Báo cáo khoa học: Flexibility and communication within the structure of the Mycobacterium smegmatis methionyl-tRNA synthetase Henrik Ingvarsson and Torsten Unge potx

Flexibility and communication within the structure of the Mycobacterium smegmatis methionyl-tRNA synthetase Henrik Ingvarsson and Torsten Unge Department of Cell and Molecular Biology, Uppsala Biomedical Center, Uppsala University, Sweden Introduction With the aim of developing a new anti-tuberculosis drug, we selected methionyl-tRNA synthetase (MetRS) as a potential target. The aminoacyl-tRNA synthetases (aaRSs) have been considered as promising targets as a result of their central role in cell metabolism, the sig- nificant sequence differences between the prokaryotic and eukaryotic enzymes, the availability of enzyme material and access to structural information [1–3]. Furthermore, the potential of aaRSs as drug targets has been illustrated by the work on isoleucyl-tRNA synthetase. The isoleucyl-tRNA synthetase-specific inhibitor mupirocin has been shown to be active against the Gram-positive pathogens Staphylococ- cus aureus and Streptococcus pyogenes and is currently used as a topical antibiotic [4]. The c harging of th e tRNA species with their corre- sponding amino acids is catalyzed b y their cognate aaRSs, one for each amino acid [5]. However, not all bacterial pathogens possess all 20 aaRSs; only the eukaryotic spe- cies and a few eubacteria have a complete set [6–8]. The aminoacylated tRNA molecules bind to the A-site of the ribosome for participation in protein biosynthesis [9]. The aminoacylation is a two-step reaction [2]. In the first step, the corresponding amino acid is activated by Keywords adenosine; methionine; methionyl-tRNA synthetase; MetRS; Mycobacterium smegmatis Correspondence T. Unge, Department of Cell and Molecular Biology, Uppsala Biomedical Center, Uppsala University, Box 596, SE-751 24, Uppsala, Sweden Fax: +46 18 53 69 71 Tel: +46 18 471 50 62 E-mail: torsten@xray.bmc.uu.se Database Structural data are available in the Protein Data Bank/BioMagResBank databases under the accession numbers 2X1L for M. smegmatis MetRS:M/A and 2X1M for M. smegmatis MetRS:M (Received 17 May 2010, revised 15 July 2010, accepted 20 July 2010) doi:10.1111/j.1742-4658.2010.07784.x Two structures of monomeric methionyl-tRNA synthetase, from Mycobac- terium smegmatis, in complex with the ligands methionine ⁄ adenosine and methionine, were analyzed by X-ray crystallography at 2.3 A ˚ and at 2.8 A ˚ , respectively. The structures demonstrated the flexibility of the multidomain enzyme. A new conformation of the structure was identified in which the connective peptide domain bound more closely to the catalytic domain than described previously. The KMSKS(301-305) loop in our structures was in an open and inactive conformation that differed from previous structures by a rotation of the loop of about 90° around hinges located at Asn297 and Val310. The binding of adenosine to the methionyl-tRNA synthetase methionine complex caused a shift in the KMSKS domain that brought it closer to the catalytic domain. The potential use of the adenosine-binding site for inhibitor binding was evaluated and a potential binding site for a specific allosteric inhibitor was identified. Abbreviations aaRS, aminoacyl-tRNA synthetase; CAPS, 3-cyclohexylaminopropane-1-sulfonic acid; CP, connective peptide; MetRS, methionyl-tRNA synthetase; MetRS:M, methionyl-tRNA synthetase in complex with methionine; MetRS:M ⁄ A, methionyl-tRNA synthetase in complex with methionine ⁄ adenosine; NCS, noncrystallographic symmetry; PDB ID, Protein Data Bank identification. FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS 3947 the formation of an aminoacyl–adenylate complex. In the second step the amino acid is linked to the 3¢-end of the cognate tRNA molecule through the formation of an ester bond. The aaRSs have been divided into two classes based on the presence of certain conserved motifs and also on the structural framework supporting the active site [10–12]. Class 1 aaRSs are characterized by the amino acid sequence motifs HIGH and KMSKS, and by having a catalytic domain with a classical Rossmann- fold topology [13–16]. Class 2 aaRSs have their active- site residues located in an antiparallel b-sheet [10]. Each class consists of about 10 aaRSs; these are fur- ther divided into three subclasses – a, b and c – based on their structures and sequences [17]. A modified clas- sification, which includes structural data, has been pro- duced [18]. MetRS belongs to class 1a, together with isoleucyl-, leucyl-, valyl-, cysteinyl- and arginyl-tRNA synthetases. The