Non-hydrolysable stable analogues of τ-pHis and π-pHis have been designed using electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole <b>8 </b>and pyridyl amino amide <b>13</b>, respectively)<b> </b>were used as haptens to generate pHis polyclonal antibodies. <a>Both τ-pHis and π-pHis conjugates in the form of a BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis</a> were synthesized and characterized by <sup>31</sup>P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the generated pHis antibodies generated using phosphopyrazole <b>8 </b>and pyridyl amino amide <b>13</b> as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated using the phosphopyrazole <b>8</b> as a hapten were found to be selective for τ-pHis, and antibodies generated using the <a>pyridyl amino amide <b>13</b> </a>were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis
A highly versatile route to oxazole-5-amides is presented. Conversion of readily accessible oxazole-5-trifluoroacetamides into their Boc-protected 5-aminooxazole derivatives provides intermediates amenable to parallel amide synthesis utilizing a reliable, one-pot, acylation−deprotection procedure. During preparation of the N-Boc compounds from trifluoroacetamides, a competing intramolecular rearrangement giving rise to novel N-(oxazol-5-yl)-2,2,2-trifluoroacetimidates was identified, the extent of which is primarily determined by the choice of reaction conditions.
Abstract DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5′-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5′-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5′polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 5′-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) n repeat expansions in vivo , implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5′-flap specificity and catalysis, preventing genomic instability.
Seismic design requirements for an open-bottom box culvert were evaluated using simplified, pseudo-static, and numerical analysis methods. The open-bottom box culvert has features that resemble both a restrained-top soldier pile wall and a box culvert but without the bottom slab that normally provides a base to the box. Therefore, the analytical design procedures included: (1) modified version of the generalized limit equilibrium (GLE) method used for seismic design of above-ground retaining walls and (2) simplified racking analysis (SRA), which is commonly used for below-grade box structures with both base and lid. Seismic demands and deformations based on these two design methods are compared to results of dynamic finite element analyses. The study results indicate that estimated structure deflections and moment demand/capacity ratios are similar for the GLE and SRA design methods, but that neither the GLE nor SRA procedures capture the deformation mode observed from dynamic finite element analyses. The observed difference in response illustrates the limitations of simplified force- and displacement-based methods when applied to certain soil-structure interaction problems.
Abstract Malaria is one of the world’s most devastating parasitic diseases, causing almost one million deaths each year. Growing resistance to classical antimalarial drugs, such as chloroquine, necessitates the discovery of new therapeutic agents for successful control of this global disease. Here, we report the synthesis of some 6‐halo‐β‐carbolines as analogues of the potent antimalarial natural product, manzamine A, retaining its heteroaromatic core whilst providing compounds with much improved synthetic accessibility. Two compounds displayed superior activity to chloroquine itself against a resistant Plasmodium falciparum strain, identifying them as promising leads for future development. Furthermore, in line with previous reports of similarities in antimalarial and antiprion effects of aminoaryl‐based antimalarial agents, the 1‐amino‐β‐carboline libraries were also found to possess significant bioactivity against a prion‐infected cell line.
DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3′-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3′-flap. This recognition event allosterically signals hydrolytic removal of the 5′-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved 'wedge' residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated 'loop–wedge' mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3′-flap verification to function.
A field study comprised of experimental testing and statistical analyses was conducted to evaluate the Caterpillar machine drive power (MDP) and Geodynamik compaction meter value (CMV) compaction monitoring technologies applied to Caterpillar rollers. The study was comprised of three projects, all of which wee conducted at the Caterpillar Edwards Demonstration facility near Peoria, IL. The first project investigated the feasibility of using MDP applied to a Caterpillar self-propelled non-vibratory 825G roller. A test strip was constructed, compacted using the prototype 825G roller, and tested with in situ test devices. The second project also consisted of experimental testing on one-dimensional test strips. This project, however, used five cohesionless base materials, which were compacted using a CS-533E vibratory smooth drum roller with both MDP and CMV measurement capabilities. The independent roller measurements were compared and described in terms of soil engineering properties. The final project was conducted with only one cohesionless material. Four test strips (three uniform strips at different moisture contents and one with variable lift thickness) were constructed and tested to develop relationships between roller measurements and soil engineering properties. Using the material of the test strips, two-dimensional test areas with variable lift thickness and moisture content were then tested. Spatial analyses of the in situ measurements were performed to identify the spatial distribution of soil properties. The interpretation of the ground condition was then compared to machine output for evaluating the roller measurement systems and the proposed roller calibration procedure.
