Multimethylationof Rickettsia …OmpB proteins (2 g each) were separated by SDS-PAGE,...

Multimethylation of Rickettsia OmpB Catalyzed by LysineMethyltransferases*□S

Received for publication, November 18, 2013, and in revised form, January 30, 2014 Published, JBC Papers in Press, February 4, 2014, DOI 10.1074/jbc.M113.535567

Amila Abeykoon‡, Guanghui Wang§, Chien-Chung Chao¶, P. Boon Chock�, Marjan Gucek§, Wei-Mei Ching¶,and David C. H. Yang‡1

From the ‡Department of Chemistry, Georgetown University, Washington, D. C. 20057, the §Proteomics Core Facility, NHLBI,National Institutes of Health, Bethesda, Maryland 20892, the ¶Viral and Rickettsial Diseases Department, Infectious DiseasesDirectorate, Naval Medical Research Center, Silver Spring, Maryland 20910, and the �Laboratory of Biochemistry, National Heart,Lung, and Blood Institute, Bethesda, Maryland 20892

Background: Methylation of OmpB has been implicated in rickettsial virulence.Results: Native OmpBs purified from Rickettsia contain mono- and trimethyllysine at specific locations that coincide with thosecatalyzed by methyltransferases in vitro.Conclusion: The number of trimethyllysine clusters in OmpBs correlates with degree of virulence.Significance: This study provides new insight into methylation of OmpB and its correlation with virulence.

Methylation of rickettsial OmpB (outer membrane protein B)has been implicated in bacterial virulence. Rickettsial methyl-transferases RP789 and RP027-028 are the first biochemicallycharacterized methyltransferases to catalyze methylation ofouter membrane protein (OMP). Methylation in OMP remainspoorly understood. Using semiquantitative integrated liquidchromatography-tandem mass spectroscopy, we characterizemethylation of (i) recombinantly expressed fragments of Rick-ettsia typhi OmpB exposed in vitro to trimethyltransferases ofRickettsia prowazekii RP027-028 and of R. typhi RT0101 and tomonomethyltransferases of R. prowazekii RP789 and of R. typhiRT0776, and (ii) native OmpBs purified from R. typhi andR. prowazekii strains Breinl, RP22, and Madrid E. We found thatin vitro trimethylation occurs at relatively specific locations inOmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F),whereas monomethylation is pervasive throughout OmpB.Native OmpB from virulent R. typhi contains mono- and tri-methyllysines at locations well correlated with methylation inrecombinant OmpB catalyzed by methyltransferases in vitro.Native OmpBs from highly virulent R. prowazekii strains Breinland RP22 contain multiple clusters of trimethyllysine in con-trast to a single cluster in OmpB from mildly virulent R. typhi.Furthermore, OmpB from the avirulent strain Madrid E con-tains mostly monomethyllysine and no trimethyllysine. Thenative OmpB from Madrid E was minimally trimethylated byRT0101 or RP027-028, consistent with a processive mechanismof trimethylation. This study provides the first in-depth charac-terization of methylation of an OMP at the molecular level andmay lead to uncovering the link between OmpB methylation andrickettsial virulence.

Rickettsiae are obligatory intracellular infectious Gram-neg-ative bacteria that are responsible for major rickettsiosis, whichincludes epidemic and endemic typhus, spotted fever, andscrub typhus (1, 2). Like all Gram-negative bacteria, rickettsiaecontain several outer membrane proteins (OMPs)2 that arefound in the outer leaflet of the outer membrane. OMPs pro-vide the first line of communication with the extracellular envi-ronment, with prominent roles in molecular transport and bac-terial infection and pathogenesis (3, 4). Rickettsial OMPs havebeen shown to participate in host cell attachment, invasion,internalization, and intracellular movement (5–7) and inducestrong host humoral and cellular immune responses (8 –10).

OmpB (outer membrane protein B) is an OMP that is presentin all species of Rickettsia and belongs to the family of OMPscalled autotransporters. The precursor of OmpB consists of asignal peptide, a large N-terminal passenger domain and aC-terminal �-barrel domain (11, 12). The passenger domain ofrickettsial OmpB has been shown to participate in adhesion tomammalian cells in vitro, suggesting this may be the role ofOmpB in the virulence of Rickettsia (5, 6, 13).

It has been known for many years that the passenger domainof OmpB of Rickettsia is methylated, and OmpBs from virulentstrains are more extensively methylated (14, 15). More recentstudies using genetic and biochemical approaches are consis-tent with the suggestion that methylation of OmpB may contrib-ute to both (i) the host immunogenic response to OmpB itselfand (ii) rickettsial virulence (16 –18). Methylation of OmpBappears to enhance its antigenicity. For example, rabbit antise-rum against recombinant OmpB is less reactive than antiserumagainst OmpB purified directly from Rickettsia (19). In addi-tion, the immunoreactivity of chemically methylated recombi-nant OmpB is enhanced against sera from infected patients(20). Thus, methylation of recombinant OmpB could increasethe efficacy of diagnostic reagents and help advance vaccinedevelopment against Rickettsia (21).

* The work was supported by Naval Medical Logistic Command AwardN62645 (to Georgetown University) and Work Unit 6000.RAD1.J.A0310 (tothe Naval Medical Research Center).

□S This article contains supplemental Tables S1–S5.1 To whom correspondence should be addressed: Dept. of Chemistry,

Georgetown University, Washington, D. C. 20057. Tel.: 202-687-6090; Fax:202-687-6209; E-mail: [email protected].

2 The abbreviations used are: OMP, outer membrane protein; MT, methyl-transferase; AdoMet, S-adenosylmethionine; PSM, peptide spectrummatch.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 11, pp. 7691–7701, March 14, 2014Published in the U.S.A.