MetRS molecules occur either in monomeric or dimeric forms. The dimeric forms contain a dimerization domain appended to the C-ter- minus [19,20]. The MetRS structure from Mycobacte- rium smegmatis studied here is monomeric, has only one knuckle in its connective peptide (CP) domain and no coordinated zinc ion. The MetRS core unit consists of four domains (Fig. 1) [21]: the catalytic domain, the CP domain, the KMSKS domain and an anticodon-binding domain (anticodon domain). The catalytic domain contains the binding pockets for the substrates methionine, ATP and the 3¢-CCA end of tRNA Met . In this study we present the unique features of the M. smegmatis MetRS in complex with methionine (MetRS:M; 2.8 A ˚ resolution) and methionine ⁄ adeno- sine (MetRS:M ⁄ A, 2.3 A ˚ resolution). Initially, our study of the mycobacterial MetRSs included the enzyme from Mycobacterium tuberculosis as well as that from M. smegmatis, but because of solubility problems with the M. tuberculosis enzyme, our study analyzed only the M. smegmatis variant. However, the sequence similarity between M. tuberculosis MetRS and M. smegmatis MetRS is high (74% identity) and the active-site resi- dues are identical, except for two residues in the ATP- binding site. We describe, in detail, the binding modes of the methionine, the adenosine and the residues involved. In order to evaluate the possibilities for designing a mycobacterial-specific competitive inhibitor, the differences in the active site relative to the human mitochondrial variant were also described. The implica- tions of the tight binding of methionine and adenosine were discussed, together with the functional significance of the structural shift of the entire KMSKS domain that is induced by addition of the adenosine molecule. The KMSKS domain of the class 1a MetRSs con- tains the conserved sequence Lys-Met-Ser-Lys-Ser [12,15]. In the M. smegmatis MetRS structure this sequence is located in a loop from residues 297 to 310. The structural information currently available shows that this loop is highly flexible and adopts different conformations (which may have different functions), depending on the composition of the aaRS complex (apo, adenylate or tRNA complex) and crystal-packing interactions. Data on the structures and sequences we used for comparisons are summarized in Table 1. The Fig. 1. An overall view of the crystallographic model of Mycobacte- rium smegmatis MetRS:M ⁄ A. The structure is divided into four domains: the catalytic domain in red, the CP domain in green, the KMSKS domain in yellow and the anticodon domain in blue. The intervening helices a2 and a8 in the catalytic domain are colored cyan. The linking p-helix a13 between the KMSKS domain and the anticodon domain is purple. The two ligands (methionine and aden- osine) bound to the active site are shown in gray. Crystal structure of M. smegmatis MetRS H. Ingvarsson and T. Unge 3948 FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS second lysine residue in the KMSKS sequence has pre- viously been predicted to be involved in coordination of the transition state [22]. Analysis of the Escherichi- a coli glutaminyl-tRNA synthetase tRNA Gln complex [E. coli GlnRS:tRNA Gln ; Protein Data Bank identifica- tion (PDB ID): 1QTQ] revealed that the MSK Lys residue (amino acid 270 in E. coli GlnRS:tRNA Gln ) hydrogen bonds to the adenylate phosphate and interacts via a water molecule with the 3¢-end of the ribose of the acceptor stem [23]. The positioning of this Lys residue is supported by the interaction of the methonine residue with the adenylate-adenine group and the hydrophobic residues making up the pocket for the methionine side chain. This arrangement of the MSK methionine residue is also present in the Aquifex aeolicus MetRS tRNA Met complex (A. aeolicus Met- RS:tRNA Met , PDB ID: 2CT8) [24]. In the A. aeolicus structure, however, the tRNA acceptor stem is disor- dered and the MSK lysine residue is not in contact with the adenylate phosphate. The structures of the class 1 aaRS enzymes show a small variation in struc- ture of the KMSKS loop and that the loop is in a con- formation that positions the MSK Lys residue close to the catalytic site. This is shown in the structural studies of E. coli MetRS [25], Thermus thermophilus leucyl-tRNA synthetase [26] and Methanocaldococcus jannaschii tyrosyl-tRNA synthetase [27]. In the M. smegmatis MetRS structures presented here, the loop containing the KMSKS sequence is trapped in an open and inactive conformation, which is the result of a 90° rotation compared with previ- ously described structures. The possible functional significance of this