Biomedical subjects
Publications and source records attributed to T A Steitz.
The recently reported crystal structures of two recombination enzymes, the catalytic domain of HIV integrase and Escherichia coli RuvC, an endonuclease, are surprisingly similar to that of ribonuclease H suggesting the possibility that they have a common enzymatic mechanism.
The superimposable dinucleotide fold domains of MetRS, GlnRS and TyrRS define structurally equivalent amino acids which have been used to constrain the sequence alignments of the 10 class I aminoacyl-tRNA synthetases (aaRS). The conservation of those residues which have been shown to be critical in some aaRS enables to predict their location and function in the other synthetases, particularly: i) a conserved negatively-charged residue which binds the alpha-amino group of the amino acid substrate; ii) conserved residues within the inserted domain bridging the two halves of the dinucleotide-binding fold; and iii) conserved residues in the second half of the fold which bind the amino acid and ATP substrate. The alignments also indicate that the class I synthetases may be partitioned into two subgroups: a) MetRS, IleRS, LeuRS, ValRS, CysRS and ArgRS; b) GlnRS, GluRS, TyrRS and TrpRS.
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Endoproteinase Asp-N cleaves the 581-amino acid Escherichia coli primase (65,564 Da) into several major fragments. One of these, a 47-kDa fragment containing the complete N terminus and the first 422 amino acids of primase, is capable of primer RNA (pRNA) synthesis in the G4oric/single-stranded DNA binding protein/primase pRNA synthesis system. A cloned 398-amino acid N-terminal fragment of primase can also synthesize pRNA. The sizes of the pRNA synthesized by these N-terminal fragments, however, are smaller than those synthesized by intact primase, suggesting that the C-terminal region of primase plays a role in processivity or regulation of pRNA synthesis. Primase mutants with the last 10 and 40 C-terminal amino acids deleted synthesize pRNA as wild-type primase, indicating that any regulatory sequences must be internal to the C terminus of primase.
BACKGROUND: Reverse transcriptase (RT) from HIV-1 is responsible for replicating the single-stranded RNA genome to double-stranded DNA. The three-dimensional structure of RT shows that it is a strikingly asymmetric heterodimer consisting of two differently folded subunits (molecular weights 66 kDa and 51 kDa) with identical amino-terminal amino acid sequences (residues 1-428). The large active site cleft is composed of subdomains named 'finger', 'palm' and 'thumb'. There is also an RNAse H domain. RESULTS: We have compared four RT structures. The structures of two independent RT heterodimers comprising the asymmetric unit of an orthorhombic crystal form have been determined by molecular replacement and are noticeably different from each other. Comparison of the molecules in this crystal form with the two previously reported RT structures shows a related pattern of variations in relative sub domain positions. The structural differences can be described as a molecular twist between the polymerase active site located on the finger and palm domains of p66 and the rest of the molecule. This twist occurs around an axis which runs from the p66 palm domain through the p66/p51 connection domain interface and which exits below the RNAse H domain. CONCLUSIONS: From the differences in the four RT structures we infer that the molecule has a specific flexibility that allows rotation of the polymerase active site relative to the rest of the molecule. The observed swivelling motion of RT may allow the polymerase to accommodate the rotational and translational movements of the growing nucleic acid duplex, which present an especial problem for RT because it uses an asymmetric molecule (tRNA(Ly3)) as a primer for first strand synthesis.
Elongation factor G (EF-G) catalyzes the translocation step of protein synthesis in bacteria, and like the other bacterial elongation factor, EF-Tu--whose structure is already known--it is a member of the GTPase superfamily. We have determined the crystal structure of EF-G--GDP from Thermus thermophilus. It is an elongated molecule whose large, N-terminal domain resembles the G domain of EF-Tu, except for a 90 residue insert, which covers a surface that is involved in nucleotide exchange in EF-Tu and other G proteins. The tertiary structures of the second domains of EF-G and EF-Tu are nearly identical, but the relative placement of the first two domains in EF-G--GDP resembles that seen in EF-Tu--GTP, not EF-Tu--GDP. The remaining three domains of EF-G look like RNA binding domains, and have no counterparts in EF-Tu.
The reverse transcriptase from human immunodeficiency virus type 1 is a heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66 subunit contains both a polymerase and an RNase H domain; proteolytic cleavage of p66 removes the RNase H domain to yield the p51 subunit. Although the polymerase domain of p66 folds into an open, extended structure containing a large active-site cleft, that of p51 is closed and compact. The connection subdomain, which lies between the polymerase and RNase H active sites in p66, plays a central role in the formation of the reverse transcriptase heterodimer. Extensive and very different intra- and intersubunit contacts are made by the connection subdomains of each of the subunits. Together, contacts between the two connection domains constitute approximately one-third of the total contacts between subunits of the heterodimer. Conversion of an open p66 polymerase domain structure to a closed p51-like structure results in a reduction in solvent-accessible surface area by 1600 A2 and the burying of an extensive hydrophobic surface. Thus, the monomeric forms of both p66 and p51 are proposed to have the same closed structure as seen in the p51 subunit of the heterodimer. The free energy required to convert p66 from a closed p51-like structure to the observed open p66 polymerase domain structure is generated by the burying of a large, predominantly hydrophobic surface area upon formation of the heterodimer. It is likely that the only kind of dimer that can form is an asymmetric one like that seen in the heterodimer structure, since one dimer interaction surface exists only in p51 and the other only in p66. We suggest that both p51 and p66 form asymmetric homodimers that are assembled from one subunit that has assumed the open conformation and one that has the closed structure.