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Protein methylation is a well established post-translationalmodification known to regulate functions of various proteins(22, 23), frequently through modulating protein-protein inter-actions (24, 25). The regulatory potential of this modificationcan be attributed in part to the multiple states of methylation atthe �-amino group of lysine, which can be mono-, di-, and tri-methylated (26). Unlike the methylation of histones, which hasbeen well established, methylation of outer membrane proteinsis not well characterized, particularly at the molecular level.

We have recently characterized two rickettsial lysine meth-yltransferases (MTs) (27). They are RP789 (PKMT1) andRP027-028 (PKMT2) from Rickettsia prowazekii. RP027-028,as well as its counterpart in Rickettsia typhi, RT0101, catalyzestrimethylation, and RP789 catalyzes monomethylation ofrOmpB fragments, monitored by incorporation of radioactive[methyl-3H] from [methyl-3H]AdoMet and Western blot anal-ysis, whereas none of these MTs can methylate either histone orEscherichia coli protein. These are the first biochemically char-acterized MTs to catalyze the methylation of OMPs and appearto belong to a novel family of protein lysine MTs. The genesequences of homologous MTs are found to be conserved inmore than 40 rickettsial species and strains. The fact that theseMTs recognize OmpB at many diverse sites suggests an unusualsubstrate recognition mechanism. Characterization of methyl-ation in rickettsial OmpB at the molecular level is essential forelucidating the roles of methylation in bacterial virulence andpathogenesis. Moreover, findings on the rickettsial OmpB methyl-ation may advance our understanding of the structure and func-tion of protein lysine methylation in general. In addition, enzy-matic methylation of recombinant OmpB may also provideimproved methods over chemical methylation in efforts toadvance diagnostic reagents and vaccine candidates.

To better understand OmpB methylation, we used semi-quantitative integrated LC-MS/MS techniques to characterizethe location, state and level of methylation. Our study providesthe first characterization of methylation in an OMP and lays thefoundation for further understanding how methylation ofOmpB may contribute to the virulence of Rickettsia.

MATERIALS AND METHODS

OmpB Proteins—Native OmpB proteins from R. typhi andR. prowazekii strains Breinl, RP22, and Madrid E were extractedusing 10 mM Tris-HCl (pH 7.6) at 45 °C for 30 min and purifiedas described previously (28). Each of the native OmpB proteinsshowed a single band at Mr 114,000 on SDS-polyacrylamidegel. Recombinant R. typhi OmpB fragments OmpB(AN) andOmpB(K), which corresponded to residues Met33 to Phe744 andArg745 to Gly1353, respectively, were expressed and purified aspreviously described (20). Briefly, the bacterial proteins thatwere expressed in inclusion bodies were dissolved in 8 M ureaand 1% deoxycholate, purified by ion exchange chromatogra-phy on DEAE-cellulose in 6 M urea. Soluble proteins wereobtained by slow dialysis at stepwise decreasing concentrationsof urea. No methylated lysine in recombinant OmpB(AN) and(K) were detected by LC-MS/MS. Protein concentrations weredetermined using Bio-Rad protein assays with bovine serumalbumin as the standard.

Rickettsial MTs—Purified recombinant R. prowazekii RP789(NP221139) and RP027-028 (ADE29537) and R. typhi RT0776(YP067714) and RT0101 (YP067069) were used in enzymaticmethylation of OmpB fragments in the present study. The MTswere prepared by the same method as previously described (27).No methyllysines in recombinant MTs were found as deter-mined by LC-MS/MS.

Deletions of N-terminal Sequences of RP789 and RT0776—The plasmids expressing RP789�N (deletion of N-terminal 28residues) and RT0776�N (deletion of N-terminal 27 residues)were constructed in pET28a by subcloning PCR-amplifiedDNA using the plasmids encoding RP789 and RT0776, respec-tively, as templates. Primer sequences are available on request.The constructs were confirmed by sequencing. Expression andpurification was performed as described previously (27).

Radioactivity Assay of MT Activity—The standard assay mix-ture contained, in 50 �l of final volume, 8.3 mM sodium phos-phate (pH 8.0), 0.16 mM [methyl-3H]AdoMet (34 mCi/mmoldiluted from 10 Ci/mmol [methyl-3H]AdoMet, which has a�97% purity obtained from PerkinElmer, with the highestpurity AdoMet purchased from New England Biolabs) and 2 �M

OmpB(AN) or 1 �M OmpB(K). The reactions were initiated byadding MT to a final concentration of 0.26 �M and incubated at37 °C. Aliquots of the reaction mixture were spotted at the indi-cated time onto Whatman 3MM cellulose filter paper discs(Fisher Scientific) and soaked briefly with 5% TCA. The paperdiscs were washed three times with 5% ice-cold TCA, followedby washing with ethanol:ether (1:1 by v/v) mixture. Theamounts of acid precipitable radioactivity were determinedusing a PerkinElmer Wallace 1410 liquid scintillation counter.The assay conditions were optimized by varying pH. DTT, KCl,and cations reduce the activity of MT, and none of them wasincluded in the assay.

Kinetic Analysis of the MTs—Initial rates of MT-catalyzedreactions were determined from the linear portions of the timecourses of methylation at varying concentrations of OmpB(AN)up to 2 �M using the radioactivity assay at 37 °C. These initialrates were used to determine the Michaelis-Menten and cat-alytic constants. The reactions were initiated by the additionof the specified MT to a final concentration of 0.26 �M.Michaelis-Menten constants and maximum velocities wereobtained by direct fit using KaleidaGraph. It should be notedthat the initial rates thus determined represent the sum ofinitial rates of numerous enzymatic methylation reactions atmultiple lysine residues in OmpB(AN) to three different meth-ylation states whose rates may vary with wide ranges. TheMichaelis-Menten and catalytic constants based on the radio-activity assay can only be considered as apparent Michaelis-Menten and catalytic constants.