Heteromeric P2X 2/3 receptors are much more sensitive than homomeric P2X 2 receptors to αβ ‐methylene‐ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the αβ and/or βγ oxygen atoms were replaced by other moieties (including –CH 2 –, –CHF–, –CHCl–, –CHBr–, –CF 2 –, –CCl 2 –, –CBr 2 –, –CHSO 3 –, –CHPO 3 –, –CFPO 3 –, –CClPO 3 –, –CH 2 –CH 2 –, –C≡C–, –NH–, –CHCOOH–). We tested their actions as agonists or antagonists by whole‐cell recording from human embryonic kidney cells expressing P2X 2 subunits alone (homomeric P2X 2 receptors), or cells expressing both P2X 2 and P2X 3 subunits, in which the current through heteromeric P2X 2/3 receptors was isolated. ADP analogues had no agonist or antagonist effect at either P2X 2 or P2X 2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X 2 receptors, except βγ ‐difluoromethylene‐ATP, which was a weak agonist. At P2X 2/3 receptors, βγ ‐imido‐ATP, βγ ‐methylene‐ATP, and βγ ‐acetylene‐ATP were weak agonists, whereas αβ , βγ ‐ and βγ , γδ ‐bismethylene‐AP 4 were potent full agonists. βγ ‐Carboxymethylene‐ATP and βγ ‐chlorophosphonomethylene‐ATP were weak antagonists at P2X 2/3 receptors (IC 50 about 10 μ M ). The results indicate (a) that the homomeric P2X 2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b) that the heteromeric P2X 2/3 receptor is much more tolerant of αβ and βγ substitution; and (c) that a P2X 2/3 ‐selective antagonist can be obtained by introduction of additional negativity at the βγ ‐methylene. British Journal of Pharmacology (2003) 140 , 1027–1034. doi: 10.1038/sj.bjp.0705531
SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-l-methionine to the lysine ϵ-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-l-methionine, and its non-reactive analogs Aza-adenosyl-l-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-l-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product ϵ-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the ϵ-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine ϵ-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation. SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-l-methionine to the lysine ϵ-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-l-methionine, and its non-reactive analogs Aza-adenosyl-l-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-l-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product ϵ-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the ϵ-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine ϵ-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation. Protein lysine methylation has emerged as a prominent post-translational modification in gene regulatory and intracellular signaling pathways. In the nucleus, site-specific methylation of lysines within histones, transcription factors, and mitotic proteins governs a diverse array of processes within the nucleus including gene expression, DNA damage checkpoint control, cell cycle progression, and mitosis (1Dillon S.C. Zhang X. Trievel R.C. Cheng X. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (547) Google Scholar). In 2000, a breakthrough in our understanding of this modification occurred with the discovery of the first histone-specific protein lysine methyltransferases (PKMTs) 4The abbreviations used are: PKMT, protein lysine methyltransferase; AdoHcy, S-adenosyl-l-homocysteine; AdoMet, S-adenosyl-l-methionine; CH···O, carbon (hydrogen)-oxygen; dco, carbon-oxygen distance; dH, hydrogen-oxygen distance; ITC, isothermal titration calorimetry; MeLys, ϵ-N-monomethyllysine; Me3Lys, ϵ-N-trimethyllysine; Rubisco, ribulose-1,5 bisphosphate carboxylase/oxygenase; LSMT, large subunit N-methyltransferase; aLSMT, A. thaliana Rubisco LSMT; pLSMT, pea Rubisco LSMT. 4The abbreviations used are: PKMT, protein lysine methyltransferase; AdoHcy, S-adenosyl-l-homocysteine; AdoMet, S-adenosyl-l-methionine; CH···O, carbon (hydrogen)-oxygen; dco, carbon-oxygen distance; dH, hydrogen-oxygen distance; ITC, isothermal titration calorimetry; MeLys, ϵ-N-monomethyllysine; Me3Lys, ϵ-N-trimethyllysine; Rubisco, ribulose-1,5 bisphosphate carboxylase/oxygenase; LSMT, large subunit N-methyltransferase; aLSMT, A. thaliana Rubisco LSMT; pLSMT, pea Rubisco LSMT. (2Rea S. Eisenhaber F. O'Carroll D. Strahl B.D. Sun Z.W. Schmid M. Opravil S. Mechtler K. Ponting C.P. Allis C.D. Jenuwein T. Nature. 