[Codases: fifty years after].

Aminoacyl-tRNA synthetases (codases) catalyze aminoacylation of a particular tRNA with the corresponding amino acid at the first step of protein biosynthesis. The review considers universal structure-functional characteristics of the largest family of enzymes partitioned into two classes. Modes of tRNA binding and recognition, and additional editing activity, which are responsible for the fidelity of aminoacyl-tRNA synthesis, are discussed. The aaRSs catalytic cores are highly relevant to the ancient metabolic reactions, namely, amino acids and cofactors biosynthesis. Thus, the biosynthetic machinery for producing amino acids had a profound effect on almost every aspect of aminoacylation reaction. The review also deals with secondary functions of synthetases in various processes of cell metabolism. Certain of these functions have to do with complex pathophysiological mechanisms involved in disease production. Their investigation may help to develop new diagnostic techniques and therapies.

Structure at 2.6 A resolution of phenylalanyl-tRNA synthetase complexed with phenylalanyl-adenylate in the presence of manganese.

The crystal structure of phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus, a class II aminoacyl-tRNA synthetase, complexed with phenylalanyl-adenylate (Phe-AMP) was determined at 2.6 A resolution. Crystals of native PheRS were soaked in a solution containing phenylalanine and ATP in the presence of Mn(2+) ions. The first step of the aminoacylation reaction proceeds within the crystals, resulting in Phe-AMP formation at the active site. Specific recognition of the phenylalanine portion of the Phe-AMP is achieved by interactions of the phenyl ring of Phe-AMP with two neighbouring residues, Phealpha258 and Phealpha260. No manganese ions were observed within the active site; their role in the formation of the transition state may be assigned to a number of polar residues and water molecules. In the anomalous Fourier difference map, a divalent metal ion was detected at the interface of the alpha- and beta-subunits at a short distance from motif 3 residues participating in the substrate binding. A sulfate ion, which was identified on the protein surface, may mediate the interactions of PheRS with DNA. Visible conformational changes were detected in the active-site area adjacent to the position of the Phe-AMP, compared with the structure of PheRS complexed with a synthetic adenylate analogue (phenylalaninyl-adenylate). Based on the known structures of the substrate-free enzyme and its complexes with various ligands, a general scheme for the phenylalanylation mechanism is proposed.

Two p16 (CDKN2A) germline mutations in 30 Israeli melanoma families.

Germline mutations in the p16 (CDKN2A) tumour suppressor gene have been linked to inherited predisposition to malignant melanoma (MM). Variable frequencies of p16 germline mutations were reported in different collections of melanoma families but it can be as high as 50%. Here we describe the results of p16 mutation screening in 30 melanoma kindreds in Israel. The entire coding region of the p16 gene, including exons 1, 2 and 3, flanking exon/intron junctions, and a portion of the 3' untranslated (UTR) region of the gene were examined by single-stranded conformation polymorphism (SSCP) analysis and direct sequencing. Two p16 germline mutations were identified: G101W, which has been previously observed in a number of melanoma kindreds, and G122V, a novel missense mutation. Thus, the frequency of mutations identified in this collection of Israeli families was 7%. Functional analysis indicated that the novel G122V variant retained some capacity to interact with cyclin dependent kinases (CDKs) in vitro, yet it was significantly impaired in its ability to cause a G1 cell cycle arrest in human diploid fibroblasts. This partial loss of function is consistent with the predicted impact of G122V substitution on the 3-dimensional structure of the p16 protein.

Preliminary crystallographic study of turkey gizzard vinculin.

Vinculin is a 117 kDa microfilament-associated protein located at the cytoplasmic aspects of focal contacts and cell-cell adherens type junctions. In both sites, vinculin participates in the formation of a submembrane 'plaque' structure which is responsible for the attachment of actin filaments to the plasma membrane. Vinculin consists of 1066 amino acids, which form a large 90 kDa globular head domain and a rod-like 29 kDa tail domain. The two domains are separated by several stretches of proline residues where the major proteolytic cleavage sites are located. The experimental procedure for isolation and purification of vinculin from smooth muscle has been developed and crystals of native vinculin suitable for X-ray analysis have been obtained. The homogeneity of the vinculin solution was analyzed prior to crystallization using dynamic light scattering. Crystals of vinculin have been obtained in buffer containing 2 mg ml(-1) protein, 0.9 M ammonium sulfate, 0.1 M MES pH 6.5 using both the hanging-drop and sitting-drop vapour-diffusion methods. The crystals have the form of rhombic plates and grow to maximal dimensions of 0.3 x 0.3 x 0.05 mm in two weeks. Preliminary X-ray data show that the crystals diffract to 3.5 A resolution at the X11 beamline of DESY and belong to the monoclinic space group P2(1). Crystal unit-cell parameters are estimated to be a = 57, b = 351, c = 70 A, alpha = 90, beta = 113, gamma = 90 degrees.

Interaction of 4,4'-dithiodipyridine with Cys(458) triggers disassembly of GroEL.

