009, 106（20）：8157-8162.
[3]Polevoda B, Sherman F. N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins[J]. Journal of Molecular Biology, 2003, 325（4）：595-622.
[4]Gromyko D, Arnesen T, Ryningen A, et al. Depletion of the human Nα-terminal acetyltransferase A induces p53-dependent apoptosis and p53-independent growth inhibition[J]. International Journal of Cancer, 2010, 127（12）：2777-2789.
[5]Starheim K, Arnesen T, Gromyko D, et al. Identification of the human Nalpha-acetyltransferase complex B（hNatB）：a complex important for cell-cycle progression[J]. Biochem J, 2008, 415：325-331.
[6]Starheim KK, Gromyko D, Evjenth R, et al. Knockdown of human Nα-terminal acetyltransferase complex C leads to p53-dependent apoptosis and aberrant human Arl8b localization[J]. Molecular and Cellular Biology, 2009, 29（13）：3569-3581.
[7]Hole K, Van Damme P, Dalva M, et al. The human N-alpha-acetyltransferase 40（hNaa40p/hNatD）is conserved from yeast and N-terminally acetylates histones H2A and H4[J]. PloS One, 2011, 6（9）：e24713.
[8]Evjenth R, Hole K, Karlsen OA, et al. Human Naa50p（Nat5/San）displays both protein Nα-and Nε-acetyltransferase activity[J]. Journal of Biological Chemistry, 2009, 284（45）：31122-31129.
[9]Van Damme P, Hole K, Pimenta-Marques A, et al. NatF contributes to an evolutionary shift in protein N-terminal acetylation and is important for normal chromosome segregation[J]. PLoS Genetics, 2011, 7（7）：e1002169.
[10]Rope AF, Wang K, Evjenth R, et al. Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency[J]. The American Journal of Human Genetics, 2011, 89（1）：28-43.
[11]Kornberg RD, Lorch Y. Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome[J]. Cell, 1999, 98（3）：285-294.
[12]Luger K, Richmond TJ. The histone tails of the nucleosome[J]. Current Opinion in Genetics & Development, 1998, 8（2）：140-146.
[13]Strahl BD, Allis CD. The language of covalent histone modificati-ons[J]. Nature, 2000, 403（6765）：41-45.
[14]Portela A, Esteller M. Epigenetic modifications and human disease[J]. Nature Biotechnology, 2010, 28（10）：1057-1068.
[15] Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human dis-ease and prospects for epigenetic therapy[J]. Nature, 2004, 429（6990）：457-463.
[16] Song OK, Wang X, Waterborg JH, Sternglanz R. An Nalpha-acetyl-transferase responsible for acetylation of the N-terminal residues of histones H4 and H2A[J]. The Journal of Biological Chemistry, 2003, 278（40）：38109-38112.
[17] Mullen JR, Kayne P, Moerschell R, et al. Identification and charac-terization of genes and mutants for an N-terminal acetyltransferase from yeast[J]. The EMBO Journal, 1989, 8（7）：2067-2075.
[18] Polevoda B, Hoskins J, Sherman F. Properties of Nat4, an N（alp-ha）-acetyltransferase of Saccharomyces cerevisiae that modifies N termini of histones H2A and H4[J]. Molecular and Cellular Biology, 2009, 29（11）：2913-2924.
[19] Polevoda B, Brown S, Cardillo TS, et al. Yeast N（alpha）-terminal acetyltransferases are associated with ribosomes[J]. Journal of Cellular Biochemistry, 2008, 103（2）：492-508.
[20] Tweedie-Cullen RY, Brunner AM, Grossmann J, et al. Identification of combinatorial patterns of post-translational modifications on individual histones in the mouse brain[J]. PloS One, 2012, 7（5）：e36980.
[21] Young NL, DiMaggio PA, Plazas-Mayorca MD, et al. High throughput characterization of combinatorial histone codes[J]. Molecular & Cellular Proteomics, 2009, 8（10）：2266-2284.
[22] Schiza V, Molina-Serrano D, Kyriakou D, et al. N-alpha-terminal acetylation of histone H4 regulates arginine methylation and ribosomal DNA silencing[J]. PLoS Genetics, 2013, 9（9）：e1003805.