2000; 406: 593-599Crossref PubMed Scopus (2152) Google Scholar). These enzymes possess a conserved catalytic SET domain, a 120-residue motif that was named for three Drosophila gene regulatory factors, SU(VAR)3-9, E(Z), and TRX (3Tschiersch B. Hofmann A. Krauss V. Dorn R. Korge G. Reuter G. EMBO J. 1994; 13: 3822-3831Crossref PubMed Scopus (469) Google Scholar). This domain shares no apparent sequence or structural homology with other S-adenosyl-l-methionine (AdoMet)-dependent enzymes, establishing the SET domain family as a novel class of methyltransferases. This seminal discovery heralded the identification of a multitude of SET domain PKMTs, which methylate histone and non-histone substrates (1Dillon S.C. Zhang X. Trievel R.C. Cheng X. Genome Biol. 2005; 6: 227Crossref PubMed Scopus (547) Google Scholar).Since their identification, crystal structures of several SET domain PKMTs in complex with various substrates and products have been reported, yielding insights into the catalytic mechanism and protein substrate specificity of this methyltransferase family. These structures include the histone methyltransferases SET7/9 (4Jacobs S.A. Harp J.M. Devarakonda S. Kim Y. Rastinejad F. Khorasanizadeh S. Nat. Struct. Biol. 2002; 9: 833-838PubMed Google Scholar, 5Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. Gamblin S.J. Nature. 2003; 421: 652-656Crossref PubMed Scopus (302) Google Scholar, 6Kwon T. Chang J.H. Kwak E. Lee C.W. Joachimiak A. Kim Y.C. Lee J. Cho Y. EMBO J. 2003; 22: 292-303Crossref PubMed Scopus (105) Google Scholar, 7Chuikov S. Kurash J.K. Wilson J.R. Xiao B. Justin N. Ivanov G.S. McKinney K. Tempst P. Prives C. Gamblin S.J. Barlev N.A. Reinberg D. 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Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar), a non-histone-specific plant enzyme that trimethylates Lys-14 in the large subunits of the Rubisco holoenzyme (14Houtz R.L. Royer M. Salvucci M.E. Plant Physiol. 1991; 97: 913-920Crossref PubMed Scopus (23) Google Scholar). Together, these structures have elucidated the SET domain's novel β-sheet architecture, which harbors the AdoMet and protein substrate binding sites. A distinctive feature of this fold is the arrangement of the cofactor and substrate binding clefts, which are located on opposing faces of the SET domain. This configuration differs significantly from other methyltransferases, which generally bind AdoMet and the methyl acceptor in adjoining clefts (15Yeates T.O. Cell. 2002; 111: 5-7Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Structures of several PKMT ternary complexes have elucidated the path of methyl transfer between the two substrate binding sites (5Xiao B. Jing C. Wilson J.R. Walker P.A. Vasisht N. Kelly G. Howell S. Taylor I.A. Blackburn G.M. Gamblin S.J. Nature. 2003; 421: 652-656Crossref PubMed Scopus (302) Google Scholar, 8Couture J.F. Collazo E. Hauk G. Trievel R.C. Nat. Struct. Mol. Biol. 2006; 13: 140-146Crossref PubMed Scopus (136) Google Scholar, 9Couture J.F. Collazo E. Brunzelle J.S. Trievel R.C. Genes Dev. 2005; 19: 1455-1465Crossref PubMed Scopus (186) Google Scholar, 10Xiao B. Jing C. Kelly G. Walker P.A. Muskett F.W. Frenkiel T.A. Martin S.R. Sarma K. Reinberg D. Gamblin S.J. Wilson J.R. Genes Dev. 2005; 19: 1444-1454Crossref PubMed Scopus (152) Google Scholar, 11Zhang X. Yang Z. Khan S.I. Horton J.R. Tamaru H. Selker E.U. Cheng X. Mol. Cell. 2003; 12: 177-185Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 13Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar). The protein substrate intercalates in the substrate binding cleft as parallel β-strand, depositing the lysyl side chain into a narrow hydrophobic channel, which traverses the core of the SET domain. This channel terminates in a narrow aperture, which we refer to as the methyl transfer pore, that opens into the AdoMet binding cleft. During methyl transfer, the cofactor's methyl group is positioned into the pore for the SN2-based transfer reaction with the ϵ-amine group. Thus, the arrangement of the substrate binding sites promotes highly specific protein substrate recognition while permitting mono-, di-, or trimethylation of the lysine ϵ-amine group through iterative rounds of catalysis.Despite the insights obtained from these structural studies, the mechanism by which the methyl group is transferred between the cofactor and lysine binding sites is poorly understood. In prior structural studies of pLSMT, we reported carbon-oxygen (CH···O) hydrogen bonding between the methyl group of N-ϵ-monomethyllysine (MeLys) and a set of structurally conserved oxygen atoms that comprise the methyl transfer pore (13Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar). This category of hydrogen bonds can occur when an aliphatic or aromatic carbon atom becomes polarized by an adjacent covalently bonded heteroatom, acidifying the CH hydrogen atoms (16Derewenda Z.S. Lee L. Derewenda U. J. Mol. Biol. 1995; 252: 248-262Crossref PubMed Scopus (497) Google Scholar). CH···O hydrogen bonds are estimated to possess half the energy of conventional hydrogen bonds (17Vargas R. Garza J. Dixon D.A. Hay B.P. J. Am. Chem. 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Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar).Our initial observation of CH···O hydrogen bonding in the active site of pLSMT prompted us to further examine methyl group interactions in the SET domain, particularly within the methyl transfer pore. To this end, we have determined the structures of pLSMT in complex with AdoMet and its nonreactive analogs Sinefungin (also known as adenosyl-l-ornithine) and AzaAdoMet (see Fig. 1) and quantified the binding of these ligands to a homolog of the enzyme. In addition, we report the structure of pLSMT bound to ϵ-N-trimethyllysine (Me3Lys) and S-adenosyl-l-homocysteine (AdoHcy), representing the terminal product complex in lysine trimethylation. Taken together, the biochemical and structural data furnish direct evidence for CH···O hydrogen bonding to methyl groups bound within the SET domain and yield mechanistic insights into the catalytic functions of these interactions in PKMTs.EXPERIMENTAL PROCEDURESChemicals and Reagents—S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, l-lysine acetate, and Sinefungin were purchased from Sigma, and l-ϵ-monomethyllysine and l-ϵ-trimethyllysine were obtained from Bachem. AdoMet was further purified by anion-exchange chromatography as reported previously (39Chirpich T.P. Zappia V. Costilow R.N. Barker H.A. J. Biol. Chem. 1970; 245: 1778-1789Abstract Full Text PDF PubMed Google Scholar). l-AzaAdoMet was synthesized and purified according to the method of Thompson et al. (40Thompson M.J. Mekhalfia A. Hornby D.P. Blackburn G.M. J. Org. Chem. 1999; 64: 7467-7473Crossref Scopus (31) Google Scholar).Protein Cloning, Expression, and Purification—The DNA encoding residues 43-477 of Arabidopsis thaliana Rubisco LSMT (aLSMT) was amplified from an Arabidopsis cDNA library (Research Genetics) and cloned into the GATEWAY vector pDEST14 (Invitrogen) with a C-terminal hexahistidine tag and a tobacco etch virus protease cleavage site. Both aLSMT and pLSMT were overexpressed and purified as reported previously (12Trievel R.C. Beach B.M. Dirk L.M. Houtz R.L. Hurley J.H. Cell. 2002; 111: 91-103Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). After