aKMT Catalyzes Extensive Protein Lysine Methylation in the Hyperthermophilic Archaeon Sulfolobus islandicus but is Dispensable for the Growth of the Organism - PubMed (original) (raw)

. 2016 Sep;15(9):2908-23.

doi: 10.1074/mcp.M115.057778. Epub 2016 Jun 21.

Yanping Zhu 2, Yuling Chen 3, Wei Li 4, Zhenfeng Zhang 1, Di Liu 4, Tongkun Wang 1, Juncai Ma 5, Haiteng Deng 3, Zhi-Jie Liu 6, Songying Ouyang 7, Li Huang 8

Affiliations

aKMT Catalyzes Extensive Protein Lysine Methylation in the Hyperthermophilic Archaeon Sulfolobus islandicus but is Dispensable for the Growth of the Organism

Yindi Chu et al. Mol Cell Proteomics. 2016 Sep.

Abstract

Protein methylation is believed to occur extensively in creanarchaea. Recently, aKMT, a highly conserved crenarchaeal protein lysine methyltransferase, was identified and shown to exhibit broad substrate specificity in vitro Here, we have constructed an aKMT deletion mutant of the hyperthermophilic crenarchaeon Sulfolobus islandicus The mutant was viable but showed a moderately slower growth rate than the parental strain under non-optimal growth conditions. Consistent with the moderate effect of the lack of aKMT on the growth of the cell, expression of a small number of genes, which encode putative functions in substrate transportation, energy metabolism, transcriptional regulation, stress response proteins, etc, was differentially regulated by more than twofold in the mutant strain, as compared with that in the parental strain. Analysis of the methylation of total cellular protein by mass spectrometry revealed that methylated proteins accounted for ∼2/3 (1,158/1,751) and ∼1/3 (591/1,757) of the identified proteins in the parental and the mutant strains, respectively, indicating that there is extensive protein methylation in S. islandicus and that aKMT is a major protein methyltransferase in this organism. No significant sequence preference was detected at the sites of methylation by aKMT. Methylated lysine residues, when visible in the structure, are all located on the surface of the proteins. The crystal structure of aKMT in complex with S-adenosyl-l-methionine (SAM) or S-adenosyl homocysteine (SAH) reveals that the protein consists of four α helices and seven β sheets, lacking a substrate recognition domain found in PrmA, a bacterial homolog of aKMT, in agreement with the broad substrate specificity of aKMT. Our results suggest that aKMT may serve a role in maintaining the methylation status of cellular proteins required for the efficient growth of the organism under certain non-optimal conditions.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

Fig. 1.

Fig. 1.

Growth curves of the parental, the mutant and the complementary strains. The three strains were grown at 65 °C (A and B) or 75 °C (C and D) in CVY-rich medium (A and C) or SCVy medium (B and D). The OD600 values of the cultures were measured. All numbers are an average of three independent measurements.

Fig. 2.

Fig. 2.

Detection of lysine methylation in total cellular proteins from the parental, the mutant and the complementary strains. Samples containing the same numbers of cells from each strain were loaded in parallel onto two 15% SDS-polyacrylamide gels. After electrophoresis, the two gels were subjected to staining with Commassie brilliant blue R-250 (A) and immunoblotting with anti-mono/dimethyllysine antibodies (B), respectively.

Fig. 3.

Fig. 3.

Representative MS/MS spectra of methylated peptides derived from trypsin-digested thermosome (SiRe_1214). The amino acid sequences of the peptides are shown with methylated lysine residues labeled. Mono, monomethylation, di, dimethylation.

Fig. 4.

Fig. 4.

Schematic representation of lysine residues that were methylated to various extents. The extents of methylation on lysine residues, as identified by mass spectrometry in parent (A) or ΔaKMT (B), are depicted in a Venn diagram. Unmethylated, monomethylated, dimethylated or trimethylated lysine residues are indicated by none, methyl, dimethyl, or trimethyl, respectively.

Fig. 5.

Fig. 5.

Sequence analysis of sites of methylation by aKMT. A, Primary structure. Different amino acid residues are shown in different colors. B, Predicted secondary structure. The secondary structures of the amino acid sequences containing the sites of methylation were predicted. Helix, sheet, turn and coil are represented by H, E, T and C, respectively. The frequencies of amino acid residues or secondary structure elements are generated by Weblogo.

Fig. 6.

Fig. 6.

The crystal structures of aKMT-SAM and aKMT-SAH. A, Stereo view of the the aKMT-SAM structure. The structure is shown as a ribbon diagram with the α helices and the β sheets colored in red and yellow, respectively. The SAM molecule is shown as gray sticks. The purple sphere represents a magnesium ion. The N- and C termini are labeled with the respective letters. B, The solvent-accessible surface of aKMT in complex with SAM, colored according to electrostatic potential. Blue, positively charged; red, negatively charged; white, neutral. Electron density of a 2Fo-Fc simulated annealing (SA) omit map for SAM bound in the catalytic pocket contoured at 1.0σ is shown. The SAM molecule is shown as gray sticks. C, Schematic diagram summarizing the interactions between aKMT and SAM in the aKMT-SAM structure generated by LIGPLOT (67). Interacting atoms are connected by green dashed lines with bonding lengths indicated (in Å). Nonligand residues involved in direct hydrophobic contacts with SAM are shown as red semicircles with radiating spokes. D, The solvent-accessible surface of aKMT in complex with SAH, colored according to electrostatic potential. Blue, positively charged; red, negatively charged; white, neutral. The SAH molecule is shown as green sticks. E, Comparison of the structures of aKMT-SAH (cylan) and aKMT-SAM (green).

Fig. 7.

Fig. 7.

Thermal stability of total cellular proteins from the parental and the mutant strains. Cells from an exponentially grown culture of the parental strain or ΔaKMT were harvested, resuspended in 50 m

m

sodium phosphate buffer, pH 7.0, and sonicated. A, The clarified cell-free extracts were heated from 50 to 95 °C at a rate of 0.2 °C/min, and the increase in absorbance at 600 nm was recorded on a Shimadzu UV-2550 spectrophotometer. B, The cell-free extracts were incubated at 65, 75, 85, or 95 °C for 15, 30, 60, and 120 min. Protein aggregation was monitored by 90° light scattering at 488 nm on a Shimadzu RF5301PC spectrofluorimeter.

Fig. 8.

Fig. 8.

A bar plot showing genes differentially expressed in the parental and the mutant strains. Transcriptional profiles for the parental strain and ΔaKMT were determined and compared. Genes whose expression differed by more than twofold (FDR < 0.001) in the two strains, are shown. Red and green bars indicate down- and up-regulated genes, respectively. Putative functions of proteins discussed in the text are indicated.

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