conformation is discussed, as is the new structural situation that this conformation opens for allosteric inhibition of the enzyme. The pos- sibilities for design of a competitive inhibitor are also discussed. These structures exhibit a new structural feature of the CP domain. In one of the complexes, the MetRS molecule is trapped in a conformation where the CP domain is in a tighter contact with the catalytic domain than has been observed earlier. By comparison with the A. aeolicus MetRS:tRNA Met anticodon domain we identified the three conserved residues Trp433, Arg363 and Asn359 (M. smegmatis MetRS) that, together with four more residues, are involved in the recognition of the anticodon triplets CAU in initiator and elongator tRNA Met (tRNA Met f and tRNA Met m , respectively). Results Crystal properties The crystals of M. smegmatis MetRS:M ⁄ A and M. smegmatis MetRS:M belong to the space groups C2 and R32, respectively. M. smegmatis MetRS:M ⁄ A has three molecules (A, B and C), and M. smegmatis MetRS:M has one molecule, in the asymmetric unit. Residues 2–512 out of 515 residues in total could be built in the A molecule, whereas in the B and C mole- cules, there was no electron density for residues 124– 160 and residues 123–158, respectively. Crystal packing contacts of the A molecule stabilize the structure of these flexible regions, which belong to the CP domain. See Table 2 for data collection and refinement statistics. Table 1. Sequence identities between MetRSs, including the catalytic domain, the CP domain, the KMSKS domain and the anticodon domain from the species referred to in the Introduction, the Results and the Discussion. The numbers denote percentage identity. The PDB IDs of the MetRSs, with their corresponding ligands, are shown. aaRS Organism PDB ID Ligands Sequence identity M. smegmatis (%) M. tuberculosis (%) MetRS M. tuberculosis Not available – 74 – A. aeolicus 2CT8 Methionyl-sulfamoyl-adenosine, tRNA Met 44 43 T. thermophilus 1A8H Zn ion 39 41 T. thermophilus 2D54 Zn ion 39 41 Human (mitochondrial) Not available – 36 38 E. coli 1F4L Met, Zn ion 25 25 P. abyssi 1RQG Zn ion 28 27 GlnRS E. coli 1QTQ Glutaminyl-sulfamoyl-adenosine, tRNA Gln sulfate ion –– LeuRS T. thermophilus 1H3N Leucine, leucyl-sulfamoyl-adenosine, sulfate ion, Zn ion –– TyrRS M. jannaschii 1J1U tRNA Tyr -tyrosine, Mg ion – – H. Ingvarsson and T. Unge Crystal structure of M. smegmatis MetRS FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS 3949 In the crystals of both M. smegmatis MetRS:M ⁄ A and M. smegmatis MetRS:M, the crystallization buffer component 3-cyclohexylaminopropane-1-sulfonic acid (CAPS), plays a crucial role for crystal formation in that it coordinates two symmetry-related molecules in the crystal lattice. The CAPS molecule binds to a shal- low pocket situated in the junction between the C-terminal end of helix a4 of the catalytic domain, the N-terminal end of strand b4 and helix a5, and the C-terminal end of strand b8 belonging to the CP domain (Figs 1 and 2). Each MetRS binds one phos- phate molecule coordinated by the main-chain amide group of Asp189 in the N-terminal part of helix a6 from the CP domain and Arg288 positioned in the N-terminal end of b10 from the catalytic domain. The catalytic domain The catalytic domain is an a ⁄ b domain which is divided into two segments: the N-terminal part (residues 1–115) and the C-terminal part (residues 229–292) (Fig. 1). The inner core of the domain consists of a Table 2. Data collection, crystal parameters and refinement statistics for Mycobacterium smegmatis MetRS. Data set M. smegmatis MetRS:M ⁄ A (PDB ID: 2X1L) M. smegmatis MetRS:M (PDB ID: 2X1M) Data collection Wavelength (A ˚ ) 1.038 1.038 Resolution range (A ˚ ) 30.0–2.3 (2.42–2.30) 20.0–2.8 (2.95–2.80) No. of measured reflections 384352 (50671) 116080 (16911) No. of unique reflections 93306 (13381) 15164 (2221) Average multiplicity 4.1 (3.8) 7.7 (7.6) Completeness (%) 97.8 (96.1) 98.7 (99.4) Mean I ⁄ r(I) 11.8 (3.2) 17.8 (5.1) R merge a (%) 9.3 (38.1) 8.6 (36.3) R p.i.m b (%) 5.1 (21.6) 3.3 (13.9) Crystal parameters Solvent content (%) 61.1 54.4 Matthews coefficient, V M (A ˚ 3 ÆDa )1 ) 3.2 2.7 No. of molecules in the asymmetric unit 3 1 Space group C2 R32:H Unit-cell lengths (A ˚ ) a = 155.9, b = 138.9, c = 123.3 a = 210.0, b = 210.0, c = 73.9 Unit-cell angles (°) a = c = 90, b = 124.8 a = b = 90, c = 120 Mosaicity (°) 0.81 0.57 Refinement statistics Resolution range (A ˚ ) 30.0–2.3 20.0–2.8 No. of reflections used in working set 88634 14405 No. of reflections used in test set 4670 756 R-factor (%) 21.8 21.0 R free (%) 24.9 24.9 No. of nonhydrogen atoms 12263 4120 