Biomedical subjects

John J Perona

Publications and source records attributed to John J Perona.

17 recordsLinked to original sources

PubMed2005

Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC.

The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.

Binding Sites↗

PubMed2005

Amino acid-dependent transfer RNA affinity in a class I aminoacyl-tRNA synthetase.

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS x tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.

Amino Acids↗

PubMed2005

tRNA-dependent aminoacyl-adenylate hydrolysis by a nonediting class I aminoacyl-tRNA synthetase.

Glutaminyl-tRNA synthetase generates Gln-tRNA(Gln) 10(7)-fold more efficiently than Glu-tRNA(Gln) and requires tRNA to synthesize the activated aminoacyl adenylate in the first step of the reaction. To examine the role of tRNA in amino acid activation more closely, several assays employing a tRNA analog in which the 2'-OH group at the 3'-terminal A76 nucleotide is replaced with hydrogen (tRNA(2'HGln)) were developed. These experiments revealed a 10(4)-fold reduction in kcat/Km in the presence of the analog, suggesting a direct catalytic role for tRNA in the activation reaction. The catalytic importance of the A76 2'-OH group in aminoacylation mirrors a similar role for this moiety that has recently been demonstrated during peptidyl transfer on the ribosome. Unexpectedly, tracking of Gln-AMP formation utilizing an alpha-32P-labeled ATP substrate in the presence of tRNA(2'HGln) showed that AMP accumulates 5-fold more rapidly than Gln-AMP. A cold-trapping experiment revealed that the nonenzymatic rate of Gln-AMP hydrolysis is too slow to account for the rapid AMP formation; hence, the hydrolysis of Gln-AMP to form glutamine and AMP must be directly catalyzed by the GlnRS x tRNA(2'HGln) complex. This hydrolysis of glutaminyl adenylate represents a novel reaction that is directly analogous to the pre-transfer editing hydrolysis of noncognate aminoacyl adenylates by editing synthetases such as isoleucyl-tRNA synthetase. Because glutaminyl-tRNA synthetase does not possess a spatially separate editing domain, these data demonstrate that a pre-transfer editing-like reaction can occur within the synthetic site of a class I tRNA synthetase.

Adenosine Monophosphate↗

PubMed2005

Two-step pathway to aminoacylated tRNA.

The 3.0 Angstrom crystal structure of a tRNA-dependent amidotransferase from the hyperthermophilic archaeon Pyrococcus abyssi provides the first detailed insight into how cells lacking canonical tRNA synthetases nonetheless carry out protein synthesis with high fidelity.

Adenosine Diphosphate↗

PubMed2004

Shape-selective RNA recognition by cysteinyl-tRNA synthetase.

The crystal structure of Escherichia coli cysteinyl-tRNA synthetase (CysRS) bound to tRNA(Cys) at a resolution of 2.3 A reveals base-specific and shape-selective interactions across an extensive protein-RNA recognition interface. The complex contains a mixed alpha/beta C-terminal domain, which is disordered in the unliganded enzyme. This domain makes specific hydrogen bonding interactions with all three bases of the GCA anticodon. The tRNA anticodon stem is bent sharply toward the enzyme as compared with its conformation when bound to elongation factor Tu, providing an essential basis for shape-selective recognition. The CysRS structure also reveals interactions of conserved enzyme groups with the sugar-phosphate backbone in the D loop, adjacent to an unusual G15.G48 tertiary base pair previously implicated in tRNA aminoacylation. A combined mutational analysis of enzyme and tRNA groups at G15.G48 supports the notion that contacts between CysRS and the sugar-phosphate backbone contribute to recognition by indirect readout.

Amino Acyl-tRNA Synthetases↗

PubMed2004

Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics.

Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex. Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k(cat). Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA(Gln). Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k(chem)) exceeds the steady-state rate by nearly 10-fold. The concentration dependence of the single-turnover reaction further reveals that the glutamine K(d) is significantly higher than the steady-state K(m) value. The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction. Mutation of U35 in the tRNA anticodon loop decreases k(chem) by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 A distant. By contrast, mutation of the adjacent G36 has very small effects on k(chem) and K(d) for glutamine. Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex.