gel filtration purification in 20 mm Tris, pH 8.0, 150 mm NaCl, and 10 mm β-mercaptoethanol, the proteins were concentrated to ∼50 mg/ml, flash-frozen in liquid nitrogen, and stored at -80 °C.Isothermal Titration Calorimetry—Cofactor binding was measured using isothermal titration calorimetry (ITC) with a VP-ITC calorimeter (MicroCal, LLC). Experiments were performed in 20 mm sodium phosphate, pH 7.5, and 100 mm NaCl at 20 °C. Due to the aggregation of pLSMT during ITC, titration experiments were performed with aLSMT, a closely related ortholog of pLSMT, which shares an overall sequence identity of 64% with the pea enzyme and is 100% identical in its active site. Titrations were performed with various concentrations of ligand (1.0-5.0 mm) and enzyme (0.1-0.4 μm). Data were then analyzed using Origin 7.0 (OriginLab Corp.) with blank injections of the ligand into the buffer subtracted from the titrations prior to data analysis. All of the calculated binding curves have binding stoichiometries (N-values) between 0.8 and 1.0. Curve fitting errors for each titration experiment are reported in Table 1.TABLE 1ITC analysis of the binding of AdoMet and its analogs to aLSMTLigandKDΔHμmkcal/molAdoMet0.29 ± 0.02aDeviations represent curve-fitting errors calculated from the binding isotherms–11.0 ± 0.1aDeviations represent curve-fitting errors calculated from the binding isothermsSinefungin4.2 ± 0.1–8.3 ± 0.1AzaAdoMet>1000 ± 100<–3.9 ± 0.8AdoHcy21.2 ± 1.6–14.7 ± 0.3a Deviations represent curve-fitting errors calculated from the binding isotherms Open table in a new tab Crystallization, Structure Determination, and Modeling—Crystals of pLSMT in complex with AdoMet, Sinefungin-MeLys, AzaAdoMet-lysine, and Me3Lys-AdoHcy were obtained in 0.9-1.7 m sodium acetate at pH 6.8 as reported previously (13Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar). Crystals were serially dehydrated by increasing the sodium acetate concentration stepwise to 3.0 m, harvested in mother liquor supplemented with 15% 1,2-propanediol, and flash-frozen in liquid nitrogen. Oscillation images were collected on a Mar165 CCD detector at the COM-CAT 32-ID beamline at the Advanced Photon Source Synchrotron, Argonne, IL. Data were integrated and scaled using d*TREK (41Messerschmidt A. Pflugrath J.W. J. Appl. Crystallogr. 1987; 20: 306-315Crossref Scopus (457) Google Scholar). The structures of the various pLSMT complexes were solved using the pLSMT-AdoHcy-lysine coordinates (1P0Y.pdb) as a starting model (13Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar). Manual model building was carried out using O (42Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar), and structures were refined with CNS using the default stereochemical restraint parameters (43Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar). Simulated annealing Fo - Fc omit maps were calculated in the absence of ligands to accurately model them into the active site in an unbiased manner. Crystallographic data and refinement statistics for each structure are summarized in Table 2. For evaluating CH···O hydrogen bonding parameters, hydrogen atoms were modeled onto AdoMet using PRODRG2 (44Schuttelkopf A.W. van Aalten D.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4142) Google Scholar), and CH···O hydrogen bond distances and angles within the methyl transfer pore were measured with Swiss-PDB Viewer (45Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9472) Google Scholar) (Table 3).TABLE 2Crystallographic data and refinement statistics for pLSMT complexesAdoMetSinefungin-MeLysAzaAdoMet-LysAdoHcy-Me3LysCrystal parametersSpace groupI222Unit cell a (Å)131.45132.37132.63131.86 b155.86156.50159.49157.55 c263.61267.59268.51267.60 α, β, γ (°)α = β = γ = 90Asymmetric unit3 moleculesData collection statisticsResolution range (Å)13.0-2.4527.8-2.416.8-2.6015.0-2.45Total reflections517485356765314052447162Unique