No. of solvent water molecules 484 16 Mean B factor for protein atoms Protein atoms (A ˚ 2 ) 23.8 47.0 Ligand atoms (A ˚ 2 ) Methionine 15.5 28.9 Adenosine 42.2 – CAPS c 32.2 46.1 Dihydrogen phosphate c 23.1 41.2 Water atoms (A ˚ 2 ) 24.2 31.8 Ramachandran plot outliers d (%) 0 0 rmsd from ideal values e Bond lengths (A ˚ ) 0.006 0.005 Bond angles (°) 0.89 0.81 a R merge = R h R l ŒI hl ) ÆI h æŒ ⁄R h R l ÆI h æ, where I hl is the lth observation of reflection h. b R p.i.m = R hkl [1 ⁄ (N ) 1)] 1 ⁄ 2 R i |I i (hkl) ) ÆI(hkl)æŒ ⁄R hkl R i I(hkl). c Component of the crystallization condition. d PROCHECK (CCP4i program suite) – disallowed regions [37]. e Calculated for the protein using ideal values [50]. Crystal structure of M. smegmatis MetRS H. Ingvarsson and T. Unge 3950 FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS b-sheet built up by five parallel b-strands: b1 (residues 4-9), b2 (residues 42–49), b3 (residues 91–94), b9 (residues 258–263) and b10 (residues 289–292). The b-strands are interconnected by seven a-helices: a1 (res- idues 19–39), a2 (residues 53–64), a3 (residues 66–85), a4 (residues 97–114), a7 (residues 228–241), a8 (resi- dues 248–255) and a9 (residues 267–282). In MetRS structures determined previously, helix a4 has been assigned to either the catalytic domain or the CP domain. Here we include a4 in the catalytic domain. Specific features for some of the class 1a synthetases are additional helices in the Rossmann-fold structure [28]. In M. smegmatis MetRS, two helices (a2 and a8) are inserted. Helix a2 connects the b2 strand with helix a3, and helix a8 connects helix a7 with strand b9 (Fig. 1). The active site is positioned at the C-terminal edge of the sheet formed by the five parallel b-strands and the N-terminal ends of the helices a1, a7 and a9. The methionine-binding site The active site in MetRS contains the binding pockets for methionine and ATP. The methionine pocket is a tight cavity that only exposes the carboxylate group of the methionine ligand. The cavity, which is composed of 12 residues, is mainly hydrophobic (Fig. 3A). The atoms close-packing (within 3.8 A ˚ ) to the sulfur atom of methionine are Ce and Ne of His270, OH of Tyr237, Cb of Ala10 and N of Ile11 – thus three polar and two hydrophobic interactions. The free carboxyl- ate oxygen atoms form hydrogen bonds to three water molecules and the 5¢-hydroxyl of the adenosine mole- cule. The Tyr13 aromatic ring covers the positively charged amino group of the methionine ligand and partly covers the carboxylate group. Residue Trp230 stacks from the side. The position of Tyr13 is guided by a tight polar interaction to Trp230 through close-packing of the OH and Ne atoms. The impor- tance of Tyr13 and Trp230 residues for the methionine binding is indicated by the fact that they are con- served, and by the safe positioning of Trp230 through face-to-face stacking against Phe269 on the opposite side, and close-packing to Glu131, Tyr228 and Ile266. The ATP-binding site In our attempts to form different active-site complexes, we managed to form a complex with methionine alone, and methionine and adenosine, but not with adenosine alone. The structure of the methionine ⁄ adenosine complex shows that methionine binds to the ribose part of adenosine, and that residues Ala10 and Ala12 have close-packing interactions with both methionine and the ribose. The ATP-binding site is a deep groove rather than a cavity. The groove comprises 14 residues in the catalytic domain, including the conserved HIGH(19– 22) sequence, and three residues from the KMSKS domain (Fig. 3B). Binding of the adenosine molecule leads to re-arrangement of the site residues. The adenine moiety is oriented through a hydrogen bond from N6 to O of Leu295, face-to-face stacking against Trp294, end-to-end stacking against the two histidines 19 and 22, and close-packing interactions to Gly21. Analysis of the catalytic-site amino acid sequences General analysis of catalytic-site sequence conservation has been carried out by Serre et al. and Landes et al. [16,29]. In the present study, in order to evaluate the catalytic site as a drug target, comparison of the Pro498 Pro379 Tyr452 Arg495 Phe382 Wat Wat Arg174 Ala170 Tyr171 Asp116 Arg167 Ala114 Gly115 Fig. 2. CAPS molecules are found in the contact points between the symmetry- related proteins in the lattices of the Mycobacterium smegmatis MetRS:M and the Mycobacterium smegmatis MetRS:M ⁄ A crystals. Green residues belong to the catalytic domain and to the CP domain. Residues in cyan belong to the anticodon domain of a symmetry-related protein. The interactions holding the CAPS in its place