Preparation of Proteins for LC-MS/MS Analysis—OmpB(AN)(10 �g) and OmpB(K) (5 �g) were methylated separately using10 �g of specified MT in 50 �l of reaction mixtures containing3.2 mM AdoMet (New England Biolabs) and 8.3 mM sodiumphosphate (pH 8.0). After overnight incubation at 37 °C, thereaction mixture was evaporated to 20 �l using SpeedVac andmixed with SDS sample buffer. The proteins were separated bySDS-PAGE, and OmpB(AN) and (K) protein bands wereexcised from the gel and subjected to in-gel digestion. Native

Methylation Profiles of OmpB in Rickettsia

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OmpB proteins (2 �g each) were separated by SDS-PAGE,excised from the gel, and processed for in-gel digestion. Multi-ple samples of enzymatically methylated rOmpB and nativeOmpB proteins were independently prepared and analyzedusing LC-MS/MS.

In-gel Digestion—In-gel digestion of proteins was carried outas described with modifications (29). Briefly, the excised pro-tein bands from SDS gels were washed using 50% methanol and5% acetic acid, followed by reduction using 10 mM DTT. Theprotein samples were alkylated with 100 mM iodoacetamide inthe dark. The gel pieces were dehydrated using acetonitrile andrehydrated with 100 mM (NH4)HCO3 twice. The gel pieceswere mixed with 1 �g of sequencing grade chymotrypsin(Roche Applied Science) in 50 mM (NH4)HCO3 and digestedovernight at 25 °C. The digested peptides were extracted with50% (v/v) acetonitrile and 5% (v/v) formic acid. The volume wasreduced to less than 20 �l by evaporation, and the final volumewas adjusted to 20 �l using 1% formic acid. The samples werepurified using Zip-Tip with C18 resin (Millipore, Billerica, MA)according to the manufacturer’s protocol.

LC-MS/MS—LC-MS/MS was performed using an EksigentnanoLC-Ultra two-dimensional system (Dublin, CA) coupledto an LTQ Orbitrap Elite mass spectrometer (Thermo Scien-tific, San Jose, CA). Peptide sample was first loaded onto a Zor-bax 300SB-C18 trap column (Agilent, Palo Alto, CA) at a flowrate of 6 �l/min for 9 min and then separated on a reversedphase BetaBasic C18 PicoFrit analytical column (New Objec-tive, Woburn, MA) using a 40-min linear gradient of 5–35%acetonitrile in 0.1% formic acid at a flow rate of 250 nl/min.Eluted peptides were sprayed into the mass spectrometerequipped with a nano-spray ionization source. Survey MS spec-tra were acquired in the Orbitrap at a resolution of 60,000. EachMS scan was followed by six data-dependent MS/MS scans inthe linear ion trap with dynamic exclusion. Other mass spec-trometry settings were as follows: spray voltage, 1.5 kV; fullMS mass range, m/z 300 –2000; and normalized collisionenergy, 35%.

Data files generated from the mass spectrometer were ana-lyzed using Proteome Discoverer v1.3 software (Thermo Scien-tific) and the Mascot search engine running on a six-processorcluster at the National Institutes of Health (version 2.3). Thesearch criteria were set to: database, Swiss-Prot (Swiss Instituteof Bioinformatics); taxonomy, Bacteria; enzyme, chymotrypsin;maximal miscleavages, 3; variable modifications, methylation(K), dimethylation (K), trimethylation (K), oxidation (M), anddeamidation (N, Q); fixed modifications, carbamidomethyla-tion (C); peptide precursor mass tolerance, 25 ppm; andMS/MS fragment mass tolerance, 0.8 Da. Peptide-spectrummatches (PSM) were filtered to achieve an estimated false dis-covery rate of 1%.

In this study, mass errors of 3 ppm or less were routinelyachieved with the Orbitrap mass spectrometer used in the pres-ent study. This high mass accuracy allowed us to differentiatebetween trimethylation and acetylation or between dimethyla-tion and formylation, which have a mass difference of 0.03639Da, equivalent to 11– 45 ppm given that most peptide precur-sors had an m/z value between 400 and 1100 with a charge stateof either �2 or �3. Even though a peptide mass tolerance of 25

ppm was used in database search, the application of a massdeviation filter (e.g., �5 ppm) established confidence that thetri- or di-methylation sites identified were not due to acetyla-tion or formylation.

Fig. 1A shows the overall scheme of this analysis. The spectralmatches of a peptide by MS/MS revealed the amino acidsequence, location, and state of methylation. In addition, LC-MS/MS of the peptides provides the number of peptide spec-trum matches of all peptides at unmethylated and mono-, di-,and trimethylated states throughout the OmpB sequence. Formultiple measurements, independently prepared samples wereused. The MS data are reproducible in types and locations ofmethylation in independently prepared samples of OmpB andits fragments. The number of PSM correlates with the abun-dance of individual peptide (30). The observed numbers of PSMvary with the behavior of various peptides in ionization, iondetection, and chromatography and with the amount of proteinsamples. The variations are progressively lower for greater val-ues of PSM. For comparison of methylations at different loca-tions in OmpB, normalized fraction of methylation (from 0 to1) is used to compare the relative levels of various methylationstates at a specific lysine residue. The peptide coverage in LC-MS/MS analyses is close to the entire sequence of OmpB or thefragments in all cases. It should be pointed out that in the chy-motryptic digests, the fragments containing Lys149 and Lys157

were consistently not detected, likely because of retaining bythe chromatographic resins.

