KMSKS Loop Research Articles

Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC50 values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.

Dynamics of the Catalytic Active Site of Isoleucyl tRNA Synthetase from Staphylococcus aureus bound with Adenylate and Mupirocin.

The development of new antimicrobial drugs is critically needed due to the alarming increase in antibiotic resistance in bacterial pathogens. The active sites of different bacterial aminoacyl tRNA synthetases (aaRS) are validated targets of antibiotics. At present, the only aaRS inhibitor approved is mupirocin (MRC) which targets bacterial isoleucyl tRNA synthetase (IleRS). The present work is aimed at understanding the lacunae of knowledge concerning the active site conformational dynamics in IleRS in the presence of inhibitor mupirocin. With this end in view, we have carried out classical molecular dynamics simulation and metadynamics simulations of the open state of IleRS from Staphylococcus aureus (SaIleRS), the closed state tripartite complex bound with cognate adenylate (Ile-AMP) and tRNA, the closed state tripartite complex bound with noncognate MRC, and the closed state tripartite complex bound with tRNA and MRC with mutated SaIleRS (V588F). The present simulation established a dynamic picture of SaIleRS complexed with cognate and the noncognate substrates which are completely consistent with crystallographic and biochemical studies and explain the existing lacunae of knowledge. The active site is significantly more compact in the Ile-AMP bound complex compared to the open state due to the closure of the KMSKS and HMGH loops and clamping down of the tRNA acceptor end near the active site gate. The present result shows that the unusual open conformational state of the KMSKS loop observed in the cognate substrate-bound complex in the crystal is due to crystallographic constraints. Although the mupirocin tightly fits the catalytic active site in the MRC-bound complex, the nonanoic acid moiety is partly exposed to the water. The KMSKS loop is pushed open in the MRC-bound complex to accommodate the noncognate MRC. New tunnels open up, extending to the editing site in the complex. Out of its three broad segments, the C12 to C17 segment, the conjugated segment, and the nonanoic moiety, the conjugated part of MRC binds most effectively with the active site of the MRC-bound complex. The aromatic residues packing around the C12 to C17 segment of MRC stabilize the tRNA hairpin conformation in a similar way as observed in the TrpRS. The V588F mutation is weakening the interaction between this region of the active site and weakens the binding of MRC in the active site. This result explains why the V588F mutation is responsible for low-level mupirocin resistance. The free energy of unbinding the conjugated segment (and C12 to C17 segment, as well) largely contributes to the total free energy of unbinding the MRC. The active site organization of IleRS from eukaryotic Candida albicans is compared with the bacterial IleRS active site to understand the low binding affinity in eukaryotic IleRS. The present study could be a starting point of future studies related to the development of effective drug binding in the SaIleRS.

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg(2+) ion in a manner that allows it to assist in transition state stabilization. The Mg(2+) ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg(2+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg(2+). Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS.

Conformational landscapes for KMSKS loop in tyrosyl-tRNA synthetases.

Protein synthesis requires accurate charging of tRNA with cognate amino acid as catalyzed by aminoacyl-tRNA synthetases. Crystal structures of tyrosyl-tRNA synthetase (YRSs) show remarkably diverse conformations for the KMSKS loop, hitherto classified as "open" and "closed". This traditional classification implied that the KMSKS loop adopts different conformations depending on occupancy of active site pocket. Our structural analyses of evolutionarily derived ensemble of differentially ligated YRSs using quantitative structural criterion demonstrate intrinsic conformational heterogeneity in KMSKS loop that is independent of occupancy of active site. Differential centroid distance analyses between KMSKS motif and Rossmann fold domain reveal an intriguing bimodal distribution. These insights have been used for a more consistent re-classification of YRS conformations as either compact or extended. Our data not only reflect inherent dynamics within the conformational landscape of KMSKS loops, but also have implications for structure-based drug design efforts.

An Appended Domain Results in an Unusual Architecture for Malaria Parasite Tryptophanyl-tRNA Synthetase

Specific activation of amino acids by aminoacyl-tRNA synthetases (aaRSs) is essential for maintaining fidelity during protein translation. Here, we present crystal structure of malaria parasite Plasmodium falciparum tryptophanyl-tRNA synthetase (Pf-WRS) catalytic domain (AAD) at 2.6 Å resolution in complex with L-tryptophan. Confocal microscopy-based localization data suggest cytoplasmic residency of this protein. Pf-WRS has an unusual N-terminal extension of AlaX-like domain (AXD) along with linker regions which together seem vital for enzymatic activity and tRNA binding. Pf-WRS is not proteolytically processed in the parasites and therefore AXD likely provides tRNA binding capability rather than editing activity. The N-terminal domain containing AXD and linker region is monomeric and would result in an unusual overall architecture for Pf-WRS where the dimeric catalytic domains have monomeric AXDs on either side. Our PDB-wide comparative analyses of 47 WRS crystal structures also provide new mechanistic insights into this enzyme family in context conserved KMSKS loop conformations.

