ABSTRACT
Here we report a 2.3-A crystal structure of scallop myosin S1 complexed with ADP.BeF(x), as well as three additional structures (at 2.8-3.8 A resolution) for this S1 complexed with ATP analogs, some of which are cross-linked by para-phenyl dimaleimide, a short intramolecular cross-linker. In all cases, the complexes are characterized by an unwound SH1 helix first seen in an unusual 2.5-A scallop myosin-MgADP structure and described as corresponding to a previously unrecognized actin-detached internally uncoupled state. The unwinding of the SH1 helix effectively uncouples the converter/lever arm module from the motor and allows cross-linking by para-phenyl dimaleimide, which has been shown to occur only in weak actin-binding states of the molecule. Mutations near the metastable SH1 helix that disable the motor can be accounted for by viewing this structural element as a clutch controlling the transmission of torque to the lever arm. We have also determined a 3.2-A nucleotide-free structure of scallop myosin S1, which suggests that in the near-rigor state there are two conformations in the switch I loop, depending on whether nucleotide is present. Analysis of the subdomain motions in the weak actin-binding states revealed by x-ray crystallography, together with recent electron microscopic results, clarify the mechanical roles of the parts of the motor in the course of the contractile cycle and suggest how strong binding to actin triggers both the power stroke and product release.
Subject(s)
Myosins/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Crystallography, X-Ray , Electrons , Models, Molecular , Mollusca , Protein Binding , Protein Conformation , Protein Structure, TertiaryABSTRACT
Shikimate kinase (SK) from Mycobacterium tuberculosis (Mt) was overexpressed in Escherichia coli, purified and cocrystallized with MgADP in hanging drops using the vapor-diffusion procedure with PEG 4000 and 2-propanol as precipitants at pH 7.5. The crystal of MtSK-MgADP, which diffracted to 2.2 A resolution, belonged to space group P3(2)21 or P3(1)21, with unit-cell parameters a = b = 64.01, c = 92.41 A. There was one MtSK molecule in the asymmetric unit. Molecular-replacement trials with the crystal structure of SK from Erwinia chrysanthemi (PDB code 1shk) and adenylate kinase (PDB code 1ake) as search models were not successful. Heavy-atom derivative screening is in progress.
Subject(s)
Adenosine Diphosphate/chemistry , Mycobacterium tuberculosis/enzymology , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Data Collection , Models, Molecular , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Conformation , Recombinant Proteins/chemistrySubject(s)
HIV Infections/epidemiology , HIV Infections/drug therapy , HIV Infections/genetics , HIV Protease Inhibitors/therapeutic use , HIV-1/genetics , HIV-1/isolation & purification , Humans , Polymerase Chain Reaction , Receptors, CCR5/genetics , Reverse Transcriptase Inhibitors/therapeutic use , Risk Factors , Russia/epidemiologyABSTRACT
The crystal structures of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) complexed with phenylalanine and phenylalaninyl-adenylate (PheOH-AMP), the synthetic analogue of phenylalanyl-adenylate, have been determined at 2.7A and 2.5A resolution, respectively. Both Phe and PheOH-AMP are engulfed in the active site cleft of the catalytic alpha-subunit of PheRS, and neither makes contact with the PheRS beta-subunit. The conformations and binding of Phe are almost identical in both complexes. The recognition of Phe by PheRS is achieved through a mixture of multiple van der Waals interactions and hydrogen bonds. The side-chain of the Phe substrate is sandwiched between the hydrophobic side-chains of Phealpha258 and Phealpha260 on one side, and the main-chain atoms of the two adjacent beta-strands on the other. The side-chains of Valalpha261 and Alaalpha314 form the back wall of the amino acid binding pocket. In addition, PheRS residues (Trpalpha149, Seralpha180, Hisalpha178, Argalpha204, Glnalpha218, and Glualpha220) form a total of seven hydrogen bonds with the main-chain atoms of Phe. The conformation of PheOH-AMP and the network of interactions of its AMP moiety with PheRS are reminiscent of the other class II synthetases. The structural similarity between PheRS and histidyl-tRNA synthetase extends to the amino acid binding site, which is normally unique for each enzyme. The complex structures suggest that the PheRS beta-subunit may affect the first step of the reaction (formation of phenylalanyl-adenylate) through the metal-mediated conserved alpha/beta-subunit interface. The modeling of tyrosine in the active site of PheRS revealed no apparent close contacts between tyrosine and the PheRS residues. This result implies that the proofreading mechanism against activated tyrosine, rather than direct recognition, may play the major role in the PheRS specificity.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Phenylalanine-tRNA Ligase/chemistry , Phenylalanine/chemistry , Adenosine Monophosphate/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Macromolecular Substances , Metals/chemistry , Models, Molecular , Molecular Sequence Data , Phenylalanine-tRNA Ligase/genetics , Phenylalanine-tRNA Ligase/metabolism , Protein Conformation , Sequence Homology, Amino Acid , Thermus thermophilus/enzymology , Thermus thermophilus/geneticsSubject(s)