Data Analysis—Custom software PTMsite was used to com-pute the numbers of PSM at unmethylated and mono-, di-, andtrimethylated states for all lysine residues throughout OmpBbased on the results of LC-MS/MS of each sample. Normalizedfractions of mono-, di-, and trimethylations of a specific lysineresidue were calculated from the observed PSM numbers ofrespective mono-, di-, and trimethylated peptides divided by thetotal number of observed PSM numbers including unmethylatedand mono-, di-, and trimethylated peptides of the correspondinglysine residue. For multiple trials of a protein, the normalizedfractions were calculated from the sums of the PSM numbers ofeach methylated state at the lysine residue from multiple trialsdivided by the sum of PSM numbers of all methylation states ofall trials at the same lysine residue.

RESULTS AND DISCUSSION

Kinetics of Methylation by Rickettsial MTs—Expression ofthe passenger domain of OmpB from R. typhi in E. coli producesinclusion bodies. However, recombinant OmpB (rOmpB) frag-ments OmpB(AN) and OmpB(K), which correspond to the N- andC-terminal halves of OmpB passenger domain (Fig. 1B), can besuccessfully purified and refolded to yield soluble protein sub-strates for analysis. Using the OmpB(AN) as the substrate of MTs,we analyzed the steady state kinetics of two MTs from R. prowa-zekii (RP789 and RP027-028) and two MTs from R. typhi(RT0766 and RT0101). Table 1 summarizes the steady statekinetic parameters based on the initial rates of the methylationof OmpB(AN) catalyzed by 0.26 �M each of the four MTs bymonitoring the incorporation of radioactive [methyl-3H] from[methyl-3H]AdoMet to OmpB(AN). MonomethyltransferasesRT0776 and RP789 exhibit an appreciably higher kcat than




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those of trimethyltransferases RT0101 and RP027-028. Inaddition, the enzyme efficiency (kcat/Km) of RP789 is higherthan that of RT0776, whereas RT0101 is more active thanRP027-028.

R. typhi RT0101- and RT0776-catalyzed Methylation of rOmpB—Because catalytic activity of MTs is relatively modest, weused 2 �M of trimethyltransferase to prepare enzymaticallymethylated rOmpB for the LC-MS/MS analysis. The timecourses for the methylation of OmpB(AN) (2 �M) andOmpB(K) (1 �M) catalyzed by 2 �M trimethyltransferaseRT0101 are shown in Fig. 2A and remain linear for at least 4 h.Similar time courses were also obtained for the methylation of

rOmpB fragments catalyzed by 2 �M monomethyltransferaseRT0776 (Fig. 2B).

To determine methylation profiles in rOmpB, OmpB(AN)and OmpB(K) were enzymatically methylated using 2 �M

RT0101 and 3.2 mM AdoMet. After overnight incubation at37 °C, the methylated OmpB(AN) and OmpB(K) were sepa-rated using SDS-PAGE followed by in-gel digestion and LC-MS/MS analysis. The numbers of PSM at four methylationstates of all lysine residues in OmpB(AN) and OmpB(K) areshown in supplemental Table S1. The LC-MS/MS analysisreveals a total of 133 trimethyllysine-containing PSM distributedamong 16 locations in OmpB. In contrast, nine monomethylly-sine-containing PSM and one dimethyllysine-containing PSMwere observed (supplemental Table S1). The predominance oftrimethyllysine-containing PSM clearly shows that RT0101 isfunctioned mainly as a trimethyltransferase. The observed mono-and dimethylations could conceivably represent intermediates oftrimethylation. Normalized fractions of trimethylation at alllysine residues in rOmpB were calculated for RT0101-catalyzedmethylation (Fig. 2C). The normalized fractions reveal that spe-cific lysine residues are trimethylated at varying levels from lessthan 1% to 36%. No single consensus sequences can be dis-cerned based on the amino acid sequences adjacent to the tri-methylated lysines. Our attempt to uncover amino acid sequencemotifs of RT0101-catalyzed trimethylation using Motif-X (31)

FIGURE 1. The overall schemes of LC-MS/MS analysis (A) and OmpB and recombinant OmpB(AN) and OmpB(K) (B). A, the state, location, and normalizedfraction of methylation in native OmpB and in enzymatically methylated rOmpB fragments catalyzed by rickettsial MTs were determined by LC-MS/MS analysisaccording to the outlined scheme. See “Materials and Methods” for details. B, the full-length R. typhi OmpB precursor consists of a signal peptide (amino acids1–32) in red, a passenger domain (amino acids 33–1353) in green, and an autotransporter domain (amino acids 1354 –1645) in blue. The corresponding residuenumbers in R. typhi OmpB for recombinant OmpB(AN) and OmpB(K) are shown.

TABLE 1Kinetic parameters of Rickettsial methyltransferasesThe steady state kinetics was monitored by the incorporation of radioactive[methyl-3H] from [methyl-3H]AdoMet into OmpB(AN) as described under “Mate-rials and Methods.” The final concentration of methyltransferase was 0.26 �M.

Methyltransferase KmAN kcat kcat/Km

�M s�1 M�1�s�1

RT0101 0.48 � 0.057 0.83 � 10�3 1.74 � 103

RP027-028a 10.1 � 1.63 0.16 � 10�3 0.015 � 103

RP789 0.29 � 0.093 5.98 � 10�3 20.6 � 103

RP789�N 1.13 � 0.17 3.32 � 10�3 2.93 � 103

RT0776 1.71 � 0.182 2.20 � 10�3 1.28 � 103

RT0776�N 3.07 � 1.55 4.45 � 10�3 1.45 � 103

a RP027-028-catalyzed methylation was analyzed at 2 �M RP027-028 and 2–20 �MOmpB(AN) because of the low enzyme efficiency.