are mainly of a close-packing type. The dotted green lines represent electrostatic or hydrogen bonds. Wat denotes water molecule. H. Ingvarsson and T. Unge Crystal structure of M. smegmatis MetRS FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS 3951 M. smegmatis sequence with the human mitochondrial (Hs-mit) and cytoplasmic sequences was performed (Fig. 4). The human cytoplasmic sequence was not included in the alignment due to too low sequence simi- larity. Comparison with the Hs-mit sequence showed high degree of identity among the methionine- and adenosine-binding residues. The residues Ala10, Ala12, Glu25 and Trp294, however, were M. smegmatis specific. Trp294 is a phenylalanine in M. tuberculosis. The KMSKS domain The KMSKS domain (residues 293-350), which has also been called the ‘stem contact fold domain’ and the b-a-a-b-a topology domain, connects the catalytic domain with the anticodon domain (Fig. 1). The sec- ondary structure elements are: b11 (residues 295-297), a10 (residues 311-319), a11 (residues 321-332), b12 (residues 338-340) and a12 (residues 341-350). This domain contains the conserved sequence KMSKS(301- 305) located in a loop (comprising residues 297–310) with a b-hairpin like structure at the tip of the domain (Fig. 5). In our structures this loop is trapped in an open and inactive conformation, with Lys304 unable to reach the active site. This arrangement of the loop is stabilized by hydrogen bonds to residues within the KMSKS domain (Fig. 5). Analysis of the crystal-pack- ing interactions verifies that this conformation is not induced by contacts made to neighbouring molecules. Comparison with the class 1a A. aeolicus Met- RS:tRNA Met and T. thermophilus MetRS (apo) struc- tures reveal that the position of the KMSKS loop in our structures is the result of a rotation of  90° around the hinges H1 and H2 located at Asn297 and at Val310, respectively. In A. aeolicus MetRS and T. thermophilus MetRS the corresponding hinge posi- tions are Val291 ⁄Val304 and Gly292 ⁄Val306 (Figs 6–8). The properties of the two hinges differ significantly. A B Fig. 3. Stereoviews of the methionine (Met) and adenosine (Ade) binding sites. The residues in the figures are all within a 4 A ˚ distance from the Met and Ade molecules. (A) The residues lining the Met molecule form a tight cavity with a mainly hydrophobic interior. An ion-pair inter- action with Od2 of Asp50 and a hydrogen bond to O of Ile11 orients the amino group of the Met. The Tyr13 aromatic ring covers the posi- tively charged amino group of the Met and partly covers the carboxylate group. Trp230 stacks from the side against the Met and is in a close-packing polar interaction with Tyr13. (B) The orientation of the ribose ring is controlled by hydrogen bonds to main-chain atoms as well as to side-chain atoms. The adenine base is oriented by hydrogen bonding to O of Leu295, by stacking against Trp294, by end-to-end stack- ing with His19 and His22, and by close packing to Gly21. Residues 293–295 belong to the KMSKS domain and residues 19–22 constitute the conserved HIGH sequence. Water molecules fill the volume between the ligands. Crystal structure of M. smegmatis MetRS H. Ingvarsson and T. Unge 3952 FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS Hinge H2 is located in the conserved sequence GNV VDP (the position of H2 is underlined) (Fig. 7). The rotation axis is around the Ca-C bond of the Val residue. Comparison with the A. aeolicus structure shows that, despite the large conformational changes accompanying the rotation at H2, the side chain of the hinge Val residue maintains its interaction with the conserved isoleucine of the HIGH sequence (leucine in A. aeolicus MetRS and in T. thermophilus MetRS). At H1, the rotation mechanism is more complex, and the result of conformational changes of the peptide chain. The changes include a rotation of the peptide plane between Asn297 and Arg298. Furthermore, the sequences are not conserved at H1, and in the T. ther- mophilus MetRS structure a proline residue has also been inserted. The open conformation of the KMSKS loop exposes the vacant binding site for the KMSKS methionine residue (Figs 8 and 9). The binding site is a cavity composed of the adenine group and hydrophobic Fig. 4. Alignment of the active-site sequences from Mycobacterium smegmatis MetRS (Ms) and Hs-mit MetRS (Hs). The adenosine-binding residues are denoted with ‘a’ and the methionine-binding residues with ‘m’. Fig. 5. The open conformation of the KMSKS loop. The structure of the KMSKS loop is stabilized by internal hydrogen bonds as well as by hydrogen bonds to the KMSKS domain. In order