Structural Insights into Substrate Recognition and ...

JBC Papers in Press. Published on July 29, 2016 as Manuscript M116.723460 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.723460

Structural Insights into Substrate Recognition and Catalysis in OmpB by Protein Lysine Methyltransferases from Rickettsia Amila H. Abeykoon1,¶, Nicholas Noinaj2,*, Bok-Eum Choi1,¶, Lindsay Wise1, Yi He3, Chien-Chung Chao4, Guanghui Wang5, Marjan Gucek5, Wei-Mei Ching4, P. Boon Chock3, Susan K. Buchanan6, and David C. H. Yang1,* 1

Department of Chemistry, Georgetown University, Washington DC, 20057 Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 3 Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, MD 20892 4 Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, Maryland 20910 5 Proteomics Core Facility, National Heart, Lung, and Blood Institute, Bethesda, MD 20892 6 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892 2

¶These authors contributed equally to this work Running Title – Structural insight into OmpB methyltransferases Key words: outer membrane protein, protein lysine methyltransferases, crystal structure, trimethylation, monomethylation, post-translational modifications, Gram-negative bacteria Abbreviations: OMP, outer membrane protein; MT, methyltransferase; PKMT, protein lysine methyltransferase; RpPKMT1, RP789 from R. prowazekii, RtPKMT2, RT0101 from R. typhi, AdoMet, Sadenosylmethionine (also known as SAM); AdoHcy, S-adenosylhomocysteine; SeMet, selenomethionine; PKMT2 are unusual in that their primary substrate appears to be limited to OmpB and both are capable of methylating multiple lysyl residues with broad sequence specificity. Here we report the crystal structures of PKMT1 from R. prowazekii and PKMT2 from R. typhi, both the apo-form and in complex with its cofactor AdoMet or AdoHcy. The structure of PKMT1 in complex with AdoHcy is solved to a resolution of 1.9 Å. Both enzymes are dimeric, with each monomer containing an AdoMet binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding and substrate interactions were examined using site-directed mutagenesis followed by steady-state kinetic analysis to ascertain their catalytic functions

ABSTRACT Rickettsia belong to a family of Gramnegative obligate-intracellular infectious bacteria that are the causative agents of typhus and spotted fever. Outer membrane protein B (OmpB) occurs in all rickettsial species, serves as a protective envelope, mediates host cell adhesion and invasion, and is a major immunodominant antigen. OmpBs from virulent strains contain multiple trimethylated lysine residues, while avirulent strain contains mainly monomethyllysine. Two protein lysine methyltransferases that catalyze methylation of recombinant OmpB at multiple sites with varying sequences have been identified and overexpressed. PKMT1 catalyzes predominantly monomethylation, while PKMT2 catalyzes mainly trimethylation. Rickettsial PKMT1 and 1

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*To whom correspondence may be addressed: David C. H. Yang, Email: [email protected] *To whom correspondence may be addressed: Nicholas Noinaj, Email: [email protected]

mediated in part by cellular proteins involved in the regulation of these processes.

in solution. Together, our data reveal new structural and mechanistic insights on how rickettsial methyltransferases catalyze OmpB methylation. INTRODUCTION Methylation of outer membrane proteins has been implicated in the rickettsial virulence. Rickettsia belong to a family of obligatory intracellular infectious bacteria that are the causative agents of typhus and spotted fever (1). The bacterial outer membrane of Rickettsia contains a major surface protein called OmpB that occurs in all rickettsial species and accounts for up to 15 % of total cellular proteins (2-4). OmpB has been shown to mediate host cell adhesion, attachment, and invasion (5-7). OmpB belongs to the family of autotransporters (8), and is a major immunodominant antigen (3). The precursor of OmpB consists of a signal peptide, a passenger domain, and a C-terminal -barrel domain (9). The passenger domain of OmpB has been shown to undergo methylation at its lysine residues and this methylation appears to associate with rickettsial pathogenicity and immunogenic response (10-15). OmpB was first found to be methylated by amino acid composition analysis and later confirmed using mass spectrometric methods. The levels of methylation of OmpB from several virulent and avirulent strains appear to correlate well with the virulence level of the strains. In-depth analysis of methylation profiles, using semi-quantitative integrated LC-MS/MS methods on the locations, states and levels of methylated lysine residues in OmpB purified from several rickettsial strains, revealed that (i) OmpBs from virulent strains contain clusters of highly trimethylated lysine residues, and (ii) OmpB from avirulent strain contains primarily monomethyllysine residues and no trimethyllysine (16). Posttranslational protein methylation has been shown to play a major role in regulating biological processes (17,18), ranging from chemotaxis to epigenetics (19-21), transcription regulation (22), translation (23) as well as cell signaling (24). If methylation of OmpB indeed causes virulence, rickettsial virulence could be

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Two classes of rickettsial protein lysine methyltransferases (PKMTs) have been identified and termed PKMT1 and PKMT2, via bioinformatic analysis, cloning, overexpression, and characterization in terms of their enzyme activity using recombinant OmpB fragments as the substrates (25). Both classes of PKMTs catalyze methylation of recombinant OmpB but show distinct levels of specificity and types of methylation. At distinct sites, PKMT1 catalyzes primarily monomethylation and producing a substantially lower levels of di- and trimethylated lysine, while PKMT2 catalyzes almost exclusively trimethylation at relatively specific sites (16). In recombinant fragments of OmpB, RpPKMT1 can catalyze monomethylation of its 39 lysyl residues and 14 of these residues can be trimethylated by RpPKMT2 (16). Homologous PKMTs have been found in more than forty different rickettsial species including RP789 from R. prowazekii (RpPKMT1), RT776 from R. typhi (RtPKMT1), RP027-028 from R. prowazekii (RpPKMT2), and RT0101 from R. typhi (RtPKMT2). The locations, states and levels of methylation in methylated recombinant OmpB fragments using these PKMTs and the methyl donor AdoMet were determined using LCMS/MS. The observed lysine methylation profiles from in vitro methylation correlate well with those found in native OmpBs purified directly from Rickettsia (16). The results indicate that virulent strains contain and express both PKMT1 and PKMT2, but the avirulent strain contains a frame shift mutation of PKMT2 gene and does not possess active PKMT2 (13). The primary substrate of rickettsial PKMTs appear to be limited to OmpB since these purified PKMTs fail to catalyze the methylation of histones or E. coli proteins (25). Furthermore, PKMT1 and PKMT2 exhibit unusually broad amino acidsequence specificity and produce distinct methylation profiles in OmpB. To our knowledge, with the exception of PrmA methyltransferase that catalyzes the trimethylation at multiple residues of ribosomal protein L11 (26) and of archaeal protein lysine methyltransferase that multi-methylates Cren7, a chromatin protein (27), there is no PKMT that

to a final resolution of 2.6Å and 3.1Å, respectively (Table 1). The RpPKMT1 structure consists of modeled residues Tyr43-Val553 of chain A and chain B, while the RtPKMT2 structure consists of modeled residues Pro14– Gly534 of chain A and chain B. For all structures, two molecules were found in the asymmetric unit related by 2-fold symmetry with significant interactions, indicating a possible homodimer.