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase.

Leucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNA(Leu) for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme-tRNA complex in a novel 'aminoacylation-like' conformation, forming novel interactions with the KMSKS loop and the tRNA 3'-end. Our findings reveal an intriguing tRNA-dependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design.

Structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase

Leucyl-tRNA synthetase (LeuRS) produces error free leucyl-tRNALeu by coordinating translocation of the 3′ end of (mis-)charged tRNAs from its synthetic site to a separate proof-reading site for editing. Here we report co-crystal structures of the Escherichia coli LeuRS-tRNALeu complex in the aminoacylation or editing conformations and show that translocation involves correlated rotations of four flexibly linked LeuRS domains. This pivots the tRNA to guide the charged tRNA 3′ end from the closed aminoacylation state to the editing site. The editing domain unexpectedly stabilizes the tRNA during aminoacylation while a large rotation of the leucine-specific domain positions the conserved KMSKS loop to bind the 3′ end of the tRNA, promoting catalysis. Our results give new insight into the structural dynamics of a molecular machine that is essential for accurate protein synthesis.

Flexibility and communication within the structure of the Mycobacterium smegmatis methionyl‐tRNA synthetase

Two structures of monomeric methionyl-tRNA synthetase, from Mycobacterium smegmatis, in complex with the ligands methionine/adenosine and methionine, were analyzed by X-ray crystallography at 2.3 Å and at 2.8 Å, 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.

Molecular trigger for pre-transfer editing pathway in Valyl-tRNA synthetase: A molecular dynamics simulation study

Pre-transfer editing pathway in Valyl-tRNA synthetase (ValRS) is a very important process to maintain the high fidelity of protein synthesis. However, molecular basis for this pathway remains unclear. Here we employed molecular dynamics (MD) simulation to study two complexes, ValRS·tRNA(val)·Val-AMP (complex V) and ValRS·tRNA(val)·Thr-AMP (complex T), and compared their simulation trajectories, in order to understand how the pre-transfer editing pathway is triggered by the noncognate substrate Thr-AMP. The MD simulations showed that the binding of Thr-AMP to ValRS led to different motions from those in complex V: clockwise rotation of the editing domain along the hinge region, and strong motions in the catalytic domain, especially in KMSKS loop. We found that the changed motion of Trp495 induced by Thr-AMP serves as a signal to discriminate Thr-AMP from Val-AMP, and the rigid (491)ILFL(494) segment then propagates this signal from Trp495 to Asp490 and induces dissociation of the salt-bridge Asp490-Arg346 and formation of the salt-bridge Glu189-Lys533. The change in salt-bridges alters the motion of KMSKS loop and the editing domain, and eventually triggers the pre-transfer editing pathway. This study provides a model for the molecular trigger of the pre-transfer editing pathway in ValRS, and is useful for further exploring this process.

A structural model for catalysis by tyrosyl‐tRNA synthetase

Twenty‐eight tyrosyl‐tRNA synthetase (TyrRS) structures were aligned and their active sites were compared. This analysis revealed that the HIGH signature sequence adopts two distinct conformations that correlate with the open and closed forms of the KMSKS loop. Based on this analysis, we propose a model in which the binding of ATP forces HIGH to adopt a closed conformation. This forces the KMSKS loop to adopt a closed, strained, conformation, bringing the KMSKS sequence into the active site and positioning it to interact with pyrophosphate in the TyrRS·[Tyr‐ATP]‡ transition state. Formation of the transition state results in cleavage of the scissile bond in ATP, releasing the KMSKS loop from the closed (strained) conformation. This mechanism provides a structural explanation for the observations that: (1) tyrosyl‐tRNA synthetase uses ATP‐binding energy to stabilize the transition state of the reaction and (2) deletion of the KMSKS sequence increases the binding affinity of the TyrRS·Tyr complex for ATP. Lastly, structural comparison of eukaryotic and archaeal tyrosyl‐tRNA synthetases revealed a ligand‐dependent conformational change that links the tyrosine binding site to the dimer interface, suggesting a possible model for the observation that tyrosyl‐tRNA synthetase displays half‐of‐the‐sites reactivity with respect to the binding of tyrosine.This research was funded NIH GM68070 and MDA 4268.