Acquired Immunodeficiency Syndrome , Research , Acquired Immunodeficiency Syndrome/diagnosis , Acquired Immunodeficiency Syndrome/epidemiology , Acquired Immunodeficiency Syndrome/therapy , Animals , Humans , Incidence , International Cooperation , Research/economics , Research/trends , Research Design , Russia/epidemiologyABSTRACT
Elongation factor Tu (EF-Tu) is a G-protein which, in its active GTP conformation, protects and carries aminoacylated tRNAs (aa-tRNAs) to the ribosome during protein biosynthesis. EF-Tu consists of three structural domains of which the N-terminal domain consists of two special regions (switch I and switch II) which are structurally dependent on the type of the bound nucleotide. Structural studies of the complete functional cycle of EF-Tu reveal that it undergoes rather spectacular conformational changes when activated from the EF-Tu.GDP form to the EF-Tu.GTP form. In its active form, EF-Tu.GTP without much further structural change interacts with aa-tRNAs in the so-called ternary complex. The conformational changes of EF-Tu involve rearrangements of the secondary structures of both the switch I and switch II regions. As the switch II region forms part of the interface between domains 1 and 3, its structural rearrangement results in a very large change of the position of domain 1 relative to domains 2 and 3. The overall shape of the ternary complex is surprisingly similar to the overall shape of elongation factor G (EF-G). Thus, three domains of the protein EF-G seem to mimic the tRNA part of the ternary complex. This macromolecular mimicry has profound implications for the function of the elongation factors on the ribosome.
Subject(s)
Molecular Mimicry , Protein Biosynthesis , Amino Acid Sequence , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Peptide Chain Elongation, Translational , Peptide Elongation Factor Tu/chemistry , Peptide Elongation Factor Tu/genetics , Peptide Elongation Factor Tu/metabolism , Protein Folding , Protein Structure, Secondary , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: In the translation of the genetic code each aminoacyl-tRNA synthetase (aaRS) must recognize its own (cognate) tRNA and attach the corresponding amino acid to the acceptor end of tRNA, discriminating all the others. The(alphabeta)2 phenylalanyl-tRNA synthetase (PheRS) is one of the most complex enzymes in the aaRS family and is characterized by anomalous charging properties. Structurally, the enzyme belongs to class II aaRSs, as its catalytic domain is built around an antiparallel beta sheet, but functionally it resembles class I as it aminoacylates the 2'OH of the terminal ribose of tRNA (class II aaRSs aminoacylate the 3'OH). With the availability of the three-dimensional structure of the complex between multisubunit PheRS and tRNAPhe, a fuller picture of the specific tRNA-aaRS interactions is beginning to emerge. RESULTS: The crystal structure of Thermus thermophilus PheRS complexed with cognate tRNA has been solved at 3.28 A resolution. It reveals that one tRNAPhe molecule binds across all four PheRS subunits. The interactions of PheRS with tRNA stabilize the flexible N-terminal part of the alpha subunit, which appeared to form the enzyme's 11th domain, comprising a coiled-coil structure (helical arm) built up of two long antiparallel alpha helices. The helical arms are similar to those observed in SerRS and are in the same relative orientation with respect to the catalytic domain. Anticodon recognition upon tRNA binding is performed by the B8 domain, the structure of which is similar to that of the RNA-binding domain (RBD) of the small spliceosomal protein U1A. The Th. thermophilus PheRS approaches the anticodon loop from the minor groove side. CONCLUSIONS: The mode of interactions with tRNA explains the absolute necessity for the (alphabeta)2 architecture of PheRS. The interactions of tRNAPhe with PheRS and particularly with the coiled-coil domain of the alpha subunit result in conformational changes in TPsiC and D loops seen by comparison with uncomplexed yeast tRNAPhe. The tRNAPhe is a newly recognized type of RNA molecule specifically interacting with the RBD fold. In addition, a new type of anticodon-binding domain emerges in the aaRS family. The uniqueness of PheRS in charging 2'OH of tRNA is dictated by the size of its adenine-binding pocket and by the local conformation of the tRNA's CCA end.
Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , RNA, Transfer, Phe/chemistry , Thermus thermophilus/enzymology , Anticodon/genetics , Base Sequence , Binding Sites , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , RNA, Transfer, Phe/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolismABSTRACT
Kirromycin inhibits bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). Complexes of the antibiotic, Phe-tRNA(Phe), the guanosine triphosphate analog GDPNP, and mesophilic (Escherichia coli), as well as thermophilic (Thermus thermophilus) EF-Tu were isolated. Crystallization was achieved at 4 degrees C, pH 6.4, using ammonium sulphate as precipitant. Crystallographic data were recorded at cryogenic temperature on crystals exposed to synchrotron radiation. Crystals of the thermophilic complex are based on a rhombohedral lattice with cell dimensions of 137.3 A, and angles of 54.0 degrees. Although related, these cell parameters are different from those found in the crystals of the recently solved structure of the ternary complex of Phe-tRNA(Phe), GDPNP, and Thermus aquaticus EF-Tu (Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F. and Nyborg, J. (1995) Science 270, 1464-1472 [1]), possibly indicating some allosteric effect caused by kirromycin. Crystals of the mesophilic complex belong to the cubic space P432, with cell axis of 196.26 A. In both cases, the crystals contain one complex per asymmetric unit.
Subject(s)
Guanosine Triphosphate/analogs & derivatives , Peptide Elongation Factor Tu/chemistry , RNA, Transfer, Amino Acyl/chemistry , Guanosine Triphosphate/chemistry , Pyridones/chemistry , X-Ray DiffractionABSTRACT
The structure of the ternary complex consisting of yeast phenylalanyl-transfer RNA (Phe-tRNAPhe), Thermus aquaticus elongation factor Tu (EF-Tu), and the guanosine triphosphate (GTP) analog GDPNP was determined by x-ray crystallography at 2.7 angstrom resolution. The ternary complex participates in placing the amino acids in their correct order when messenger RNA is translated into a protein sequence on the ribosome. The EF-Tu-GDPNP component binds to one side of the acceptor helix of Phe-tRNAPhe involving all three domains of EF-Tu. Binding sites for the phenylalanylated CCA end and the phosphorylated 5' end are located at domain interfaces, whereas the T stem interacts with the surface of the beta-barrel domain 3. The binding involves many conserved residues in EF-Tu. The overall shape of the ternary complex is similar to that of the translocation factor, EF-G-GDP, and this suggests a novel mechanism involving "molecular mimicry" in the translational apparatus.