In this study, we determined the crystal structures of RpPKMT1 and RtPKMT2, as well as the complexes with the cofactor, AdoMet or AdoHcy. Our structural data provide the first look of this family of PKMTs that specifically target OmpB, allowing us to identify conserved active site residues and a putative substrate binding cleft, and revealed their catalytic roles using sidedirected mutagenesis and enzyme kinetic analyses. Together, our data provide structural and mechanistic insights of rickettsial PKMTs and enable us to propose a model that describes the molecular basis for how the rickettsial PKMTs interact with AdoMet and OmpB for substrate specificity and catalysis.

The crystal structures of RpPKMT1 and RtPKMT2 are shown in Figure 2. The overall three dimensional folds of RpPKMT1 and RtPKMT2 are well conserved with an RMSD of 1.51 Å (Figures 2A-2C). The RpPKMT1 monomer contains four subdomains consisting of an N-terminal AdoMet binding domain (Tyr43Asn179, Tyr261-Tyr286, and Arg318-Ile331), a dimerization domain (Thr180-Phe260, Leu287Arg317), a middle domain (Asn332-Arg447), and a C-terminal domain (Ser448-Val553) (Figures 1, 2B). The alignments of the secondary structure and the domain boundaries revealed in the crystal structure of RpPKMT1 with its amino acid sequence are shown in Figure 1. Similar to RpPKMT1, the RtPKMT2 monomer consists of an N-terminal AdoMet binding domain (Ala14Thr152, Thr232-Phe258, Arg289-Lys306), a 153 231 259 dimerization domain (Leu -Gly , Ile Lys290), a middle domain (Ile307-Thr416), and a Cterminal domain (Lys417-Gly534). The four domains are spatially arranged as an open-palm where the AdoMet binding domain occupies the center of the palm, the dimerization domain orients as the thumb, and the middle and Cterminal domains together position as the fingers (Figure 2B, right panel). The AdoMet binding domain in both structures contains a well conserved core Rossmann fold, characteristic of type 1 AdoMet-dependent MTs (31).

RESULTS The RpPKMT1 and RtPKMT2 crystal structures Crystal structural analysis was employed to investigate the catalytic action of RpPKMT1 and RtPKMT2 in their methylation of OmpB. Figure 1 shows the amino acid sequence alignment of RpPKMT1 and RtPKMT2. The sequence of RpPKMT1 exhibits a 44% identity with that of RtPKMT2, and an N-terminal extension of 28 residues. RpPKMT1 catalyzes predominantly lysine monomethylation, while RtPKMT2 catalyzes almost exclusively trimethylation (16,25). To determine the crystal structure, the purified recombinant RpPKMT1, SeMet-labeled RpPKMT1, and RtPKMT2 were prepared and crystallized. All structures were solved in space group P21. Se-SAD (single wavelength anomalous dispersion) phasing was used to solve the structures of SeMet substituted RpPKMT1 to 2.9Å resolution. This structure was then used as a search model to solve the native structures of RpPKMT1 and RtPKMT2

RpPKMT1 and RtPKMT2 share an open cleft located along the base of the AdoMet binding domain, which may serve as the putative protein substrate binding site. A number of loops from each of the adjacent domains are ideally positioned to interact with protein substrates. The most prominent structural difference between RpPKMT1 and RtPKMT2 occurs in the C-terminal domain, where RtPKMT2 contains an elongated loop (His440 to Ser451) (Figures 2C and 3


catalyzes methylation at multiple sites of a protein in a manner similar to that exhibited by rickettsial PKMTs has been reported. Structural comparison of PKMT1 and PKMT2 may uncover insights as to their different product specificity. Comparative structural analyses with known structures of PKMTs (28-30) could reveal physical and chemical bases that underlie their unusual catalytic properties of rickettsial PKMTs.

2D). This extension is mediated by an insertion of six amino acid residues (Asn444-Met449) in RtPKMT2, as indicated in the sequence alignment in Figure 1. Interestingly, this loop is located in close proximity to the AdoMet binding domain and may play a role in enzyme-substrate interactions.

interior face interacts with the AdoMet binding domain. The structures of AdoHcy and AdoMet superimpose well and therefore they both bind at essentially the same site in RpPKMT1. Furthermore, structural comparison of apoRpPKMT1 with the AdoMet- and AdoHcybound complexes reveals few overall structural changes upon binding of the cofactor. Slight conformational changes in the side chains of Ser129 and Leu103 are observed moving towards the ligand as well as some slight changes along the dimerization interface. Overall no large domain shifts or conformational changes were observed between the apo and holo structures. Same results as RpPKMT1 were observed for RtPKMT2 in complex with AdoHcy. Catalytic and structural effects due to elongated loop insertion into RpPKMT1 and deletion from RtPKMT2 Structural analysis of RpPKMT1 and RtPKMT2 revealed the six-residue insertion, Asn444 to Met449, presents as an elongated loop in the C-terminal domain of RtPKMT2 (Figures 1 and 2D). The location of the elongated loop in RtPKMT2 and the amino acid residues in the stick model are shown in Figures 4A and 4B, respectively. This loop is ideally positioned along the AdoMet binding site and can also potentially interact with the protein substrate (Figure 4C). Since RpPKMT1 functions primarily as a monomethyltransferase and RtPKMT2 mainly catalyzes the trimethylation reaction, we hypothesized that this loop may account for the different methylation reactions catalyzed by the two PKMTs. Therefore, we explored the catalytic function of this loop first, by deleting residues Asn444 to Met449 in RtPKMT2 and second, by inserting the Asn444 to Met449 loop of RtPKMT2 into the corresponding location in RpPKMT1. Inserting the six-residue loop into RpPKMT1 showed a faster initial increase for the methylation process relative to that observed with the WT enzyme under the indicated experimental conditions (Figure 5A). Figure 5B shows that deletion of residues Asn444 to Met449 in RtPKMT2 led to an increase in the initial rate for the incorporation of the radioactive methyl group into the recombinant OmpB(AN) from [3H-Me]AdoMet. The apparent catalytic and Michaelis-