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analysis reveals the presence of two motifs: (i) KX(G/A/V/I)(present at Lys205, Lys232, Lys279, Lys667, Lys711, and Lys723) and(ii) KT(I/L/F) (present at Lys226, Lys232, Lys624, Lys635, and

Lys667). The sequence at Lys232 and Lys667, KTIN, conforms toboth motifs. The remaining sequences do not occur more thanthree times among the 16 sequences that are trimethylated by

FIGURE 2. Time courses and normalized fractions of methylation in OmpB(AN) and OmpB(K) catalyzed by four rickettsial methyltransferases. A and B,time courses of RT0101- (A) and RT0776-catalyzed (B) methylation of 2 �M OmpB(AN) (●) or 1 �M OmpB(K) (f) in the presence of 0.16 mM [methyl-3H]AdoMet(34 mCi/mmol in A and 68 mCi/mmol in B), and 8.3 mM sodium phosphate (pH 8.0) were monitored using the radioactivity assay as described under “Materialsand Methods.” The reaction was initiated by the addition of RT0101 or RT0776 to a final concentration of 2 �M. The control that contained MT alone (�) is alsoshown. C, the normalized fractions of trimethylation at indicated lysine residues in OmpB(AN) and (K) that are catalyzed by RT0101 (open bars) and RP027-028(solid bars) at 2 �M each are shown. The enzymatically methylated OmpB(AN) and (K) were prepared, and the methylated OmpB(AN) and (K) were analyzedusing LC-MS/MS as described under “Materials and Methods.” The PSM values were combined from three independent trials each for RT0101 and RP027-028and shown in supplemental Table S1. The correlation coefficient of the normalized fractions of trimethylations catalyzed by RT0101 and RP027-028 is 0.65. D,the normalized fractions of monomethylation at lysine residues in OmpB(AN) and (K) catalyzed by RT0776 (open bars) and RP789 (closed bars) at 2 �M are shown.Normalized fractions were determined as described for Fig. 2C. The PSM values were combined from three independent trials each for RT0776 and RP789 andshown in supplemental Table S2. The correlation coefficient of normalized fractions of monomethylation catalyzed by RT0776 and RP789 is 0.45.




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RT0101. It should be noted that our preliminary results showthat RT0101 is unable to catalyze methylation of two synthetic20-mer peptides, which contain either one or both of theseconserved trimethylation motifs (data not shown), indicatingthat the conformation of OmpB or other unknown factors mayalso contribute to substrate recognition.

Supplemental Table S1 shows that RT0101 catalyzed meth-ylation yields a disproportionally low number of PSM, whichcontains mono- and dimethylated lysine at 9 and 1, respec-tively, relative to 133 PSM for trimethylated lysine. This obser-vation indicates that the trimethylation reaction likely proceedsvia a processive instead of a distributive mechanism. With theformer mechanism, the monomethyllysine formed remainsenzyme-bound, and the release of product occurs when thelysine is fully trimethylated. This notion is supported by thetime courses, monitored during the first 2 h, of the methylationof 2 �M OmpB(AN) catalyzed by 2 �M RT0101. The time courseshowed the PSM of trimethylated peptides increased steadily to13, 28, and 56 at 30, 60, and 120 min, respectively, whereas thenumbers of PSM of monomethylated peptides were 1, 1, and 6at 30, 60, and 120 min, respectively. Thus, these data indicatethat RT0101-catalyzed trimethylation of OmpB may proceedvia a processive mechanism.

LC-MS/MS analysis of RT0776-catalyzed methylation ofOmpB(AN) and OmpB(K) was also performed. As shown insupplemental Table S2, the total number of PSM containingmonomethyllysine is 379 distributed among 34 lysine residuesof a total 45 lysines, although the number of PSM containing di-and trimethyllysines is 6 and 2, respectively. The observation ofextensive and predominant monomethylation (Fig. 2D) clearlyindicates that RT0776 is mainly functioned as a monomethyl-transferase that possesses an active site that can accommodatea wide variety of recognition sequences.

R. prowazekii RP789- and RP027-028-catalyzed Methylationof rOmpB—Unlike R. typhi, R. prowazekii causes epidemic typhus,which is transmitted by lice. MTs from heterologous species mayproduce products different from those catalyzed by enzymes fromhomologous species. To characterize the methylation of rOmpBcatalyzed by MTs from the heterologous species, we analyzedmethylation of R. typhi rOmpB using R. prowazekii RP027-028and RP789, which possess 94 and 93% identities to their corre-sponding R. typhi orthologs RT0101 and RT0776, respectively.The locations and the PSM numbers of methylated peptides inrOmpB catalyzed by RP027-028 are summarized in supple-mental Table S1, which are very similar to those observed withthe methylation of rOmpB catalyzed by RT0101 (Fig. 2C). Sim-ilarly, the locations and PSM numbers of methylated peptidesin rOmpB catalyzed by RP789 are shown in supplemental TableS2, which reveals 743, 47, and 46 PSM with mono-, di-, andtrimethyllysines, respectively. These results show that RP027-028 and RP789 function essentially as a trimethyltransferaseand monomethyltransferase, respectively, agreeing with theresults observed for their R. typhi orthologs. However, unlikeRT0776, RP789 catalyzes mono-, di-, and trimethylations tosignificantly higher normalized fractions than those catalyzedby RT0776, likely because RP789 and rOmpB are from twodifferent species, whereas RT0776 and rOmpB are from thesame species (Fig. 2D and supplemental Table S2). The appre-

ciable differences of the products catalyzed by RT0776 andRP789 for the same substrate are unusual for enzymes sharinghigh identity. Clearly, heterologous monomethyltransferasedoes not accurately methylate rOmpB. However, the fact thatboth RP789 and RT0776 can catalyze methylation at manydiverse sites suggests that they both possess active sites that canaccommodate a variety of recognition sequences.