Subject(s)
Guanosine Triphosphate/analogs & derivatives , Peptide Elongation Factor Tu/chemistry , RNA, Transfer, Amino Acyl/chemistry , Amino Acid Sequence , Anticodon , Base Sequence , Binding Sites , Crystallography, X-Ray , Guanosine Diphosphate/chemistry , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Histidine/metabolism , Lysine/metabolism , Models, Molecular , Molecular Mimicry , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Elongation Factor G , Peptide Elongation Factor Tu/metabolism , Peptide Elongation Factors/chemistry , Peptide Elongation Factors/metabolism , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Peptide Termination Factors/chemistry , Peptide Termination Factors/metabolism , Prokaryotic Initiation Factor-2 , Protein Biosynthesis , Protein Conformation , Protein Structure, Secondary , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/metabolism , ThermusSubject(s)
Acquired Immunodeficiency Syndrome , Acquired Immunodeficiency Syndrome/epidemiology , Acquired Immunodeficiency Syndrome/prevention & control , Acquired Immunodeficiency Syndrome/therapy , HIV/genetics , HIV/physiology , HIV Infections/epidemiology , HIV Infections/prevention & control , HIV Infections/therapy , Humans , Membrane Fusion , Russia/epidemiologySubject(s)
Acquired Immunodeficiency Syndrome , HIV-1 , HIV-2 , Acquired Immunodeficiency Syndrome/diagnosis , Acquired Immunodeficiency Syndrome/etiology , Acquired Immunodeficiency Syndrome/prevention & control , Acquired Immunodeficiency Syndrome/therapy , Acquired Immunodeficiency Syndrome/virology , HIV Infections/diagnosis , HIV Infections/etiology , HIV Infections/prevention & control , HIV Infections/therapy , HIV Infections/virology , HIV-1/isolation & purification , HIV-2/isolation & purification , Humans , Research , RussiaABSTRACT
The crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus, solved at 2.9 A resolution, displays (alpha beta)2 subunit organization. Unexpectedly, both the catalytic alpha- and the non-catalytic beta-subunits comprise the characteristic fold of the class II active-site domains. The alpha beta heterodimer contains most of the building blocks so far identified in the class II synthetases. The presence of an RNA-binding domain, similar to that of the U1A spliceosomal protein, in the beta-subunit is indicative of structural relationships among different families of RNA-binding proteins. The structure suggests a plausible catalytic mechanism which explains why the primary site of tRNA aminoacylation is different from that of the other class II enzymes.
Subject(s)
Phenylalanine-tRNA Ligase/ultrastructure , Thermus thermophilus/enzymology , Adenosine Triphosphate/metabolism , Bacillus subtilis/enzymology , Bacterial Proteins/ultrastructure , Base Sequence , Binding Sites , Biological Evolution , Crystallography, X-Ray , Escherichia coli/enzymology , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The crystal structure of a recombinant form of the proteinase encoded by the feline immunodeficiency virus (FIV PR) has been solved at 2 A resolution and refined to an R-factor of 0.148. The refined structure includes a peptidomimetic, statine-based inhibitor, LP-149, which is an even more potent inhibitor of HIV PR. Kinetic parameters were obtained for the cleavage of five substrates by FIV PR, and inhibition constants were measured for four inhibitors. The structure of FIV PR resembles other related retroviral enzymes although few inhibitors of HIV PR are capable of inhibiting FIV PR. The structure of FIV PR will enhance our knowledge of this class of enzymes, and will direct testing of new proteinase inhibitors in a feline animal model.
Subject(s)
Aspartic Acid Endopeptidases/chemistry , Endopeptidases/chemistry , Immunodeficiency Virus, Feline/enzymology , Oligopeptides/chemistry , Protease Inhibitors/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/isolation & purification , Aspartic Acid Endopeptidases/metabolism , Binding Sites/physiology , HIV Protease/chemistry , HIV Protease/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Molecular Structure , Oligopeptides/metabolism , Peptides/metabolism , Protease Inhibitors/metabolism , Protein Conformation , Sequence Alignment , Statistics as Topic , Substrate Specificity , Viral Proteins/metabolism , X-Ray DiffractionABSTRACT
Elongation factor Tu (EF-Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G-proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF-Tu binds aminoacylated tRNA (aa-tRNA) forming the ternary complex EF-Tu:GTP:aa-tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF-Tu:GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF-Tu:GDPNP and yeast Phe-tRNA(Phe) after its purification by HPLC.