Crystal structures of RpPKMT1 in complex with AdoMet or AdoHcy The crystal structures of RpPKMT1 bound with AdoMet or AdoHcy were solved by crystal soaking and co-crystallization experiments, respectively. Both ligands were found to bind within the AdoMet binding domain along the conserved Rossmann fold (Figure 3A). The complex with AdoMet yielded a lower resolution structure with the AdoMet ligand showing disorder at the binding site, as evidenced by poor density along the carboxyl end of the AdoMet molecule (Figure 3B). However, the complex with AdoHcy was solved at 1.9 Å resolution with significantly more order in the ligand (Figure 3C) and surrounding residues. Figure 3D shows the network of amino acid residues in RpPKMT1 that interact with AdoHcy. Both ligands were found in extended conformations within the deep narrow elongated pocket formed along the base of the AdoMet binding domain (Figure 3A). The methyl-donor side of AdoHcy faces the open cleft, while the 4


The RpPKMT1 and RtPKMT2 crystal structures are arranged as a 2-fold symmetric dimer mediated by the mostly helical dimerization domain (Figure 2E). To determine whether the dimers observed in the crystal structures were the biological unit, SEC-MALS analysis was performed. The data show both PKMTs were found to also exist as dimers in solution (Figure 2F). The buried surface area of the dimers is 1540.6 Å2 and 1451.3 Å2 for RpPKMT1 and RtPKMT2, respectively, with further stabilization from multiple hydrogen bonds along the dimer interface. The overall electrostatics of the two structures differ in various regions and may contribute to interactions with substrate, however, further work is needed to confirm this hypothesis (Figure 2G).

Menten constants of RpPKMT1, the loopinserted RpPKMT1, RtPKMT2 and the loopdeleted RtPKMT2 were analyzed, using much lower enzyme concentrations, and the results are shown in Table 2. These data reveal that loop insertion into RpPKMT1 did not alter much the values of kcat, while the value of Km reduced noticeably. When the elongated loop was deleted from RtPKMT2, the values of both kcat and Km were elevated. Table 2 also shows that the catalytic efficiency (kcat/Km) for the loopcontaining methyltransferases is about two-fold higher than that observed in the absence of the elongated loop. Note that the comparative analysis shown in Figures 5A and 5B, and the observed values of kcat shown in Table 2 indicate that the rate of RpPKMT1-catalyzed monomethylation reaction is about two hundred folds faster than that of RtPKMT2-catalyzed trimethylation reaction.

localized perturbation of conformation induced by the removal of the loop. Catalytically essential residues at the AdoMet binding site

To resolve if the increase in the rate of methylation observed with the loop-deleted RtPKMT2 is attributed to the possibility that deletion of the elongated loop may partially convert the trimethyltransferase to catalyze the formation of monomethylated OmpB, we analyzed the methylation profiles of OmpB using LC-MS/MS methods (16). Figure 5C showed that the loop-insertion to RpPKMT1 did not elevate the production of trimethyllysine residues. Similarly the loop-deletion of RtPKMT2 also did not convert the enzyme from a tri- to a monomethyltransferase (Figure 5D). In essence, the elongated-loop does not play an important role in switching the enzyme from a mono- to a trimethyltransferase or vice versa. Circular dichroism and intrinsic fluorescence were used to monitor whether the loop mutations could induce significant conformational changes of PKMTs. Figure 6A shows that there is no appreciable CD spectral changes caused by the loop deletion from RtPKMT2 or insertion into RpPKMT1. However, notable changes of the intensity and the emission maximum wavelength of the intrinsic fluorescence of RtPKMT2 (Figure 6B) were detected due to loop deletion, suggesting that the observed changes in kcat and Km of the loopdeleted RtPKMT2 could be attributed to the

Similar to RpPKMT1, the crystal structure of RtPKMT2 reveals that Asn57, Asp74, Leu75, Ile102, and His118 are located at cofactor binding site found in the RpPKMT1-AdoHcy complex (Figure 4C), suggesting these residues may interact with AdoMet during catalysis. Consistent with this notion, alanine mutation of 5


Crystal structural analysis revealed that the cofactor AdoMet/AdoHcy forms a network of interactions with RpPKMT1 (Figure 3D). They include hydrogen bonds with residues Tyr48 (OH to carboxyl moiety of Hcy), Asp102 (β-carboxyl to cis-diol of ribose), His146 (backbone carbonyl to α-NH2 of Hcy), Gly79 (backbone carbonyl to αNH2 of Hcy), Ser129 (OH to N4 of adenine), and Ile130 (backbone N to N3 of adenine). Side chains of Ile130 and Leu103 stack from opposite sides of the adenine ring. The catalytic roles of these residues at the AdoMet binding site in RpPKMT1, were probed by mutating each of these residues to Ala. The single point mutants Y48A, E77A, N85A, D102A and H146A all exhibited no PKMT activity. The loss of PKMT activity in D102A and H146A mutants is likely due to the loss of critical hydrogen bonds between RpPKMT1 and AdoMet (Figure 3D). The apparent Km for AdoMet and OmpB(AN) and apparent kcat were determined for those mutants that retained their PKMT activity including, Y48F, L103A, I130A and C145A. As shown in Table 3, these mutations affect both the values of Km and kcat. Interestingly, the mutant Y48F exhibited a significant decrease in the kcat value, and increase in Km for AdoMet, revealing the effect of eliminating the hydrogen bond formation between OH of tyrosine residue and the carboxyl moiety of AdoMet. In addition, substituting Ile130 with Ala also yielded an increase in KmAdoMet and a moderate decrease in kcat. Together, these observations indicate that the amino acid residues located at the AdoMet binding site of RpPKMT1 are involved in both the binding of AdoMet as well as participating in the catalytic action of RpPKMT1.

by monitoring the change in its PKMT activity by site-directed mutagenesis (Figure 7B, Table 4). Mutation of Trp123, Ser149, Asn151, Arg163 and Phe291 to alanine eliminated their PKMT activity detectable under standard assay conditions, while mutants W156A, Y219A, and E208A retained low MT activity. These eight residues are located along the flat base of the AdoMet binding domain (Figure 7B). The observed inactivation and inhibition caused by mutation to Ala of these residues, distant to the methyl donor, imply that they interact with OmpB during the catalytic action of PKMT2. To further investigate the mechanism by which site-directed mutagenesis alters catalytic activity, the Km for OmpB(AN) and kcat of the highly active mutant, L211A, and the moderately active Y219A mutant were determined (Table 4). In addition, residues Tyr342, Tyr343 and Glu348 in the middle domain were also mutated to Ala to probe the possible involvement of the middle domain during the catalytic action of RtPKMT2. Our results (Table 4) show mutants Y219A and Y343A induced a significant and specific reduction in the catalytic rate constant, while mutants L211A, E348A and Y342A altered the values for both Km for OmpB(AN) and kcat.