for the loop to adopt the closed active conformation, the bonds Asn314 in a10 to Ile306 and Asn308, Glu342 in a12 to Ser303 and Ser305, and Tyr340 to Val309, have to be broken. H2 H1 Fig. 6. Least-squares superimpositioning of the KMSKS domains from Mycobacterium smegmatis (yellow), Aquifex aeolicus (magenta) and Thermus thermophilus (gray). Hinges (H1 and H2) located at sequence positions 297 ⁄ 310 (M. smegmatis), 291 ⁄ 304 (A. aeolicus) and 292 ⁄ 306 (T. thermophilus) allow rotation of the KMSKS loop. The two positions of the loop correspond to a rotation of  90°. H. Ingvarsson and T. Unge Crystal structure of M. smegmatis MetRS FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS 3953 residues. The hydrophobic nature or this site makes it attractive for binding the side chain of an inhibitor. The majority of the residues in the site are identical between the M. smegmatis and the human enzymes, but at two positions – residues Leu295 and Leu296 – the sequences differ, and the human sequence has instead a trypyophan and a threonine (Fig. 7). The M. smegmatis MetRS:M ⁄ A structure reveals a shift of the entire KMSKS domain relative to its posi- tion in the M. smegmatis MetRS:M structure that Fig. 7. Alignment of the MetRS KMSKS domain sequences from Mycobacerium smegmatis, Aquifex aeolicus, Thermus thermophilus, and Hs-mit. The hinges H1 and H2 are indicated. H2 is located in the conserved sequence GNV VDP (Val310 is underlined). At H1, however, the sequences are not conserved. The LL residues of the LLR sequence are important for the stability of the hydrophobic core below the ade- nine group. Fig. 8. A stereoview of the conformation shift of the KMSKS loop. In the Mycobacerium smegmatis MetRS structures, the KMSKS loop is rotated  90° compared with previous structures. Even though the loop is not rotated exactly as a rigid body, two hinges could be identified. The M. smegmatis MetRS loop is shown in yellow, the Aquifex aeolicus MetRS loop (PDB ID: 2C8T) in magenta and the Thermus thermo- philus MetRS loop (PDB ID: 2D54) in gray. At hinge H2, which is located at Val310 in M. smegmatis, at Val304 in A. aeolicus and at Val306 in T. thermophilus, the rotation is around the Ca-C bond at the conserved Val residue. At hinge H1, which is located at Asn297 in M. smegmatis, at Val291 in A. aeolicus and at Gly292 in T. thermophilus, the rotation is more complex than a simple hinge rotation and the peptide chain makes new turns compared with the closed structure, including a rotation of the peptide plane between Asn297 and Gly298. Crystal structure of M. smegmatis MetRS H. Ingvarsson and T. Unge 3954 FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS seems to be induced by the interactions with the ade- nine moiety of the adenosine group. The entire KMSKS domain is moved towards the catalytic site through the interactions between adenine and the resi- due His292, the KMSKS domain residues Gly293, Trp294 and Leu295, and the HIGH sequence residues His19 and Gly21. At the tip of the KMSKS domain the greatest structural shifts are about 1.8 A ˚ (Fig. 10). Structural shifts of the same size (up to 1.7 A ˚ ) are also observed in the anticodon domain. The CP domain The CP domain covers the active site in the catalytic domain (Fig. 1). It consists of two subdomains: one mainly b and one mainly a. A distinctive feature of the mainly b subdomain is the arched antiparallel b-strands, b4 (residues 117-127) and b8 (residues 156- 166). On the sides of the arch there is one flanking b-strand b5 (residues 132-134) and an arched tip with the antiparallel strands b6 (residues 139-141) and b7 (residues 147-149) (Fig. 1). The mainly a subdomain consists of the helices a5 (residues 168-182) and a6 (residues 189-200), which pack against helix a9 and b10 in the catalytic domain. The mainly b subdomain of the M. smegmatis Met- RS structure is trapped in a conformation which differs significantly from that of the previously determined homologus enzymes. This is illustrated in Fig. 11, which shows a superimposition of the M. smegmatis CP domain on the homologus CP domains from E. coli (the rmsd is  1.4 A ˚ for 243 Ca atoms), Pyrococcus abyssi (the rmsd is  1.3 A ˚ for 236 Ca atoms) and T. thermophilus (the rmsd is  1.2 A ˚ for 254 Ca atoms). The comparison shows that the mainly b subdomain in the M. smegmatis CP domain is shifted downwards towards the active site. The tip of the subdomain located between b4 and b8 has a unique conformation. It shows the special features of a representative of the ‘one knuckle’ without a metal motif of the CP domain. It consists of a concave b- sheet, b6 and b7, flanked by two