reflections9780710831185610101323Rsym (%)6.54.810.35.6I/σ (I)12.713.77.210.7Completeness (%)99.397.698.299.6Refinement statisticsResolution range (Å)13.0-2.4527.8-2.4516.7-2.6015.0-2.45Reflections (Fo > 2σ)94670965958527099453Final model Protein atoms10483104831048310483 Ligands81114111117 Water559450385390R-factorsaR-factor: Rworking = Σ||Fo| – |Fc||/Σ|Fo|; Rfree = ΣT||Fo| – |Fc||/ΣT|Fo|, where T is a test data set of 5% of the total reflections randomly chosen and set aside before refinement Rworking24.825.326.225.3 Rfree28.829.229.828.8Luzzati coordinate error (Å)0.420.420.470.42Cross-validated LuzzatiCoordinate error (Å)0.490.480.510.48r.m.s.br.m.s., root mean square Bond length (Å)0.0070.0070.0080.008 Bond angles (°)1.21.31.31.2Average B-factors (Å2) Protein68.170.971.766.7 Ligands52.558.692.981.3 Water62.063.970.060.2a R-factor: Rworking = Σ||Fo| – |Fc||/Σ|Fo|; Rfree = ΣT||Fo| – |Fc||/ΣT|Fo|, where T is a test data set of 5% of the total reflections randomly chosen and set aside before refinementb r.m.s., root mean square Open table in a new tab TABLE 3CH···O hydrogen bond parameters for the AdoMet methyl group within the three pLSMT molecules in the asymmetric unitCH···O H-bond acceptorMean dCOMean dHMean θÅÅdegreesSer-221 carbonyl oxygen3.2 ± 0.22.3 ± 0.2147 ± 3Asp-239 carbonyl oxygen3.2 ± 0.22.4 ± 0.2139 ± 4Tyr-287 hydroxyl group3.3 ± 0.12.3 ± 0.1155 ± 4 Open table in a new tab RESULTSCalorimetric Analysis of AdoMet, AdoHcy, AzaAdoMet, and Sinefungin Binding to aLSMT—To investigate the interactions of different chemical moieties within the methyl transfer pore of pLSMT, we determined the thermodynamic parameters for the binding of AdoMet, Sinefungin, and AzaAdoMet to a homolog of the enzyme using ITC. The latter two cofactor analogs were selected for analysis because they possess chemical moieties that differ with respect to the methyl group and sulfonium cation of AdoMet but are otherwise isosteric (Fig. 1). In AzaAdoMet, a tertiary nitrogen is substituted for the AdoMet sulfonium cation, rendering the methyl group inert for transfer. In Sinefungin, a δ-amine and a methylene moiety are incorporated in place of the cofactor's methyl group and sulfonium cation, respectively. The structural variations in these cofactor analogs provide a direct means for thermodynamically quantifying the interactions of distinct chemical groups within the enzyme's methyl transfer pore. Unfortunately, attempts to measure the binding of these ligands to pLSMT were unsuccessful due to its propensity to aggregate during ITC. To circumvent this issue, we determined the equilibrium dissociation constants (KD) and binding enthalpies (ΔH) using aLSMT, a closely related ortholog of the pea enzyme, which is not prone to aggregation during calorimetry experiments (Fig. 2).FIGURE 2ITC analysis of AdoMet binding to aLSMT. The top panel represents the titration of AdoMet into an aLSMT solution with the heat evolved (μcal/s) plotted versus time (min). The bottom panel illustrates the binding isotherm with the fitted binding curve.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Based on our knowledge of the active site, we postulated that Sinefungin would exhibit the greatest affinity for aLSMT due to conventional NH···O hydrogen bonding between the ligand's primary δ-amine group and the oxygens comprising the methyl transfer pore. Surprisingly, AdoMet binds to aLSMT with a 14-fold higher affinity than Sinefungin and exhibits a correspondingly higher enthalpy of binding to the enzyme (Table 1). Due to the isosteric nature of the ligands, these data suggest that the methyl transfer pore preferentially interacts with cofactor's methyl group in comparison with the δ-amine of Sinefungin. Conversely, AzaAdoMet binds over 3000-fold more weakly to aLSMT than AdoMet, indicating that the cofactor's sulfonium cation is essential for high affinity