Identification of the putative protein substrate binding site in RtPKMT2 To identify those residues that are important for protein substrate binding, we first aligned the amino acid sequences of four PKMTs (RpPKMT1, RpPKMT2, RtPKMT1 and RtPKMT2), which are known to methylate OmpB (Figure 1). We then mapped all fully conserved residues to the surface of the RtPKMT2 crystal structure with the rationale that those residues that are important for binding OmpB would be fully conserved in all four PKMTs. Based on this analysis, we were able to identify a number of solvent-exposed residues in proximity to the AdoMet binding site that we hypothesized may serve as the substrate binding site. Among them, residues in the elongated loop of RtPKMT2, which could potentially involve in OmpB interaction, are shown in Figure 7A. In addition, crystal structural analysis also shows that residues Tyr219, Trp123, Trp156, Phe291, and Tyr224 form a hydrophobic pocket facing the methyl group of AdoMet. This hydrophobic environment is expected to lower the local pKa and to enhance the nucleophilicity of the amino group to facilitate the methylation reaction.

Taken together, the catalytic effects from alanine scanning of amino acid residues along the base of the AdoMet binding domain (Figure 4C and 7B), in the middle domain (depicted in Table 4), and in the elongated loop of RtPKMT2 (Figure 4B) (shown in Table 2), imply that each of the mutated residues has the potential for either directly interacting with the enzyme-bound OmpB, or the mutated residues could induce an allosteric effect to alter OmpB binding affinity and/or the catalytic activity.

DISCUSSION Here we present the first crystal structures of PKMTs known to target specifically an OMP in Gram-negative bacteria. Our structural studies reveal a number of features shared between RpPKMT1 and RtPKMT2, and likely with other members of this family. This family has a unique overall fold compared to other families of MTs, albeit the AdoMet binding

The potential catalytic function of the amino acid residues in RtPKMT2 was examined 6


these residues led to drastic changes in the activity of RtPKMT2. Our experiment revealed that the point mutants of RtPKMT2, D74A, and H118A, exhibited no PKMT activity (results not shown), while the mutants, L75A and I102A remained active (Table 3). Steady state kinetics revealed that both Km and kcat of the active mutants were altered significantly from those found with the wild type RtPKMT2. With the exception for the relatively inactive RpPKMT1(Y48F), the effects on Km caused by point mutations on the residues at the AdoMet binding domain were comparable for RpPKMT1 and RtPKMT2. However, the value of kcat decreases with the RpPKMT1 mutants while increases with RtPKMT2 mutants. Clearly, mutation studies show these residues play critical roles in the methylation catalysis, in accord with their interactions with the cofactor at the AdoMet binding site found in the crystal structures.

domain is well conserved. PKMT1 and PKMT2 share four structural domains consisting of a core AdoMet binding domain, a dimerization domain, a middle domain, and a C-terminal domain. In comparison to other known structures of MTs in the Protein Data Bank, PKMT1 and PKMT2 possess a Rossmann fold, characteristic of type 1 MTs, while most structures of protein lysine MTs in the Protein Data Bank using SET domain in their active site, with the exception of PrmA (26) and the DOT1 family (29,31,32). Unlike known PKMTs, PKMT1 and PKMT2 contain the middle and C-terminal domains. Furthermore, both enzymes are dimers and this is likely a shared feature of this family.

modulates the formation of multimethylated OmpB. It should be pointed out that the crystal structures for both RpPKMT1 and RtPKMT2 reveal that their N-terminal sequences exist as disordered, and provide no structural basis of altering products.

RpPKMT1 catalyzes primarily monomethylation while RtPKMT2 catalyzes trimethylation (16), commonly known as product specificity. Our study reveals that the three dimensional structures of these enzymes are highly conserved. Given the structural similarity between the two PKMTs, it is intriguing that they exhibit such distinct product specificity. One major structural difference between RpPKMT1 and RtPKMT2 is the presence of a six residue insertion (Asn444 to Met449) in a Cterminal loop in RtPKMT2 that projects across the top of the open cleft as an elongated loop over where the AdoMet binding site is located. In our attempt to investigate whether this elongated loop of RtPKMT2 may participate in mediating the trimethyltransferase activity of RtPKMT2, we analyzed the methylation profiles of the OmpB catalyzed by RtPKMT2, loop-deleted RtPKMT2, RpPKMT1, and loop-inserted RpPKMT1. The results (see Figures 5C and 5D) reveal that this elongated loop does not play an important role in switching the enzyme from a mono- to a trimethyltransferase or vice versa.

Based on the sequence alignments of PKMTs that target OmpB, we identified the fully conserved residues that may mediate substrate binding. We then mapped these residues to the surface of the RtPKMT2 structure and used mutagenesis studies to identify a putative substrate binding site that locates in close proximity along the AdoMet binding site (Figure 7A). The AdoMet binding site is within the open cleft. Interestingly, the Ala mutations at the AdoMet binding site, Leu103, and Ile130, affects the Km value for OmpB(AN) (Table 3). In addition, RtPKMT2 mutants such as W123A, S149A, N151A, R163A, Y219A and F291A, shown in Figure 7B, drastically reduce their catalytic activity for OmpB(AN) methylation. Thus, mutation of conserved residues along this

RpPKMT1 contains an additional 28 Nterminal residues in the aligned sequences with RtPKMT2 (Figure 1). Deletion of the N-terminal residues from RpPKMT1 (RP789) or RtPKMT1 (RT776) does not inactivate its PKMT activity and causes a limited elevation of di- and trimethylation as monitored using the LCMS/MS method (16). This observation indicates that the N-terminal extension in PKMT1 partially 7


In addition to the elongated loop in RtPKMT2 and the N-terminal extension in RpPKMT1, it is intriguing to note that the two PKMTs also differ in their substitutions of five conserved Tyr residues in PKMT1 to Phe in PKMT2. Mechanistic studies of the SET domain in histone MTs using site-directed mutagenesis coupled with crystal structural analysis reveal that substituting a Tyr residue at the active site with a Phe attenuates hydrogen bonding to a structurally conserved water molecule adjacent to the Phe/Tyr switch and facilitates its dissociation. When this water molecule dissociates, it enables the side chain of monomethyllysyl residue to adopt a catalytically competent conformation and space to accommodate the formation of di- or trimethyllysine (33,34). The present crystal structures of PKMTs reveal that three of the five substituted Tyr residues, Tyr46, Tyr48, and Tyr175, are located in close proximity to bound AdoMet in PKMT1. Thus the crystal structures from the present study advance the likelihood that these Tyr and Phe residues may modulate their catalyzed reactions toward mono- and trimethylation by PKMT1 and PKMT2, respectively.