tRNA(2Gln) made in vitro by transcription with T7 RNA polymerase does not contain the pseudouridines at positions 38, 39, and 55, the 4-thiouridine at position 8, or any of the methylated bases found in the tRNA(2Gln) made in vivo. Cocrystals of unmodified tRNA(2Gln) complexed with glutaminyl-tRNA synthetase from Escherichia coli are isomorphous with those of the complex with modified tRNA(2Gln). A difference electron density map between the complexes with modified and unmodified tRNAs calculated at 2.5-A resolution shows no differences in the protein or tRNA structures, except for some very small shifts in atoms contacting the thiol at the 4 position of uridine 8 that are required to accommodate the smaller oxygen in the unmodified tRNA. Perhaps the most functionally significant change in the unmodified tRNA is the absence of the specifically bound water molecules that are observed to cross-link the N5 of the pseudo-uridines to their 5' phosphate. This suggests a possible role for pseudouridinylation in stabilization of the tRNA through water-mediated linking of these modified bases to the backbone, which is consistent with the lower thermal stability of the unmodified tRNA. An identical water-bridging structure is possible at four of the five other psuedo-uridines in known tRNA structures.
BACKGROUND: gamma delta resolvase is a 20.5 kDa enzyme that catalyzes a site-specific recombination in the second step of the transposition of the gamma delta transposon and requires no cofactors other than Mg2+ for activity. Dimers of resolvase bind cooperatively to DNA at three inverted repeat sequences of differing geometry but catalyze recombination at only one site. RESULTS: The structure of the catalytic domain of gamma delta resolvase, which provides the protein-protein interactions in the synaptic complex, has been refined to an R-factor of 20% at 2.3 A resolution. The structures of the three independent monomers in the asymmetric unit are similar but not identical. Differences occur in the positions of surface loops and in the overall twist of the central beta-sheet of the molecule. The crystal also gives two independent structures for the dimeric form of the molecule, which also show significant differences in the relative orientations of their subunits. CONCLUSION: Resolvase is an unusually flexible protein. This conformational adaptability may be necessary to allow each of the 12 resolvase subunits in the synaptic complex to play a different but specific role in wrapping DNA, binding sites of differing geometry and catalyzing recombination.
The dipyridodiazepinone Nevirapine is a potent and highly specific inhibitor of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). It is a member of an important class of nonnucleoside drugs that appear to share part or all of the same binding site on the enzyme but are susceptible to a variety of spontaneous drug-resistance mutations. The co-crystal-structure of HIV-1 RT and Nevirapine has been solved previously at 3.5-A resolution and now is partially refined against data extending to 2.9-A spacing. The drug is bound in a hydrophobic pocket and in contact with some 38 protein atoms from the p66 palm and thumb subdomains. Most, but not all, nonnucleoside drug-resistance mutations map to residues in close contact with Nevirapine. The major effects of these mutations are to introduce steric clashes with the drug molecule or to remove favorable protein-drug contacts. Additionally, four residues (Phe-227, Trp-229, Leu-234, and Tyr-319) in contact with Nevirapine have not been selected as sites of drug-resistance mutations, implying that there may be limitations on the number and types of resistance mutations that yield viable virus. Strategies of inhibitor design that target interactions with these conserved residues may yield drugs that are less vulnerable to escape mutations.
The packing arrangement of the 12 subunits of intact gamma delta resolvase in the unit cell of a hexagonal crystal form suggests a model for site-specific recombination that involves a DNA-mediated synaptic intermediate. The crystal structure has been determined by molecular replacement and partially refined at 2.8/3.5 A resolution. Although the small DNA-binding domain is disordered in these crystals, packing considerations show that only a small region of space in the crystal could accommodate a domain of its size. A family of related models for a synaptic complex between two DNA duplexes and 12 monomers that are arranged as situated in the crystal is consistent with the known topology of the complex and the distances between the three resolvase dimer-binding sites per DNA; further, these models place the two DNA recombination sites in contact with each other between two resolvase dimers, implying that strand exchange is accomplished through direct DNA-DNA interaction. A major role postulated, then, for the resolvase protein assembly is to stabilize a res DNA structure that is close to the topological transition state of the reaction.