Amino Acyl-tRNA Synthetases↗

PubMed2004

DNA cleavage by EcoRV endonuclease: two metal ions in three metal ion binding sites.

Four crystal structures of EcoRV endonuclease mutants K92A and K38A provide new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The removal of a key active site positive charge in the uncleaved K92A-DNA-M(2+) substrate complex results in binding of a sodium ion in the position of the amine nitrogen, suggesting a key role for a positive charge at this position in stabilizing the sharp DNA bend prior to cleavage. By contrast, two structures of K38A cocrystallized with DNA and Mn(2+) ions in different lattice environments reveal cleaved product complexes featuring a common, novel conformation of the scissile phosphate group as compared to all previous EcoRV structures. In these structures, the released 5'-phosphate and 3'-OH groups remain in close juxtaposition with each other and with two Mn(2+) ions that bridge the conserved active site carboxylates. The scissile phosphates are found midway between their positions in the prereactive substrate and postreactive product complexes of the wild-type enzyme. Mn(2+) ions occupy two of the three sites previously described in the prereactive complexes and are plausibly positioned to generate the nucleophilic hydroxide ion, to compensate for the incipient additional negative charge in the transition state, and to ionize a second water for protonation of the 3'-oxyanion. Reconciliation of these findings with earlier X-ray and fluorescence studies suggests a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft. This reconfigures the local environment to permit binding of the second metal ion followed by movement toward the pentacovalent transition state. The new mechanism suggested here embodies key features of previously proposed two- and three-metal catalytic models, and offers a view of the stereochemical pathway that integrates much of the copious structural and functional data that are available from exhaustive studies in many laboratories.

Binding Sites↗

PubMed2003

Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence.

The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M(-1) s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg(2+) ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg(2+) or Ca(2+), while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.

Calcium↗

PubMed2003

Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase.

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves high amino acid specificity without the need for an editing reaction. Crystallographic and spectroscopic studies have previously demonstrated that a major determinant of the specificity is an active site zinc ion that recognizes the substrate cysteine through a strong zinc-thiolate interaction. The active site cleft of CysRS is composed of highly or strictly conserved amino acids, including four inner-sphere zinc ligands, five histidine imidazoles at the base of the cleft, and a tryptophan that flips down upon cysteine binding to complete formation of the binding pocket. Here we establish the significance of each of these major features of the active site cleft by mutational analysis. Substitutions generally lead to substantially deleterious effects on K(m) and k(cat) parameters with respect to each of the cysteine, ATP, and tRNA(Cys) substrates. These findings emphasize the importance of the highly differentiated nature of the active site and provide new insights into the origins of selectivity without editing. Most mutants are less attenuated in tRNA aminoacylation than in adenylate synthesis, suggesting that tRNA binding drives a conformational change to help assemble the active site.

Adenosine Triphosphate↗

PubMed2003

Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants.

The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.

Adenosine Triphosphate↗

PubMed2003

Zinc-mediated amino acid discrimination in cysteinyl-tRNA synthetase.

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves a high level of amino acid specificity without an editing reaction. The crystal structure of CysRS bound to substrate cysteine suggested that direct thiol coordination to a tightly bound zinc ion at the base of the active site is the primary determinant of selectivity against non-cognate amino acids. This hypothesis has now been supported by spectroscopic studies of cobalt-substituted CysRS. Binding of cysteine, but not non-cognate amino acids, induces high absorption in the ligand-to-metal charge transfer region, providing evidence for formation of a metal-thiolate bond. In addition, mutations in the zinc ligands alter the absorption spectrum without reducing the discrimination against non-cognate amino acids. These results argue strongly for a major role for the zinc ion in amino acid discrimination by CysRS, where the tight zinc-thiolate interaction and the strict structural geometry of the metal ion are sufficient to reject serine by more than 20,000-fold at the binding step.