PKMT1 and RtPKMT2 is not known and remains to be investigated. In conclusion, rickettsial PKMT1 and PKMT2 catalyze multi-site methylation of OmpB. We determined the crystal structures of OmpB mono-methyltransferase RpPKMT1 and tri-methyltransferase RtPKMT2 and their respective complexes with the cofactor AdoMet/AdoHcy and carried out site-directed mutagenesis and biochemical studies to illuminate substrate binding and catalysis. The structural determination revealed that both PKMT1 and PKMT2 are dimers, contain a sevenstrand Rossmann fold for AdoMet-binding, and enfold a cleft. We identified residues that are essential for the cofactor binding and methyl transfer. Mutagenesis studies of PKMTs revealed that the elongated loop does not play an important role in switching the enzyme from a mono- to a tri-methyltransferase. This work provides an example of structurally distinct proteins that carry out a common function of AdoMet-dependent methyl transfer to lysyl residues in proteins. Together, these results could provide new insights for structure-based design of inhibitors against PKMTs (42,43), and facilitate the development of novel drugs against virulent Rickettsia.

PKMT1 and PKMT2 target a large number of lysyl residues with diverse amino acid sequences for multi-site methylation (16). With the exception of PrmA (26) and an archaeal Cren7 (27) PKMTs, most PKMTs with known crystal structures methylate a unique Lys residue located in specific sequence motifs. PKMTs have an open cleft at bound AdoMet, while the SET domain in histone MTs has a well-shielded AdoMet and employs a narrow channel for the side chain of Lys to relay the -amino group of methylating Lys residue (39-41). On the one hand, DOT1 histone MT, which is also a type 1 MT, catalyzes specifically the methylation of Lys79 of histone H3 (29). On the other hand, PrmA shows large domain movements mediated through a flexible linker between the catalytic and the substrate binding domains, which provides the structural bases for multisite methylation (26), OmpB PKMTs exhibit little or no conformational changes upon AdoMet binding. The mechanism of the multi-site methylation at diverse sequences catalyzed by

EXPERIMETNAL PROCEDURES Protein expression and purification The cloning, expression, and purification of RpPKMT1, RtPKMT2, OmpB(AN) (R. typhi OmpB residues 33-744), and OmpB(K) (R. typhi OmpB residues 745-1353) were performed as previously reported (25) with modifications. Briefly, codon-optimized RpPKMT1 and RtPKMT2 gene sequences carrying sequence encoding a TEV protease recognition site immediately upstream to the start codon, were subcloned at Nde1/Xho1 restriction sites into the pET28a expression vector (Novagen) downstream of the 6xHis tag coding sequence, and the plasmid was transformed into E. coli BL21(DE3) cells (Agilent Technologies). The cultures were induced with 0.04 mM IPTG and the mixtures were incubated overnight at 22°C with shaking at 250 rpm. Purification was 8


putative OmpB binding site would inactivate RtPKMT2. Together these results lead us to propose a working model for RtPKMT2 depicted in Figure 7C. It shows that the AdoMet binding site and an OmpB fragment threaded along its putative binding site. A fully extended substrate fragment peptide is positioned along this binding site to illustrate how OmpB may bind into the cleft. Crystallization attempts to capture a complex with a peptide-mimetic substrate have so far been unsuccessful. Figure 7C is our working model for PKMT2 showing the peptide substrate is present in an extended conformation that binds to the cleft in PKMT2. Note that examples of peptides in extended conformation binding to proteins have been reported in the literature; e.g. binding of SNAP-25 to the light chain of botulinum neurotoxin (35), complex formation between the octapeptides repeat of prion protein and the Fab fragment of the POM2 antibody (36), peptide substrate at the binding cleft of Hsp70 chaperone DnaK (36,37), and a number of histone peptides in complex with histone lysine methyltransferases (34,38). It is conceivable that PKMT binding to an extended conformation of OmpB could facilitate enzymatic methylation at multiple lysyl residues.

Initial crystals were screened using an inhouse X-ray diffractometer (Rigaku MicroMax007 HF microfocus x-ray generator, Raxis IV++ detector) with final data sets collected at either the GM/CA-CAT or SER-CAT beamline at the Advanced Photon Source at Argonne National Laboratory. All data were processed using HKL2000 (44) and statistics are summarized in Table 1. For experimental phasing, SeMet substituted RpPKMT1 was prepared using B834(DE3) cells (Merck Millipore) grown in SelenoMet Media (Molecular Dimensions) supplemented with SeMet (40 mg/l) and purified using the same protocol as for the native protein sample. The initial RpPKMT1 structure was solved by Se-SAD (AutoSol) and a starting model automatically built (AutoBuild) and refined using PHENIX (45,46). Molecular replacement was then used to solve the native and complex structures of RpPKMT1 and the RtPKMT2 native structure. A difference map was used to locate the AdoMet and AdoHcy ligands. All model building was performed using COOT (46,47), and final refinement performed using PHENIX and CCP4 (48). Figures were prepared using PyMOL (Schrödinger) and final editing performed with Adobe Illustrator. Root mean square deviation (RMSD) analysis was carried out using PyMOL (Schrödinger).

Protein Crystallization For crystallization, purified PKMTs were concentrated to 10 mg/ml, and a broad matrix screening performed with a Mosquito crystallization robot (TTP LabTech) using hanging drop vapor diffusion method and plates incubated at 21 °C. The final crystallization conditions were as follows: RpPKMT1 (0.1 M HEPES-NaOH, pH 7.5, 10 % v/v isopropanol, 20 % w/v polyethylene glycol 4000); RpPKMT1 complex with AdoMet (4 % v/v isopropanol, 0.1 M Bis-Tris propane, pH 9.0, 20% w/v polyethylene glycol monomethyl ether 5000); RpPKMT1 complex with AdoHcy (0.1 M Tris, pH 8.5, 20 % w/v polyethylene glycol 1000); SeMet-labeled RpPKMT1 (0.2 M sodium formate, 0.1 M Bis-Tris propane, pH 8.5, 20 % w/v polyethylene glycol 3350); RtPKMT2 and RtPKMT2 complex with AdoHcy (0.19 M CaCl2, 0.095 M HEPES-NaOH, pH 7.5, 26.6 % polyethylene glycol 400, 5 % v/v glycerol;). Crystals were harvested directly from the crystallization drop.

Site-directed Mutagenesis Mutagenesis was carried out using QuikChange Lightning mutagenesis kits (Agilent) in accord with the manufacture’s protocol. Primers for site-directed mutagenesis were designed using QuikChange Primer Design online tool (Agilent). Sequences of primers are available at request. Mutant plasmids were purified from overnight culture using a Qiaprep spin miniprep kit (Qiagen) and DNA sequences were verified by sequencing analysis (Genewiz Inc). PKMT activity assay PKMT activity was assayed by the incorporation of [3H-Me] into protein substrate OmpB(AN) using [3H-Me]-AdoMet as previously described (25).