Chaperonin GroEL, consisting of two seven-subunit rings stacked back-to-back, is disassembled by interaction of 4, 4'-dithiodipyridine (DTP) with Cys(458) located close to the intersubunit contacts within and between the rings. The thiol group of Cys(458) is inaccessible to the probe being buried into the pocket locked by segment Asn(475)-Asn(487). Flexibility of this segment is proposed to induce the "open" state of the pocket and accommodate the bulky probe inside so that the consequential irreversible shifts in the pocket constituents disassemble GroEL. This scheme is supported by the finding that DTP-induced disassembly of GroEL is facilitated by ATP, which specifically stimulates a local shift of the segment Asn(475)-Asn(487) into solution.

Human phenylalanyl-tRNA synthetase: cloning, characterization of the deduced amino acid sequences in terms of the structural domains and coordinately regulated expression of the alpha and beta subunits in chronic myeloid leukemia cells.

Unlike the catalytic alpha-subunit, the beta-subunit of heterodimeric (alphabeta)2 phenylalanyl-tRNA synthetase (PheRS) has no invariant functional amino acids directly involved in the aminoacylation process as it is evident from the crystal structure of the T. thermophilus enzyme complexed with tRNAPhe. Having no catalytic function, the prokaryotic beta-subunit comprises OB-, RNP-, SH3-, and DNA-binding-like domains involved in a variety of biological functions in other proteins. It was shown that the mRNA of the human alpha-subunit overexpressed in the tumorigenic versus the nontumorigenic variant of the same acute-phase chronic myeloid leukemia cell line (CML). We cloned, sequenced, and expressed human PheRS. The layout of the human sequence indicates that the general tRNA binding mode and anticodon recognition differ between prokaryotes and eukaryotes for the phenylalanine system. Northern blot hybridization analysis from malignant and normal human tissues enabled us to assess the relative expression levels of the alpha- and beta-subunits independently, in view of the additional cellular role proposed for the beta-subunit in tumorigenic events. The levels of mRNA corresponding to the alpha- and beta-subunits were remarkably similar in all cell types and tissues examined, thus indicating the implication of the entire (alphabeta)2 heterodimer in tumorigenic events.

The crystal structure of phenylalanyl-tRNA synthetase from thermus thermophilus complexed with cognate tRNAPhe.

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.

Structural similarities in the noncatalytic domains of phenylalanyl-tRNA and biotin synthetases.

Detailed comparison between the structures of the Escherichia coli biotin synthetase/repressor protein (BirA) and the recently solved Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) reveals significant similarities outside their respective catalytic domains. These comprise a DNA-binding alpha+beta domain and an Src-homology 3 (SH3)-like domain that were observed in both enzymes. This similarity provides a novel example in which all domains of one multidomain protein appear to be constituents of the other multidomain protein and supports a concept of a common ancestor for two different synthetase families.

Structure of phenylalanyl-tRNA synthetase from Thermus thermophilus.

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.

Aminoacyl-tRNA synthetases from Haloarcula marismortui: an evidence for a multienzyme complex in a procaryotic system.

Aminoacyl-tRNA synthetases in halophilic archaebacterium Haloarcula marismortui form a high molecular weight multienzyme complex which is resistant to dissociation when subjected to gel filtration, ion exchange, ammonium sulfate mediated and hydroxyapatite chromatography. Functional and structural aspects of the aminoacyl-tRNA synthetase complex formation are discussed.

Crystals of the phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 complexed with tRNA(Phe).

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) from the extreme thermophile Thermus thermophilus HB8 has been crystallized with its cognate tRNA. Compared with the native crystals, the crystals of the complex are more stable to radiation damage and diffract to 3.0 A resolution. They are of space group P3(2)21, with a = b = 175 A, c = 142.1 A, gamma = 120 degrees, almost identical with the crystal parameters of the native synthetase.

Phenylalanyl-tRNA synthetase from Thermus thermophilus has four antiparallel folds of which only two are catalytically functional.

Phenylalanyl-tRNA synthetase from Thermus thermophilus has an alpha 2 beta 2 type quaternary structure and is one of the most complicated members of the synthetase family. Identification of PheRSTT as a member of class II aaRSs was based only on sequence alignment of the small alpha-subunit with other synthetases. The three-dimensional crystal structure of the catalytic and 'catalytic-like' domains at 2.9 A resolution in PheRSTT is described. The alpha-subunit contains an antiparallel fold which includes signature motifs 1, 2 and 3, characteristic of class II synthetases. One of the three structural domains of the beta-subunit (alpha'-domain) is formed by a seven-stranded antiparallel beta-sheet surrounded by alpha-helices similar to catalytic domains in SerRS, AspRS and the alpha-subunit of PheRSTT. The alpha beta heterodimer (alpha and alpha') exhibits essentially the same topology in the intersubunit region as in the known alpha 2 structures of class II aaRS's. The multimerization area of whole PheRSTT molecule comprises a quasi-tetrahedral four-helix bundle.

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.

X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin.