loops on both N- and C-terminal ends of the sheet, which extends the concave shape. The concave area faces the solution. In order to show the unique structural features of the ‘one knuckle’ without a metal compared to with a metal, a superimposition of the M. smegmatis tip structure (residues 127–155) on the T. thermophilus tip (residues 127–152) was made (Fig. 11D). In the T. thermophilus structure, a zinc ion is tetrahedrally coordinated by three cysteines and one histidine. Fig. 9. The binding site of the KMSKS methionine (Met) residue. The open conformation of the KMSKS loop exposes the binding site of the KMSKS loop Met residue in the closed conformation. Through an alignment with complexes of Aquifex aeolicus MetRS:tRNA Met and Escherichia coli GlnRS:tRNA Gln , the binding- site residues shown in the figure were identified. The residues Leu295 and Leu296 are tryptophan and threonine, respectively, in the Hs-mit sequence (Fig. 7). Fig. 10. The binding of adenosine (Ade) (green) to the My cobacterium smegmatis MetRS:M complex causes a shift in the KMSKS domain and in the HIGH sequence of the catalytic domain. The superim- positions shown here are made between all Ca atoms of the catalytic domains of the M. smegmatis MetRS structures (the rmsd is  0.3 A ˚ for all proteins). The three proteins A, B and C of the asymmetric unit of the M. smegmatis MetRS:M ⁄ A crystal (purple) and the protein M. smegmatis MetRS:M (gray) are included. Molecules A, B and C are grouped together. This super- imposition clearly shows that the binding of Ade causes a shift in the KMSKS domain. H. Ingvarsson and T. Unge Crystal structure of M. smegmatis MetRS FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS 3955 However, in the M. smegmatis structure there is a water molecule close to the metal position. This water molecule is coordinated through a network of hydro- gen bonds to the carbonyl oxygen of Pro155, the main-chain amide groups of Ile128 and Arg129. Despite these differences in the coordination of the res- idues in the tip, the fold of the loop is strikingly simi- lar. The rmsd for the Ca atoms of the residues 127 to 138 was  0.5 A ˚ . The mainly b subdomain is anchored to the catalytic domain through an extensive hydrogen-bond network (Fig. 12A). Three water molecules participate in this interaction. The position and curvature of the b8 and the b4 strands is stabilized by an ion-pair interaction between Glu161 and Arg210, which also forms hydro- gen bonds to Gln54 and a water molecule (Fig. 12A). The stabilizing interactions involve hydrophobic stack- ing interactions through the aromatic side chains of Tyr122, Tyr126, Phe133 and Tyr228. In the B and C molecules in the MetRS:M ⁄ A (with both methionine and adenosine) crystal, these interactions are not pres- ent and the tip of the mainly b subdomain is not visi- ble (Figs 12A,B). The anticodon domain By comparison with the A. aeolicus MetRS:tRNA Met complex, the anticodon-binding domain of M. smegma- tis MetRS was assigned to include residues 358–515 (Fig. 1) [24]. This domain is mainly a-helical. The helices a15 (residues 383–406), a16 (residues 408–430) and a18 (residues 441–465) form a bundle, and the heli- ces a14 (residues 357–372), a19 (residues 470–480) and the short 3 10 helix a20 (residues 488–493) interact with a18 at an angle (Fig. 1). The same arrangement of the helices is found in the structures of T. thermophilus MetRS, Pyrococcus abyssi MetRS, A. aeolicus MetRS and E. coli MetRS. A p-helix a13 (residues 351–357) links the KMSKS domain with the anticodon domain. By alignment of the anticodon-binding domains of the M. smegmatis holo structure to the A. aeolicus Met- RS:tRNA Met complex (the rmsd  1.4 A ˚ ) we identified residues Asn359, Arg363, Trp433, Ile508 and Phe509 as primarily the most important residues involved in the binding of the tRNA anticodon loop. Asn359 and Arg363 are located on helix a14. In our structure, Trp433 interacts with Arg363, but in the A. aeolicus AB CD Fig. 11. Structural comparisons of CP domains from homologus structures to Mycobacterium smegmatis MetRS (green): (A) Escherichia coli MetRS in blue (PDB ID: 1F4L), (B) Pyrococcus abyssi MetRS in pink (PDB ID: 1RQG) and (C) Thermus thermo- philus MetRS in gray (PDB ID: 2D54). M. smegmatis MetRS was found to have a significant shift (highlighted in the oval in Fig. 11A) of the protruding arm in com- parison with the other homologus structures. (D) The tip of the protruding arm in T. thermophilus MetRS (residues 127–152) was superimposed onto the counterpart of M. smegmatis MetRS (residues 127–155). The zinc atom in yellow and the water molecule in