Data collection and structure determination

Steady State Kinetics Analysis 9


performed using Ni-NTA affinity chromatography. The 6xHis tag was cleaved by incubating TEV-6xHis protease at 4°C overnight, and PKMT was flowed through of a second NiNTA column. The sample was then concentrated and chromatographed on a Sephacryl S100 HR gel filtration column (GE Healthcare) in the crystallization buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl). Protein in the peak fractions was concentrated to a final concentration of 10 mg/ml, and TCEP-HCl (Tris(2-carboxyethyl)-phosphine hydrochloride) was added to a final concentration of 5 mM. For SeMet labeled RpPKMT1, B834 competent cells (NEB) were used along with SelenoMethionine Medium Base plus Nutrient Mix (Athena ES, Baltimore, MD). Protein concentration was determined using a NanoDrop UV-Vis spectrophotometer. For the complexes of RpPKMT1 or RtPKMT2 with AdoMet or AdoHcy, PKMT was incubated with 5 mM AdoMet or AdoHcy (Sigma) in crystallization buffer for at least 60 min on ice prior to crystallization.

Initial rates of methylation catalyzed by WT or mutants of RpPKMT1 and of RtPKMT2 were determined from the linear portions of the time courses obtained with the indicated concentration of OmpB(AN) and varying concentrations of AdoMet, or at a constant AdoMet and varying concentrations of OmpB(AN). The reaction was monitored under standard assay conditions using the PKMT radioactivity assay at 37°C (25). The values of Km and kcat were determined by direct-curve fitting using KaleidaGraph (Synergy) and Sigmaplot. Recombinant OmpB(AN) contains multiple lysyl residues that are methylated under the assay conditions. In addition, multiple methyl groups can be incorporated into each ε-amino moiety of lysyl residues. The values of Km and kcat of various methylation reactions at different lysyl residues may vary in a wide range. Thus, the kinetic constants so obtained are the weighted averages of multiple methylation reactions catalyzed by PKMT and can only be considered as apparent Km and kcat. It should be pointed out that we could not use very high concentrations of OmpB(AN) for our kinetic measurements because OmpB(AN) is a membrane-binding protein, and it tends to form aggregates at high protein concentration in the absence of its membrane counterpart.

Biolabs) and 8.3 mM sodium phosphate at pH 8.0. After overnight incubation at 37oC, the reaction mixture was evaporated to 20 µl using SpeedVac and mixed with SDS sample buffer. The proteins were separated by SDS-PAGE, and the OmpB(AN) and OmpB(K) protein bands were excised from the gel and subjected to in-gel digestion followed with LC-MS/MS analysis as described (16). Circular dichroism measurements

and

fluorescence

Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

Mass spectrometry Integrated liquid chromatographytandem mass spectrometry and in-gel digestion with chymotrypsin were performed as previously described (16). Note the LTQ Orbitrap Elite mass spectrometer (Thermo Scientific, San Jose, CA) used in this study routinely yielded a mass error range of 3 ppm or less. This high mass accuracy allowed us to differentiate between trimethylation and acetylation or between dimethylation and formylation in our peptide mass analysis (16). Data analysis and calculation of normalized fraction were carried out as described (16)

Wild type RpPKMT1 and RtPKMT2 were tested for their oligomeric state using size exclusion chromatography with multi-angle light scattering (SEC-MALS) analysis (Wyatt Technology Corp). Protein samples in 1x PBS supplemented with TCEP or DTT were separated by HPLC (Agilent 1200 series) using a size exclusion column (Shodex KW-803) equilibrated in 1x PBS and with in-line UV, MALS, and refractive index (RI) detectors (DAWN Heleos II and Optilab reX, respectively, Wyatt Technology Corp) for molecular weight characterization. For each run, the protein sample in 100 μl (2 mg/ml) was injected and eluted at a flow rate of 0.5 ml/min. UV and MALS data were collected and analyzed using ASTRA software (Wyatt Technology Corp). The calculated molecular weights of the peaks are presented as mean with a 95% confidence interval (lower limit, upper

For LC-MS/MS analysis, OmpB(AN) (10 µg) and OmpB(K) (5 µg) were methylated separately using 10 µg of specified methyltransferase in 50 µl of reaction mixtures containing 3.2 mM AdoMet (New England 10


Protein samples for CD and fluorescence measurements were prepared in 5 mM NaH2PO4, and 50 mM KF, at pH7.5. All CD measurements were performed between 180 and 280 nm at 25°C using a 1 mm cuvette and a Jasco J710 CD spectropolarimeter. The scanning speed used was 50 nm/min, with an 8 sec response time, and 5 nm bandwidth. Each spectrum represents the average from 5 scans. The intrinsic fluorescence was measured using a FluoroMax-2 fluorimeter at 25°C. Protein emission spectra were obtained with the excitation wavelength set at 280 nm using a cuvette with 10 mm path-length. Each spectrum obtained was the averaged of five scans between 300-450 nm, with both the excitation and emission slits set at 5 nm.

limit). Bovine serum albumin (BSA, 66.5 kDa) was run as a control.

Conflict of Interest The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgements

Author Contributions AA performed mutagenesis and steady state kinetics, NN, AA and SB conducted crystallization and determined the crystal structures, YH expressed proteins for crystallography, BEC designed and analyzed steady state kinetics, LW made the CD and fluorescence measurements, CC and WC constructed the MT clones and prepared OmpB, GW and MG conducted LC-MS/MS analysis, DY, NN, SK and PBC designed experiments and DY, NN and PBC wrote and edited the manuscript. All authors reviewed and approved the final version of the manuscript.

11


The work was supported by Naval Medical Logistic Command award N62645 (to DY) and Work Unit Number (WUN) 6000.RAD1.J.A0310 (to NMRC). N.N. and S.K.B are supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases. Data were collected at SER-CAT beamline 22-ID and the GM/CA-CAT beamline 23-ID at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at http://www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

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Figure Legends

Figure 1. Amino acid sequence alignment of RpPKMT1, RpPKMT2, RtPKMT1 and RtPKMT2. Amino acid sequence alignment was obtained using ClusterW and ESPript (49). Identical residues are highlighted in blue and conserved residues are shown in red. The secondary structure shown above the sequences is based on the crystal structure of the RpPKMT1-AdoHcy complex. The structural domains consist of the AdoMet binding domain (green), the dimerization domain (yellow), the middle domain (blue) and the C-terminal domain (magenta).