Amino Acids↗

PubMed2003

tRNA-dependent active site assembly in a class I aminoacyl-tRNA synthetase.

The crystal structure of ligand-free E. coli glutaminyl-tRNA synthetase (GlnRS) at 2.4 A resolution shows that substrate binding is essential to construction of a catalytically proficient active site. tRNA binding generates structural changes throughout the enzyme, repositioning key active site peptides that bind glutamine and ATP. The structure gives insight into longstanding questions regarding the tRNA dependence of glutaminyl adenylate formation, the coupling of amino acid and tRNA selectivities, and the roles of specific pathways for transmission of tRNA binding signals to the active site. Comparative analysis of the unliganded and tRNA-bound structures shows, in detail, how flexibility is built into the enzyme architecture and suggests that the induced-fit transitions are a key underlying determinant of both amino acid and tRNA specificity.

Amino Acid Sequence↗

PubMed2002

Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease.

Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 A of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 10(3)-10(5)-fold compared to that of wild-type EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 A from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small alpha-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.

Alanine↗

PubMed2002

Structural origins of amino acid selection without editing by cysteinyl-tRNA synthetase.

Cysteinyl-tRNA synthetase (CysRS) is highly specific for synthesis of cysteinyl adenylate, yet does not possess the amino acid editing activity characteristic of many other tRNA synthetases. To elucidate how CysRS is able to distinguish cysteine from non-cognate amino acids, crystal structures of the Escherichia coli enzyme were determined in apo and cysteine-bound states. The structures reveal that the substrate cysteine thiolate forms a single direct interaction with a zinc ion bound at the base of the active site cleft, in a trigonal bipyramidal geometry together with four highly conserved protein side chains. Cysteine binding induces movement of the zinc ion towards substrate, as well as flipping of the conserved Trp205 indole ring to pack on the thiol side chain. The imidazole groups of five conserved histidines lie adjacent to the zinc ion, forming a unique arrangement suggestive of functional significance. Thus, amino acid discrimination without editing arises most directly from the favorable zinc-thiolate interaction, which is not possible for non-cognate substrates. Additional selectivity may be generated during the induced-fit conformational changes that help assemble the active site.

Amino Acid Sequence↗

PubMed2002

Type II restriction endonucleases.

Type II restriction endonucleases have emerged as important paradigms for the study of protein-nucleic acid interactions. This is due to their ability to catalyse phosphodiester bond cleavage with very large rate enhancements while also maintaining exquisite sequence selectivities. The principles and methods developed to analyze site-specific binding and catalysis for restriction endonucleases can be applied to other enzymes which also operate on nucleic acids. This paper reviews biochemical and structural approaches to characterization of these enzymes, with particular attention to the multiple crucial roles of divalent metal ions, the possibilities for use of alternative substrates in binding and catalytic experiments, the strategies for exploring the detailed chemistry of phosphoryl transfer, and the use of X-ray crystallography to provide descriptions of conformational pathways at specific, nonspecific, and noncognate DNA sites.

Crystallography↗

PubMed2002

Aminoacyl-tRNA synthetases: versatile players in the changing theater of translation.

Aminoacyl-tRNA synthetases attach amino acids to the 3' termini of cognate tRNAs to establish the specificity of protein synthesis. A recent Asilomar conference (California, January 13-18, 2002) discussed new research into the structure-function relationship of these crucial enzymes, as well as a multitude of novel functions, including participation in amino acid biosynthesis, cell cycle control, RNA splicing, and export of tRNAs from nucleus to cytoplasm in eukaryotic cells. Together with the discovery of their role in the cellular synthesis of proteins to incorporate selenocysteine and pyrrolysine, these diverse functions of aminoacyl-tRNA synthetases underscore the flexibility and adaptability of these ancient enzymes and stimulate the development of new concepts and methods for expanding the genetic code.

Amino Acids↗

PubMed2002

Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation.

The crystal structure of the HincII restriction endonuclease-DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine-purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base-stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.

Base Sequence↗