The 1.6 Å Crystal Structure of Mycobacterium smegmatis MshC: The Penultimate Enzyme in the Mycothiol Biosynthetic Pathway

Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and l-cysteine to form l-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5'-O-[N-(l-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 A. The refined structure exhibited electron density corresponding to the entire 47 kDa MshC molecule, with the exception of the KMSKS loop (residues 285-297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions.

Investigateion of Requirements for the KMSKS Loop in Aminoacyl-tRNA Synthetase by Random PCR Method

Investigateion of Requirements for the KMSKS Loop in Aminoacyl-tRNA Synthetase by Random PCR Method

Comparative structural dynamics of Tyrosyl-tRNA synthetase complexed with different substrates explored by molecular dynamics

Several molecular dynamics simulations of S. aureus Tyrosyl-tRNA synthetase (TyrRS) in its free form and complexed with Tyr, ATP, tyrosyl adenylate and inhibitor respectively have been carried out to investigate the ligand-linked conformational stability changes associated with its catalytic cycle. The results show that unliganded S. aureus TyrRS samples a more relaxed conformational space than substrate-bound TyrRS. There are three high flexibility regions encompassing residues 114-118, 128-133, and 226-238 respectively. The region which includes the KMSKS motif (KFGKS in S. aureus TyrRS) shows the highest difference in fluctuations. Hydrogen bond network formed by Tyr, ATP, tyrosyl adenylate and inhibitor with S. aureus TyrRS is discussed. Our simulations suggest the induced-fit conformational changes of the KMSKS loop as follows: the KMSKS loop of substrate-free S. aureus TyrRS adopts an open conformation. The tyrosine binds in the pocket with the KMSKS loop balancing between semi-open and open forms. The ATP binding induces the KMSKS loop to the open form. After the Tyr-AMP is formed, the first two residues of KMSKS loop twists in an anticlockwise direction and drives the loop in a conformation similar to the closed one, while those of the last three GKS residues adopt a conformation between semi-open and open conformation. This conformational change may probably be involved in the initial tRNA binding.

Crystal Structure of Tryptophanyl-tRNA Synthetase Complexed with Adenosine-5′ Tetraphosphate: Evidence for Distributed Use of Catalytic Binding Energy in Amino Acid Activation by Class I Aminoacyl-tRNA Synthetases

Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5′ tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 Å structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the γ and δd phosphate groups to occupy positions equivalent to those occupied by the β and γ phosphates of ATP. The β-phosphate effects a 1.11 Å extension that relocates the α-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Pα as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.

Computational Studies of Tryptophanyl-tRNA Synthetase: Activation of ATP by Induced-Fit

Catalysis of amino acid activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase involves three allosteric states: (1) Open; (2) closed pre-transition state (PreTS); and (3) closed products (Product). The interconversions of these states entail significant domain motions driven by ligand binding. We explore the application of molecular dynamics simulations to investigate ligand-linked conformational stability changes associated with this catalytic cycle. Multiple molecular dynamics trajectories (5 ns) for 11 distinct liganded and unliganded monomer configurations show that the PreTS conformation is unstable in the absence of ATP, reverting within ∼600 ps nearly to the Open conformation. In contrast, Open and Product state trajectories were stable, even without ligands, confirming the previous suggestion that catalysis entails destabilization of the protein conformation, driven by ATP-binding energies developed as the PreTS state assembles during induced-fit. The simulations suggest novel mechanistic details associated with both induced-fit (Open-PreTS) and catalysis (PreTS-Product). Notably, Mg 2+–ATP interactions are coupled to interactions between ATP and active-site lysine side-chains via mechanisms that cannot be captured under the molecular mechanics approximations, and which therefore require restraining potentials for stable simulation. Simulations of Mg 2+·ATP-bound PreTS complexes with restraining potentials and with a virtual K111A mutant confirm that these coupling interactions are necessary to sustain the PreTS conformation and, in turn, provide a new model for how the PreTS conformation activates ATP for catalysis. These results emphasize the central role of the PreTS state as a high-energy intermediate structure along the catalytic pathway and suggest that Mg 2+ and the KMSKS loop function cooperatively during catalysis.