Figure 3. The AdoMet binding site in RpPKMT1. (A) Rossmann fold in RpPKMT1 (in red) and the orthogonal view with a 90º rotation; (B) the electron density map of AdoMet in the RpPKMT1-AdoMet complex; (C) the electron density map of AdoHcy in the RpPKMT1-AdoHcy complex. In B and C, the electron density shown as blue mesh is of a A-weighted 2FO-FC map contoured at 1.0  and the electron density shown in magenta is of an FO-FC SA-omit map contoured at 3.0 . (D) The residues that interact directly with AdoHcy in the RpPKMT1-AdoHcy complex are shown using Maestro (Schrodinger). His residue with protonated -N is abbreviated as HIE. Figure 4. Spatial arrangements of mutated residues in the elongated loop and AdoMet binding site of RtPKMT2. (A) Superimposition of the C-terminal and AdoMet binding domains of RpPKMT1 (green) and those of RtPKMT2 (grey). (B) Depicts the elongated loop in RtPKMT2 with the amino acid residues shown in the stick model. The catalytic effects due to loop-deletion of RtPKMT2, and loop-insertion in RpPKMT1 are shown in Table 2. (C) Mutated residues in the AdoMet binding site are shown in the stick model (RpPKMT1 in green and RtPKMT2 in gray). The resulting effects of mutants that retained MT activity on the methylation of OmpB(AN) are shown in Table 3. Figure 5. Time courses of methylation and methylation profiles of OmpB catalyzed by PKMTs and their loop mutants. A. Time courses of methylation catalyzed by the RpPKMT1 and loop-inserted RpPKMT1. Time courses of OmpB(AN) methylation catalyzed by RpPKMT1 (●) or loop-inserted RpPKMT1 (○) were monitored by the incorporation of radioactive [3H-Me] AdoMet (0.16 mM) into 2 µM OmpB(AN) in 8.3 mM phosphate buffer (pH 8.3) at 37°C. The reactions were initiated by the addition of MT to a final concentration of 2 µM. The control contained all the reaction mixtures in the absence of OmpB(AN) (▼). Each time point represents the mean from two independent measurements with the error bar indicating S.D. B. Time courses of methylation catalyzed by the RtPKMT2 and loop-deleted RtPKMT2. Time courses of OmpB(AN) methylation catalyzed by RtPKMT2 (●) or loopdeleted RtPKMT2 (○) were monitored under the conditions described in A. The reactions were initiated by the addition of MT to a final concentration of 2 µM. The control all the reaction mixtures in the absence of enzyme (▼). Each time point represents the mean from two independent measurements with the error bar indicating S.D. C. LC-MS/MS analysis of methylation profiles in OmpB catalyzed by 15


Figure 2. The structural overview of RpPKMT1 and RtPKMT2. (A) Representative electron density (2Fo-Fc map at 1) for the RpPKMT1 structure; (B) Ribbon presentation of RpPKMT1 showing the AdoMet binding domain (green), the dimerization domain (yellow), the middle domain (blue), and the Cterminal domain (magenta), and the ribbon presentation with 90° rotation; (C) Superimposition of RpPKMT1 and RtPKMT2 (grey) monomers in ribbon presentation. The dashed box is enlarged in D. (D) Enlarged graph of the inset in (C) shows the elongated loop in the C-terminal domain of RtPKMT2 (grey), which is absent in RpPKMT1; (E) Superimposition of dimers of RpPKMT1 and RtPKMT2 (grey), and the 90° rotation view; (F) SEC-MALS of RpPKMT1 and RtPKMT2, which have expected monomeric molecular weights of 63345 and 61275, respectively; (G) Electrostatic potentials on the surfaces of RpPKMT1 and RtPKMT2.

RpPKMT1 and loop-inserted RpPKMT1. The normalized fractions of PSM (peptide spectral matches) values of mono- (top), di- (middle) and tri- (lower) methylated peptides from chymotrypsin in-gel digest of OmpB(AN) and (K) at indicated lysine residues on the x-axis in OmpB(AN) and (K) that were methylated by WT RpPKMT1 (blue) or loop-inserted RpPKMT1 (orange) as described in the Experimental Procedures are shown. The enzymatically methylated OmpB(AN) and (K) were analyzed using LC-MS/MS as described in Experimental Procedures. The PSM values were combined from 3 independent trials for each PKMT. (D) LC-MS/MS analysis of methylation profiles of OmpB catalyzed by RtPKMT2 and loop-deleted RtPKMT2. The same procedures as described in 5C legend were used to obtain the methylation profiles in OmpB catalyzed by RtPKMT2 (blue) and loop-deleted RtPKMT2 (orange) are shown.

Figure 7. A working model of the substrate binding in RtPKMT2. (A) Conserved residues are highlighted in blue. Residues in Site A may involve in substrate binding. The dashed circle indicates the location of the elongated loop. (B) Mutation of seven residues in Site A (in stick model) to Ala caused the substantial loss of enzyme activity, and Y219A retained 5% activity. (C) The working model of RtPKMT2-OmpB peptide complex showing the potential binding site with an extended peptide fragment in green. The AdoMet binding site and the putative OmpB binding site are indicated. The dashed circle indicates the location of the elongated loop in RtPKMT2.

16


Figure 6. Comparison of circular dichroism and fluorescence spectra of RpPKMT1, RtPKMT2, loop-inserted RpPKMT1, and loop-deleted RtPKMT2 mutants. (A) CD spectra were obtained with 1 M of RpPKMT1, loop-inserted RpPKMT1, RtPKMT2, or loop-deleted RtPKMT2 as described in the Experimental Procedures. The intensity is shown in mean residue ellipticity (MRE) in degcm2dmol-1. (B) Fluorescence spectra of 0.5 M of RpPKMT1, loop-inserted RpPKMT1, RtPKMT2, and loop-deleted RtPKMT2 were obtained as described in the Experimental Procedures.

Table 1: Crystallographic data collection and refinement summary. Rp PKMT1/

Rp PKMT1

Rp PKMT1/

Rp PKMT1/

Rt PKMT2/

Rt PKMT2/

Se-SAD

/Native

AdoMet

AdoHcy

Native

AdoHcy

0.979 P21 2

1 P21 2

1 P21 2

1 P21 2

1 P21 2

1 P21 2

Data Collection λ (Å) Space group Mol/ASU a, b, c (Å) a, , g (°) Resolution (Å) Completeness (%)* Redundancy

*

Rsym†* *

98.0, 62.9, 107.6 90, 100.42, 90 50-3.0 (3.11-3.0)

98.0, 62.1, 107.4 97.2, 66.9, 222.6 90, 100.88, 90 90, 89.82, 90 50-1.9 (1.97-1.9) 50-3.1 (3.21-3.1)

83.4, 91.0, 105.8 90, 112.3, 90 50-3.2 (3.31-3.2)

99.8 (99.9)

99.5 (96.9)

95.0 (89.7)

99.4 (97.3)

95.6 (96.0)

97.5 (90.4)

6.3 (6.2)

3.7 (2.9)

2.4 (2.0)

3.3 (2.8)

3.7 (3.6)

2.8 (2.4)

0.20 (0.91)

0.16 (0.69)

0.12 (0.52)

0.12 (0.72)

0.11 (0.77)

0.14 (0.69)

12.5 (2.3)

9.5 (1.9)

8.4 (1.5)

13.9 (1.5)

15.1 (1.7)

7.0 (1.4)

Refinement Resolution (Å)