N-terminal Sequences in RT0776 and RP789 Contribute toCatalysis and Substrate Recognition—RT0776 shares 45%sequence identity with RT0101 but contains additional 27N-terminal amino acid residues, which, as analyzed usingSignalP 4.1 (32), are not conformed as a signal peptide thathave been found in Gram-negative bacteria. Deleting the N-ter-minal 27 residues in RT0776, we generated RT0776�N, whichwas found to be enzymatically active as monitored by the incor-poration of the radioactivity assay (Table 1). Unexpectedly, di-and trimethylation in rOmpB catalyzed by RT0776�N is ele-vated to 32 and 15 PSM, respectively, versus 6 and 2 by RT0776(comparing supplemental Tables S3 and S2). In addition, thenormalized fractions of monomethylation are significantlyhigher in the RT0776�N-catalyzed methylation than those inthe RT0776-catalyzed methylation (Fig. 3A). Although theincrease in observed mono-, di-, and trimethylation catalyzedby RT0776�N (versus the full-length construct) is surprising,the results suggest that the N-terminal sequence in RT0776may participate in modulating both the catalytic activity andthe states of methylation.

RP789 contains additional N-terminal 28 residues relative toRP027-028. An N-terminal 28 residue-deleted RP789, RP789�N,was also constructed. Similar to RT0776�N, RP789�N is enzy-matically active (Table 1) and also catalyzed additional mono-, di-,and trimethylation in rOmpB as shown in supplemental Table S3.Fig. 3B compares the location and the normalized fraction ofmonomethylation catalyzed by RP789 and RP789�N.

The normalized fractions of mono-, di-, and trimethylationin rOmpB catalyzed by RT0776, RT0776�N, RP789, andRP789�N are summarized in Table 2. Together, bothRT0776�N and RP789�N produce higher normalized frac-tions of mono-, di-, and trimethylation than their full-lengthcounterparts, indicating that the N-terminal domain in bothRT0776 and RP789 may alter the products of monomethyl-transferases by inhibiting their capacity to catalyze di- andtrimethylation. Exactly how the N-terminal domain accom-plishes this effect remains to be determined and will likelyrequire structural studies of these enzymes. Interestingly, theplasticity exhibited by N-terminal truncated RT0776�N andRP789�N resembles the alteration of products noted earlierwhen rOmpB from R. typhi was used as a substrate of heter-ologous MT from R. prowazekii. Although the physiologicalfunction of the N-terminal domain remains to be estab-lished, the present results raise the possibility that the N-ter-minal sequence in rickettsial monomethyltransferases couldbe processed in vivo to alter its catalytic property and yieldadditional cellular function.

Native OmpB from Virulent R. typhi Contains a Cluster ofHighly Trimethylated Lysine Residues—OmpB from Rickettsiais methylated in vivo as revealed previously by amino acid com-position analyses (14, 15). When the methylation profile in




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native OmpB purified directly from R. typhi was analyzed byLC-MS/MS, the numbers of PSM with mono-, di-, and tri-methylation were found to be 300, 164, and 215, respectively(supplemental Table S4). The locations of trimethyllysines innative OmpB coincide with those observed for RT0101-cata-lyzed trimethylation of rOmpB (Fig. 4, A and B). A cluster offour residues at Lys667, Lys676, Lys711, and Lys723 show normal-ized fraction of trimethylation close to 100%. The same res-idues are also trimethylated by RT0101 and RP027-028 invitro. However, the normalized fractions of trimethylationin native OmpB are appreciably higher than those observed invitro with enzymatically methylated rOmpB using RT0101(Figs. 2C and 4B).

The high normalized fraction of trimethylation at specificlysine residues observed in native OmpB suggests the existenceof a highly efficient cellular system in vivo through which thoselysine residues in OmpB can be fully trimethylated. At present,little is known about the mechanism of in vivo trimethylation.Studying the trimethyltransferase catalyzed trimethylation ofrOmpB in vitro would provide an approach to reconstitute thein vivo system. To this end, the evidence indicating the presence

of a highly efficient in vivo trimethylation system appeared to becontrary to the observation that a high concentration of MTs isrequired to achieve an appreciable level of methylated rOmpB.This observation is consistent with the presence of productinhibition because of a slow release of the trimethylated lysinefrom the MT. However, the presence of cellular proteins orfactors, which may interact with the methylated OmpB, wouldshift the equilibrium toward the methylated form of OmpB byfacilitating the release of methylated lysine from its MT com-plex and/or reducing the population of methylated lysine-MTcomplex. Alternatively, the reduction of methylated MT popu-lation can also be accomplished via subcellular translocation(e.g., cytoplasm or periplasmic space), an effect of allosteric fac-tors that could facilitate the post-translational modification. Inaddition, it is possible that in vivo methylation could occurbefore or during folding of the OmpB passenger domain wheremethylation sites could be more accessible than in the case ofthe fully folded OmpB.

Methylation in Native OmpBs from Virulent and AvirulentStrains of R. prowazekii—We next examined the methylation innative OmpBs from three strains of R. prowazekii (Madrid E,RP22, and Brein1). R. prowazekii Madrid E is known to be avir-ulent, whereas R. prowazekii RP22 and Breinl are both highlyvirulent. PSM numbers of peptides in the three native OmpBsas determined by LC-MS/MS are shown in supplemental TableS5. This table shows that the total numbers of PSM of trimeth-yllysine-containing peptides in native OmpB from Madrid E,RP22, and Breinl are 0, 271, and 139, respectively (supplementalTable S5). The absence of trimethyllysine in R. prowazekiiMadrid E is consistent with the fact that the gene encodingRP027-028 is interrupted by a frameshift mutation, which gen-erates the inactive RP027 and RP028 fragments (17, 27). Fig. 5shows that the high normalized fraction of trimethylation

FIGURE 3. The normalized fractions of RT0776�N- and RP789�N-catalyzed monomethylation at lysine residues of OmpB(AN) and (K). The normalizedfractions of monomethylation at indicated Lys residues in OmpB(AN) and (K) that are catalyzed by RT0776�N (A, solid bars) and full-length RP0776 (A, open bars)and RP789�N (B, solid bars) and full-length RP789 (B, open bars) are shown. The enzymatically methylated OmpB(AN) and (K) were prepared and analyzed usingLC-MS/MS as described under “Materials and Methods.” The PSM numbers were combined from three independent trials each for RP789�N and one trial forRT0776�N and shown in supplemental Table S3. The correlation coefficient of the normalized fractions of monomethylation catalyzed by RT0776 andRT0776�N is 0.68, and that by RP789 and RP789�N is 0.75.