Crystal Structures of Tyrosyl-tRNA Synthetases from Archaea

Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNA(Tyr) in a two-step reaction. TyrRS has the "HIGH" and "KMSKS" motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8A and 2.2A resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 A. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound "closed" form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 3(10) helix, which appears to correspond to the "semi-closed" form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs.

Structures of Tryptophanyl-tRNA Synthetase II from Deinococcus radiodurans Bound to ATP and Tryptophan: INSIGHT INTO SUBUNIT COOPERATIVITY AND DOMAIN MOTIONS LINKED TO CATALYSIS

An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radiation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. Crystal structures of drTrpRS II, empty of ligands or bound to either Trp or ATP, reveal that drTrpRS II has an overall structure similar to standard bacterial TrpRSs but undergoes smaller amplitude motions of the helical tRNA anti-codon binding (TAB) domain on binding substrates. TAB domain loop conformations that more closely resemble those of human TrpRS than those of Bacillus stearothermophilus TrpRS (bsTrpRS) indicate different modes of tRNA recognition by subclasses of bacterial TrpRSs. A compact state of drTrpRS II binds ATP, from which only minimal TAB domain movement is necessary to bring nucleotide in contact with Trp. However, the signature KMSKS loop of class I synthetases does not completely engage the ATP phosphates, and the adenine ring is not well ordered in the absence of Trp. Thus, progression of the KMSKS loop to a high energy conformation that stages acyl-adenylation requires binding of both substrates. In an asymmetric drTrpRS II dimer, the closed subunit binds ATP, whereas the open subunit binds Trp. A crystallographically symmetric dimer binds no ligands. Half-site reactivity for Trp binding is confirmed by thermodynamic measurements and explained by an asymmetric shift of the dimer interface toward the occupied active site. Upon Trp binding, Asp68 propagates structural changes between subunits by switching its hydrogen bonding partner from dimer interface residue Tyr139 to active site residue Arg30. Since TrpRS IIs are resistant to inhibitors of standard TrpRSs, and pathogens contain drTrpRS II homologs, the structure of drTrpRS II provides a framework for the design of potentially useful antibiotics.

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine.

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.

Structural Snapshots of the KMSKS Loop Rearrangement for Amino Acid Activation by Bacterial Tyrosyl-tRNA Synthetase

Tyrosyl-tRNA synthetase (TyrRS) has been studied extensively by mutational and structural analyses to elucidate its catalytic mechanism. TyrRS has the HIGH and KMSKS motifs that catalyze the amino acid activation with ATP. In the present study, the crystal structures of the Escherichia coli TyrRS catalytic domain, in complexes with l-tyrosine and a l-tyrosyladenylate analogue, Tyr-AMS, were solved at 2.0A and 2.7A resolution, respectively. In the Tyr-AMS-bound structure, the 2'-OH group and adenine ring of the Tyr-AMS are strictly recognized by hydrogen bonds. This manner of hydrogen-bond recognition is conserved among the class I synthetases. Moreover, a comparison between the two structures revealed that the KMSKS loop is rearranged in response to adenine moiety binding and hydrogen-bond formation, and the KMSKS loop adopts the more compact ("semi-open") form, rather than the flexible, open form. The HIGH motif initially recognizes the gamma-phosphate, and then the alpha and gamma-phosphates of ATP, with a slight rearrangement of the residues. The other residues around the substrate also accommodate the Tyr-AMS. This induced-fit form presents a novel "snapshot" of the amino acid activation step in the aminoacylation reaction by TyrRS. The present structures and the T.thermophilus TyrRS ATP-free and bound structures revealed that the extensive induced-fit conformational changes of the KMSKS loop and the local conformational changes within the substrate binding site form the basis for driving the amino acid activation step: the KMSKS loop adopts the open form, transiently shifts to the semi-open conformation according to the adenosyl moiety binding, and finally assumes the rigid ATP-bound, closed form. After the amino acid activation, the KMSKS loop adopts the semi-open form again to accept the CCA end of tRNA for the aminoacyl transfer reaction.

Interconversion of ATP Binding and Conformational Free Energies by Tryptophanyl-tRNA Synthetase: Structures of ATP Bound to Open and Closed, Pre-Transition-state Conformations

Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at ∼8 mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range (∼5 mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1 mM ATP and a low-affinity complex grown at 10 mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7 Å. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2 Å) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the α and γ-phosphates; interactions of the K111 side-chain with the γ-phosphate; and a water molecule bridging the consensus sequence residue T15 to the β-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP i group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.