48-2.6

20-3.0

20-1.9

48-3.1

48-3.2

0.21/0.24

0.20/0.23

0.20/0.25

0.23/0.28

0.24/0.28

Bonds (Å)

0.004

0.007

0.007

0.005

0.005

Angles (º) Protein atoms Ligand atoms Waters

0.856 8012 333

1.245 8069 54 -

1.037 8050 52 928

0.983 8165 2 -

1.166 8025 54 -

45.4 34.7

60.73 61.5 -

36.1 21 39.1

78.9 -

72.9 73.3 -

96.7 2.9 0.4 5DO0

92.6 6.8 0.6 5DPD

97.5 2.4 0.1 5DNK

90.1 9.1 0.8 5DOO

86.6 12.5 0.9 5DPL

§

R /Rfree

¶

B-factors Protein Ligand Waters Ramachandran Analysis ¥ Favored (%) Allowed (%) Outliers (%) PDB ID

Rsym = hkl,j (|Ihkl-|) / hkl,j Ihkl, where is the average intensity for a set of j symmetry related reflections and Ihkl is the value of the intensity for a single reflection within a set of symmetry-related reflections. § R factor = hkl(||Fo| - |Fc||)/hkl|Fo| where Fo is the observed structure factor amplitude and Fc is the calculated structure factor amplitude. ¶ Rfree = hkl,T(||Fo| - |Fc||)/hkl,T|Fo|, where a test set, T (5% of the data), is omitted from the refinement. ¥ Performed using Molprobity. * Indicates statistics for last resolution shell shown in parenthesis. †

17


I / σ (I)

100.1, 65.0, 108.8 98.5, 62.5, 107.9 90, 101.18, 90 90, 100.78, 90 50-2.9 (3.0 2.9) 50-2.6 (2.69-2.6)

Table 2. Apparent catalytic and Michaelis-Menten constants of RpPKMT1, the loop-inserted RpPKMT1, RtPKMT2 and the loop-deleted RtPKMT2. Initial rates of methylation catalyzed by PKMT were determined from the linear portions of the time courses obtained at 0.2 M WT, or loop-inserted PKMT1, and 0.5 M WT or loop-deleted PKMT2. The concentration of OmpB(AN) was varied from 1 µM to 10 µM at 0.16 mM AdoMet. The reaction was monitored under standard assay conditions for PKMT at 37°C.

KmAN (μM)

kcat (s-1)

kcat/KmAN (M-1s-1)

RpPKMT1 0.93 ± 0.42

(24 ± 2.8) × 10-3

26 × 103

Loop-inserted

0.51 ± 0.35

(26 ± 3.4) × 10-3

51 × 103

RtPKMT2

0.89 ± 0.56

(0.10 ± 0.08) × 10-3

112

Loop-deleted

3.2 ± 1.8

(0.16 ± 0.04) × 10-3

50

RtPKMT2

18


RpPKMT1

Table 3: Effects on the catalytic activity mediated by site-directed mutagenesis of selected residues at the AdoMet binding domain of RpPKMT1 and RtPKMT2. Apparent Michaelis-Menten and catalytic constants of active mutants, Y48F, C145A, L103A, I130A of RpPKMT1 and L75A and I102A of RtPKMT2 were evaluated using steady state kinetic analysis. The initial rates of methylation catalyzed by RpPKMT1 and its mutant enzymes were measured at 0.1 M of enzyme. The values of KmAdoMet were determined by varying [AdoMet] from 0.01 to 5 mM, at 5 M OmpB(AN). The values of KmAN and kcat were evaluated by varying [OmpB(AN)] from 1 to 10 M at 0.16 mM AdoMet. The apparent KmAdoMet, KmAN and kcat for RtPKMT2 were evaluated with 0.5 M enzymes. To determine the apparent KmAdoMet, the initial rates were measured by varying [AdoMet] from 0.04 to 0.4 mM and [OmpB(AN)] maintained at 2 M. To obtain the apparent KmAN and kcat, the initial rates were monitored by varying [OmpB(AN)] from 0.5 to 4 M at 0.16 mM of AdoMet.

KmAN (M)

0.04 ± 0.01

1.1 ± 0.34

(40 ± 7.4)x10-3

36x103

Y48F

0.48 ± 0.04

0.22 ± 0.02

(0.75 ± 0.01)x10-3

3.4x103

C145A

0.022 ± 0.015

1.5 ± 0.52

(32 ± 3.1)x10-3

21x103

L103A

0.04 ± 0.01

2.3 ± 0.75

(37 ± 4.0)x10-3

16x103

I130A

0.11 ± 0.04

1.6 ± 0.63

(13 ± 1.5)x10-3

8.1x103

0.16 ± 0.032

0.89 ± 0.56

(0.10 ± 0.08) × 10-3

112

-3

217 270

RpPKMT1

RtPKMT2

kcat (s-1)

kcat/KmAN (M-1s-1)

L75A

0.36 ± 0.069

0.83 ± 0.075

(0.18 ± 0.020) x10

I102A

0.35 ± 0.080

0.52 ± 0.075

(0.14 ± 0.022) x10-3

19


KmAdoMet (mM)

Table 4. Apparent kinetic constants of Ala mutants of RtPKMT2. The initial rates of methylation catalyzed by RtPKMT2 mutants, L211A, Y219A, E348A, Y342A, and Y343A were analyzed using 0.5 M mutant enzymes. The concentrations of OmpB(AN) were varied from 0.5 to 4.0 M at 0.16 mM AdoMet..

KmAN (M)

kcat (s-1)

kcat/KmAN (M-1s-1)

4.0 ± 1.6

(1.1 ± 0.52) x10-3

275

Y219A

0.39 ± 0.06

(0.047 ± 0.0017) x10-3

121

E348A

0.95 ± 0.20

(0.12 ± 0.032) x10-3

126

Y342A

2.0 ± 0.59

(0.29 ± 0.044) x10-3

145

Y343A

0.87 ± 0.06

(0.040 ± 0.001) x10-3

46

20


L211A

Figure 1


21

Figure 2


22

Figure 3


23

Figure 4


24

Figure 5

A


B

25

Figure 5 C


D

26

Figure 6

A


B

27

Figure 7


28

Structural Insights into Substrate Recognition and Catalysis in OmpB by Protein Lysine Methyltransferases from Rickettsia Amila H. Abeykoon, Nicholas Noinaj, Bok-Eum Choi, Lindsay Wise, Yi He, Chien-Chung Chao, Guanghui Wang, Marjan Gucek, Wei-Mei Ching, P. Boon Chock, Susan K. Buchanan and David C. H. Yang J. Biol. Chem. published online July 29, 2016

Access the most updated version of this article at doi: 10.1074/jbc.M116.723460 Alerts: • When this article is cited • When a correction for this article is posted

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