TABLE 2Normalized fractions of methylated peptides in OmpB(AN) and (K) cat-alyzed by RT0776, RT0776�N, RP789, and RP789�NThe normalized fractions of unmethylated, mono-, di-, and trimethyllysines werecalculated from sums of the numbers of PSM for all lysine residues as describedunder “Materials and Methods.” Supplemental Tables S2 and S3 show the numbersof PSM of all lysyl residues as unmethylated, mono-, di-, and trimethyllysines inOmpB(AN) and (K) that were catalyzed by RT0776, RP789, RT0776�N, andRP789�N.

Monomethylation Dimethylation TrimethylationNo

methylation

RT0776 0.093 0.001 0 0.883RT0776�N 0.324 0.024 0.011 0.641RP789 0.237 0.015 0.015 0.734RP789�N 0.313 0.034 0.036 0.617




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occurs at specific locations in OmpBs from R. prowazekiistrains RP22 and Breinl, and the high normalized fraction ofmonomethylation occurs in OmpB from R. prowazekii strainMadrid E, which is devoid of any trimethylation. The location,the state, and the normalized fraction of trimethylation andmonomethylation in OmpBs from the two strains RP22 andBreinl are remarkably similar (Fig. 5, A and C). The correlationcoefficient of the normalized fractions of trimethylations innative OmpBs from strains Breinl and RP22 is 0.90.

The amino acid sequences at trimethylation sites abideclosely to the recognition motifs found earlier based on the invitro methylation of rOmpB catalyzed by RT0101 and RP027-028. For example, Lys130 (KILN), Lys204 (KIVN), and Lys231

(KTIN) in OmpB of R. prowazekii conform to the KX(G/A/V/I)N motif (Fig. 2C). No other motifs were found using Prosite inExPASy (33). Similar to R. typhi OmpB, OmpBs from R. prowa-zekii strains RP22 and Breinl contain a cluster of highly tri-methylated lysine residues at Lys623, Lys634, Lys666, Lys710, andLys722. However, an additional cluster of highly trimethylatedsites occurs at Lys120 to Lys231 and two doublets of highly tri-methylated lysine residues in Lys309 to Lys414 with the tri-methylation normalized fraction reaching nearly 100% at thesesites. The amino acid sequences of OmpBs from R. prowazekiiand R. typhi differ at these lysine residues and may account forthe additional trimethyllysine in R. prowazekii. It is known thatR. prowazekii strains RP22 and Breinl are highly virulent,whereas R. typhi is mildly virulent (34, 35).

Both RP789 and RP027-028 are active in RP22 and Breinl asshown by the extensive mono- and trimethylation observed innative OmpBs. The nearly 100% normalized fraction of tri-methylation in OmpB from Breinl and RP22 supports the

notion that in vivo OmpB trimethylation and monomethylationare catalyzed by trimethyltransferase, likely via a processivemechanism, and by monomethyltransferase, respectively. Inthe trimethyltransferase-catalyzed reaction, the reaction inter-mediates monomethyllysine and dimethyllysine remain enzyme-bound. Under this situation, the monomethyltransferase does notparticipate in the formation of trimethyllysine.

In comparison to the OmpB from R. prowazekii Madrid E,which is devoid of trimethyllysine, OmpB from R. typhi con-tains a single cluster of highly trimethylated lysines. The obser-vation that trimethylation occurs in OmpBs from virulentstrains but not in OmpB from avirulent strain is in agreementwith earlier studies based on amino acid composition analysis(14). However, the present LC-MS/MS analyses show the loca-tion, state, and normalized fractions of the modified lysine res-idues. With this method we found multiple clusters of trimeth-yllysines in the highly virulent R. prowazekii strains RP22 andBreinl, a single cluster of trimethyllysines in the mildly virulentR. typhi, and none in the avirulent R. prowazekii Madrid E. Thenumber of cluster of highly trimethylated lysines in OmpBsclearly correlates with the degree of virulence of the four strainsof Rickettsia.

The correlation between the number of trimethyllysine clus-ters in OmpBs with the degree of virulence of the four differentrickettsial strains suggests that most likely the trimethyllysineclusters in OmpB are associated with rickettsial virulence. It isknown that the trimethylation of lysines may enhance cation-�electrostatic (36) and charge independent interactions (37). Forexample, trimethylation in calmodulin has been shown to mod-ulate NAD kinase (38). Similarly, trimethylation was shown topromote interaction with polynucleotides (39) and participate

FIGURE 4. Locations of trimethylation in native OmpB purified from R. typhi Wilmington coincide with those in RT0101-catalyzed trimethylation inrOmpB. A, normalized fractions of mono- (red), di- (green), and trimethylations (purple) at indicated lysine residues in native OmpB from R. typhi are shown.Native OmpB protein (2 �g) from R. typhi Wilmington was prepared and analyzed by LC-MS/MS as described under “Materials and Methods.” The PSM valueswere combined from two independent trials and are shown in supplemental Table S4. B, the locations and normalized fractions of trimethylation in nativeOmpB purified from R. typhi (solid bars, panel B) are compared with those of the trimethylation in OmpB(AN) and (K) catalyzed by RT0101 (open bars; as in Fig.2C). The correlation coefficient of the normalized fractions of trimethylation in native OmpB and those catalyzed by RT0101 is 0.76.