red belong to T. thermophilus MetRS and M. smegmatis MetRS:M ⁄ A, respectively. Crystal structure of M. smegmatis MetRS H. Ingvarsson and T. Unge 3956 FEBS Journal 277 (2010) 3947–3962 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... methionine and adenylate complexes of the 3958 homologus E coli enzyme showed that the re-arrangement of the active-site residues accompanying the coordination of the methionine ligand is a prerequisite for this compact conformation [25,29] The presence of two molecules (B and C) in the M smegmatis MetRS:M ⁄ A crystals with the CP domain in the relaxed conformation, verifies the flexibility of this domains The. .. ⁄ A structure The CP domain adopted a conformation where it was firmly anchored to the catalytic domain The biological significance of this conformation is not clear, but the structure shows that a conformation of the enzyme exists where the CP domain, by binding tightly to the catalytic domain, increases the rigidity of the complex between the enzyme and the substrate methionine Comparisons of the apo,... relationships and the classification of aminoacyl-tRNA synthetases J Biol Chem 266, 16965–16968 13 Barker DG & Winter G (1982) Conserved cysteine and histidine residues in the structures of the tyrosyl and methionyl-tRNA synthetases FEBS Lett 145, 191–193 14 Webster T, Tsai H, Kula M, Mackie GA & Schimmel P (1984) Specific sequence homology and three-dimensional structure of an aminoacyl transfer RNA synthetase. .. Ingvarsson and T Unge Crystal structure of M smegmatis MetRS Glu161 A Tyr122 W748 β4 β4 2 Arg210 70 β6 Phe133 W β7 β8 β8 Gln54 β5 Tyr228 Glu131 W 71 2 Fig 12 Anchoring of the protruding arm of the CP domain (green) to the catalytic domain (yellow) (A) A network of hydrogen bonds between Gln54, Lys55 and Met56 in the catalytic domain and Glu131, Glu161 and Arg210 in the CP domain tightly associate the. .. C34 and A35 that, together with U36, form the anticodon triplet (CAU) (Fig 13) There is a kink involving residues 394–396 in helix a15 on the back of the molecule relative to the tRNA-binding site This kink is of importance for the positioning of the helix a16 and the residue Asn424 The potentially nucleotide-binding residues Asn359, Ile508 and Phe509 all hydrogen bond to water molecules One of these... makes the loop change from the open to the closed conformation, but because our enzyme is active in the presence of tRNA, it is not unlikely that it is the interaction with the tRNA that releases the loop We speculate that the KMSKS loop could assist in the positioning of the acceptor stem in the catalytic site The exact steering and positioning of the loop, and particularly the catalytic lysine residue,... showed f that the material was biologically active Our structural analysis confirms that the MetRS molecule is flexible [29,30,32] The mobility of the domains reflects the structural changes required for binding, catalysis, communication and release of the reaction partners A new conformation of the CP domain was identified in our structures – in the M smegmatis MetRS:M structure as well as in the M smegmatis. .. (Val310) in M smegmatis MetRS is located in the conserved sequence GNVVDP (Val310 is underlined) The side chain of Val310 is anchored against the conserved Ile20 in the HIGH sequence and remains so also after the rotation, according to the comparison The sequence conservation at H2 and the rotation mechanism might indicate the biological relevance of the open conformation The properties of hinge H1 (located... expose the 2¢- or 3¢-hydroxyl of the 3¢-end ribose of the tRNA to the catalytic centre, break the anhydride bond and link the amino acid to the tRNA by formation of an ester bond These functions require a relatively large molecule with specialized domains, which in addition should be able to communicate with each other [29–31] MetRS is of special importance among the aaRS enzymes, because of the fact... structure, in close contact with the catalytic domain In all of our M smegmatis MetRS structures, however, the KMSKS loop was in an open conformation with the MSK Lys residue out of reach of the catalytic site This conformation of the KMSKS loop has not been observed previously The X-ray structures do not throw any light on the mechanism responsible for the structural change, but it is clear that the . Flexibility and communication within the structure of the Mycobacterium smegmatis methionyl-tRNA synthetase Henrik Ingvarsson and Torsten Unge Department. rotation of the KMSKS loop. The two positions of the loop correspond to a rotation of  90°. H. Ingvarsson and T. Unge Crystal structure of M. smegmatis