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Crystal structures of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli complexed with deoxynucleoside triphosphate (dNTP) or with pyrophosphate (PPi) determined to 3.9-A resolution by X-ray crystallography show these molecules binding within the cleft of the polymerase domain and surrounded by residues previously implicated in dNTP binding. The dNTP binds adjacent to the O-helix [Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G., & Steitz, T. A. (1985a) Nature 313, 762-766] with its triphosphate moiety anchored by three positively charged residues, Arg 754, Arg 682, and Lys 758, plus His 734 and Gln 708. The dNTP binding site observed in the crystal is consistent with the results of chemical modification including cross-linking and is also near many of the amino acid residues whose mutation affects catalysis [Polesky, A. H., Steitz, T. A., Grindley, N. D. F., & Joyce, C. M. (1990) J. Biol. Chem. 265, 14579-14591; Polesky, A. H., Dahlberg, M. E., Benkovic, S. J., Grindley, N. D. F., & Joyce, C. M. (1992) J. Biol. Chem. 267, 8417-8428]. However, we conclude that the position of at least the dNMP moiety of dNTP in the binary complex is not likely to be the same as in its catalytically relevant complex with primer-template DNA.
The structure of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA2Gln and ATP refined at 2.5-A resolution reveals structural details of the catalytic center and allows description of the specific roles of individual amino acid residues in substrate binding and catalysis. The reactive moieties of the ATP and tRNA substrates are positioned within hydrogen-bonding distance of each other. Model-building has been used to position the glutamine substrate in an adjacent cavity with its reactive carboxylate adjacent to the alpha-phosphate of ATP; the interactions of the carboxyamide side chain suggest a structural rationale for the way in which the enzyme discriminates against glutamate. The binding site for a manganese ion has also been identified bridging the beta- and gamma-phosphates of the ATP. The well-known HIGH and KMSKS sequence motifs interact directly with each other as well as with the ATP, providing a structural rationale for their simultaneous conservation in all class I synthetases. The KMSKS loop adopts a well-ordered and catalytically productive conformation as a consequence of interactions made with the proximal beta-barrel domain. While there are no protein side chains near the reaction site that might function in acid-base catalysis, the side chains of two residues, His43 and Lys270, are positioned to assist in stabilizing the expected pentacovalent intermediate at the alpha-phosphate. Transfer of glutamine to the 3'-terminal tRNA ribose may well proceed by intramolecular catalysis involving proton abstraction by a phosphate oxygen atom of glutaminyl adenylate. Catalytic competence of the crystalline enzyme is directly shown by its ability to hydrolyze ATP and release pyrophosphate when crystals of the ternary complex are soaked in mother liquor containing glutamine.
A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites.
The structure of d(CGCGAAAAAACG)/d(CGTTTTTTCGCG) was determined at 2.3 A resolution in order to deduce the local structural features that give rise to DNA bending by adenine tracts. Whereas all previously reported B-DNA dodecamers have crystallized isomorphously (spacegroup P2(1)2(1)2(1) with unit cell dimensions of a = 24.5 A, b = 40.3 A, c = 65.9 A), the duplex reported here crystallizes in a different lattice (spacegroup P2(1)2(1)2 with unit cell dimensions of a = 44.8 A, b = 66.1 A, c = 42.9 A). The DNA exhibits a 30 degree bend in the helix axis that is 180 degrees away from the 20 degree bend exhibited by the adenine tract DNA crystal structures that have been previously determined. This bend is 90 degrees away from the bend predicted for an adenine tract by solution and gel experiments. The adenine tract is straight and bending occurs in the G+C-rich regions. Comparison of the various adenine tract DNA crystal structures reveals that in all cases the adenine tracts have nearly identical structures, even though the overall bends of the helix axes are quite different. This implies that the structure of the adenine tract is robust, at least under the conditions of crystallization. The base-pairs in the adenine tracts exhibit a large propeller twist that leads to the formation of bifurcated hydrogen bonds and a narrow minor groove. In the crystal structure of d(CGCGAAAAAACG)/d(CGTTTTTTCGCG), a minor groove spine of hydration is observed that probably stabilizes the straight structure of the adenine tract. This straight structure of the A-tract is not consistent with the results of fiber diffraction, gel experiments, and NMR studies. Although this may imply that the results of solution experiments need to be reinterpreted, the conditions under which the crystals were grown are different from those under which the solution experiments were done. The possibility remains that the 2-methyl-2,4-pentanediol necessary for crystal growth may facilitate formation of the spine of hydration that stabilizes the straight A-tract, although recent NMR results show the presence of the spine of hydration in aqueous solution. We have also extended our previously reported observation that non-self-complementary DNA structures can exist in the crystal lattice in two orientations. A dodecamer brominated on one strand provides experimental evidence that d(CGCAAAAAAGCG)/d(CGCTTTTTTG CG) is positioned in two orientations in the crystal lattice that are related by a 180 degree rotation around the pseudo-dyad axis of the sequence.(ABSTRACT TRUNCATED AT 400 WORDS)
Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove. A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site. Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site.