binding to the enzyme. The impaired binding of AzaAdoMet to aLSMT is most likely due to the replacement of the sulfonium group by a tertiary amine (Fig. 1), which exhibits a pH-dependent ionization state (see below). Finally, we measured the binding of AdoHcy to aLSMT and observed that it exhibits an ∼70-fold lower affinity for aLSMT when compared with AdoMet despite the greater binding enthalpy of the product versus the substrate. These differences imply that an entropic penalty is involved in the association of AdoHcy with the enzyme, which may derive from losses in the rotational freedom along the Cγ-Sδ and Sδ-C5′ bonds in the product upon binding. AdoMet does not incur this entropic penalty to the same degree because of the presence of its Cϵ-methyl group, which limits rotation around these bond axes. In summary, these data reveal that AdoMet is selectively recognized by aLSMT through specific interactions with the methyl and sulfonium groups of the cofactor.Structures of pLSMT Bound to AdoMet, AzaAdoMet, and Sinefungin—To elucidate the determinants of cofactor recognition by pLSMT, we have solved the crystal structures of the enzyme bound to AdoMet and its analogs. AdoMet was crystallized in a binary complex with pLSMT, whereas AzaAdoMet and Sinefungin were co-crystallized in ternary complexes with lysine and MeLys, respectively. These free amino acids serve as minimally competent substrates for pLSMT (13Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar) and facilitated crystallization of the Sinefungin and AzaAdoMet complexes. The pLSMT binary and ternary complexes crystallized in the same space group with similar unit cell dimensions and three molecules in the asymmetric unit, as reported previously (12Trievel R.C. Beach B.M. Dirk L.M. Houtz R.L. Hurley J.H. Cell. 2002; 111: 91-103Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 13Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar).In each of the pLSMT complexes, the cofactor adopts a horseshoe-shaped conformation within the AdoMet binding pocket, inserting either an amine or a methyl group into the methyl transfer pore (Fig. 3). This binding mode is structurally conserved in AdoHcy complexes of the enzyme (12Trievel R.C. Beach B.M. Dirk L.M. Houtz R.L. Hurley J.H. Cell. 2002; 111: 91-103Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 13Trievel R.C. Flynn E.M. Houtz R.L. Hurley J.H. Nat. Struct. Biol. 2003; 10: 545-552Crossref PubMed Scopus (99) Google Scholar) and other cofactor-bound SET domain structures (46Xiao B. Wilson J.R. Gamblin S.J. Curr. Opin. Struct. Biol. 2003; 13: 699-705Crossref PubMed Scopus (133) Google Scholar), illustrating that the variations in affinity exhibited by Sinefungin, AzaAdoMet, and AdoMet are not due to overt differences in their respective binding conformations. In the pLSMT-AdoMet complex, the cofactor's methyl group is coordinated in the methyl transfer pore through short distance interactions with the carbonyl oxygens of Ser-221 (3.3 Å) and Asp-239 (3.0 Å) and the hydroxyl group of Tyr-287 (3.3 Å), which is an invariant residue in SET domain PKMTs (Fig. 3A). Similarly, the AzaAdoMet methyl group engages in short range contacts with the carbonyl oxygen of Ser-221 (3.0 Å) and the hydroxyl group of Tyr-287 (3.0 Å) (Fig. 3B). In the Sinefungin complex, the ligand's δ-amine group is bound in the methyl transfer pore through NH···O hydrogen bonds with the hydroxyl group of Tyr-287 and the carbonyl oxygen of Asp-239, whereas the carbonyl oxygen of Ser-221 is too distant (3.6 Å) to engage conventional hydrogen bonding (Fig. 3C). To ascertain whether short range carbon-oxygen interactions are present in the active sites of other SET domain PKMTs, we examined the 1.7 Å resolution structure of the histone H3 Lys-4-specific methyltransferase SET7/9 bound to AdoMet (6Kwon T. Chang J.H
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