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in histone lysine methylation-mediated chromatin remodeling(40). A number of trimethyllysine-binding domains have beenfound in recent years (41). Association of methylated H3K9with HP1 mediates the condensation of nucleosomes to hetero-chromatin in gene silencing (42). Thus, the presence of clusterof trimethyllysine in a given protein would elevate its valencyand significantly enhance its affinity for protein-protein inter-action. Additionally, multiple trimethyllysine clusters couldfurther enhance protein-protein interactions. Therefore, it willbe of interest to identify the putative rickettsial and humanproteins that interact with the trimethyllysine clusters in OmpBthat in turn may lead to additional molecular links on rickettsialvirulence. It should be noted that the three-dimensional struc-ture of OmpB, either from crystallographic study or frommolecular modeling analysis, is not known at present. Thus, wecannot address the methylation profile in term of the structuralfeatures of OmpB.

OmpB Purified from the Avirulent Strain Madrid E Is Mini-mally Methylated by RT0101 and RP027-028—The observeddifferences in the normalized fraction of methylation betweennative OmpBs and in vitro methylated rOmpB prompted us toask whether the native OmpBs can be further methylated byMTs in vitro. We chose native OmpB of R. prowazekii Madrid E

as the substrate that contains predominantly monomethylly-sine and is devoid of trimethyllysine. We observed that nativeOmpB from Madrid E is minimally trimethylated by eitherRT0101 or RP027-028 (Fig. 6). Both trimethyltransferases cangenerate only a few trimethyllysines in native OmpB fromMadrid E. Our results show that only Lys352 in native OmpBwas significantly trimethylated by RP027-028. This observationindicates that the preexisting methylation of lysine in the nativeOmpB prevents further methylation of these lysine residues toform trimethyllysine that is otherwise readily produced inrOmpB catalyzed by RT0101 or RP027-028. Together, theseresults are consistent with the earlier suggestion that the OmpBtrimethylation catalyzed by RT0101 and by RP027-028 mayproceed via a processive mechanism in which its reaction inter-mediates monomethyllysine and dimethyllysine are present asenzyme-bound complexes.

Concluding Remarks—Methylation of OMPs has been impli-cated in contributing to bacterial virulence and pathogenesis.To investigate this further and to characterize methylation ofrickettsial OmpB, we carried out an in-depth analysis compar-ing methylation profiles in rOmpB catalyzed by four differentMTs and in native OmpB purified directly from virulent andavirulent bacteria, showing their profiles agree closely in loca-

FIGURE 5. Methylation in native OmpBs purified from R. prowazekii strains Breinl, RP22, and Madrid E. The locations and normalized fractions of tri- (A),di- (B), and monomethylation (C) in native OmpBs from R. prowazekii strains Breinl (blue), RP22 (red), and Madrid E (green) are shown. Native OmpBs purifiedfrom respective strains (2 �g each) were prepared and analyzed by LC-MS/MS as described under “Materials and Methods.” The PSM numbers of unmethylatedand mono-, di-, and trimethyllysine-containing peptides in OmpBs from R. prowazekii strains Breinl, RP22, and Madrid E are shown in supplemental Table S5.The PSM numbers were obtained from two, two, and three independent trials of OmpBs from Breinl, RP22, and Madrid E, respectively.




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tion and state of methylation. Our results suggest that mono-methylation of OmpB is carried out by monomethyltrans-ferases (RT0776 and RP789), and trimethylation is carried outby trimethyltransferases (RT0101 and RP027-028). The nearquantitative trimethylation of specific lysine residues found innative OmpB of virulent Rickettsia is consistent with the exist-ence of an efficient cellular system of trimethylation of OmpBin vivo. Unlike typical lysine MTs, rickettsial MTs can recognizeand methylate a diverse set of amino acid sequences. Our studyreveals recognition motifs of rickettsial trimethyltransferases,which could be used to predict methylation sites in otherOMPs. Additionally, heterologous MTs and N-terminal trun-cations of MTs significantly alter methylation profiles pro-duced in OmpB. Interestingly, the number of cluster of tri-methyllysines in OmpB correlates with the increasing virulenceof four rickettsial strains. Furthermore, our study reveals thattrimethylation may proceed via a processive mechanism suchthat monomethylation in OmpB could have an antagonisticeffect on trimethylation.

In summary, our study provides the first characterization ofmethylation of OMPs from Gram-negative bacteria. The newfindings on OmpB methylation may bring forward the develop-ment of new approaches of investigating the plausible linkbetween OMP methylation and bacterial virulence and raisethe possibility of targeting MT (43, 44) to advance new thera-peutic strategy against Rickettsia.

Acknowledgments—We acknowledge Dr. Nicholas Noinaj (NIDDK,National Institutes of Health) for many valuable comments duringthe manuscript preparation and thank Drs. Zhiwen Zhang and Hua-Wei Chen (Naval Medical Research Center) for help in preparingOmpB(AN) and OmpB(K).

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Wei-Mei Ching and David C. H. YangAmila Abeykoon, Guanghui Wang, Chien-Chung Chao, P. Boon Chock, Marjan Gucek,

OmpB Catalyzed by Lysine MethyltransferasesRickettsiaMultimethylation of

doi: 10.1074/jbc.M113.535567 originally published online February 4, 20142014, 289:7691-7701.J. Biol. Chem.

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Multimethylationof Rickettsia …OmpB proteins (2 g each) were separated by SDS-PAGE,...

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Transcript of Multimethylationof Rickettsia …OmpB proteins (2 g each) were separated by SDS-PAGE,...