The structure of calf chymosin (EC 3.4.23.3), the aspartic proteinase from the gastric mucosa, was solved using the technique of molecular replacement. We describe the use of different search models based on distantly related fungal aspartic proteinases and investigate the effect of using only structurally conserved regions. The structure has been refined to a crystallographic R-factor of 17% at 2.2 A resolution with an estimated co-ordinate error of 0.21 A. In all, 136 water molecules have been located of which eight are internal. The structure of chymosin resembles that of pepsin and other aspartic proteinases. However, there is a considerable rearrangement of the active-site "flap" and, in particular, Tyr75 (pepsin numbering), which forms part of the specificity pockets S1 and S1'. This is probably a consequence of crystal packing. Electrostatic interactions on the edge of the substrate binding cleft appear to account for the restricted proteolysis of the natural substrate kappa-casein by chymosin. The local environment of invariant residues is examined, showing that structural constraints and side-chain hydrogen bonding can play an important role in the conservation of particular amino acids.

On the role of peripheral interactions in specificity of chymosin.

Chymosin is distinguished by a high level of milk-clotting activity which is the consequence of the specific cleavage of the Phe(105)-Met(106) bond of kappa-casein. Based on modelling considerations it was proposed that milk-clotting activity of chymosin is associated with electrostatic interactions of a charged segment His-Pro-His-Pro-His (98-102) of casein and the outer loop of the enzyme containing Glu-244,Asp-246 and Asp-248.

[Various aspects of structural studies of aspartate proteinases]. / Nekotorye aspekty strukturnykh issledovanii aspartatnykh proteinaz.

The paper is a brief account of aspartic proteinases' structural studies developed in V.A. Engelhardt Institute of Molecular Biology during the last 3 years. The work on porcine pepsin has been finalized after the refinement of the monoclinic crystal form at 1.8 A resolution performed in collaboration with the group of protein structure and function studies of the University of Alberta in Canada. An important structural property of chymosin which explains the enzyme specificity has been found. Protein engineering work on chymosin is being developed. The structural template for aspartic proteinases has been elucidated and on the basis of this template the model of HIV-1 protease molecule has been built. Some approaches to the design of HIV-1 protease inhibitors were elucidated.

Preliminary crystallographic study of the phenylalanyl-tRNA synthetase from Thermus thermophilus HB8.

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) from the extreme thermophile Thermus thermophilus HB8 has been isolated and crystallized. The enzyme was found to consist of two types of subunits with molecular masses 38 X 10(3) (alpha) and 94 X 10(3) (beta) and is likely to be a tetrameric protein with a molecular mass of about 260 X 10(3) (alpha 2 beta 2). Crystals of phenylalanyl-tRNA synthetase were grown by the hanging-drop technique at 4 degrees C in the presence of ammonium sulfate. Trigonal crystals, space group P3(1)21, with cell dimensions a = b = 176 A and c = 142 A (1 A = 0.1 nm), are suitable for medium-resolution X-ray analysis.

[The role of peripheral interactions in chymosin specificity]. / O roli perifericheskikh vzaimodeistvii v spetsifichnosti khimozina.

Kinetic parameters for the splitting of model peptide substrates and chi-casein with chymosin have been interpreted on the basis of the three-dimensional structure of chymosin. Model peptide substrates contain a fragment of the chi-casein sequence in the region of the bond Phe-105--Met-106 splitted with the enzyme. It was shown that the possible reason of the enormous milk-clotting efficiency of chymosin may be partly associated with the electrostatic interaction of the positive charged segment 98-102 (His-Pro-His-Pro-His) of the substrate and outer loop of the enzyme which contains Glu-245, Asp-247, Asp-249, Asp-251.

[X-ray study of chymosin. I. Molecular replacement at a 3 angstroms resolution]. / Rentgenostrukturnye issledovaniia khimozina. I. Molekuliarnoe zameshchenie pri razreshenii 3 angstrom.

The determination of the three dimensional structure of chymosin at 3 A resolution by molecular replacement method is described. The rotation functions for various aspartic proteases were calculated and combined results were used for the refinement of orientational parameters of chymosin molecules in the unit cell. The interpretation of Crowther-Blow translation function map with packing consideration enable to place correctly the molecules in the chymosin unit cell. Several difference Fourier syntheses for chymosin were calculated and differences between pepsin and chymosin structures were detected.

[X-ray structural analysis of pepsin. V. Conformation of the main chain of the enzyme]. / Rentgenostrukturnyi analiz pepsina. V. Konformatsiia glavnoi tsepi fermenta.

A detailed description of structural investigations of pepsin from 3.5 to 2.7 A resolution are given. The main attention is drawn to the conformation of the polypeptide backbone of the enzyme. The numbers of amino acid residues involved in the formation of various structural elements are listed. The structure of pepsin is similar to that of acid proteases isolated from lower organisms. The two domain structure of all acid proteases has a periodicity in the sequence of beta-segments and helices and a very specific symmetrical structure of each domain. This makes it possible to describe the structure of acid proteases in simple terms.