Ludmila Reshetnikova

Structure of phenylalanyl-tRNA synthetase from Thermus thermophilus.

The crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus, solved at 2.9 Å resolution, displays (αβ)2 subunit organization. Unexpectedly, both the catalytic α- and the non-catalytic β-subunits comprise the characteristic fold of the class II active-site domains. The αβ heterodimer contains most of the building blocks so far identified in the class II synthetases. The presence of an RNA-binding domain, similiar to that of the U1A spliceosomal protein, in the β-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 tRIMA aminoacylation is different from that of the other class II enzymes.

Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis.

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.

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.

Crystallographic study of γ-crystallins from calf lens

Crystals of γ-crystallins II and IIIb from calf eye lens have been obtained. They are suitable for X-ray structure analysis with a resolution of about 1·8 and 2·8 A, respectively. The crystal form of γ-crystallin II belongs to space group P 4 1 2 1 2 or P 4 3 2 1 2 with unit cell dimensions a = b =58·0 A, and c =98·6 A. The assumed number of protein molecules per asymmetric part of the unit cell is one. The crystal form of γ-crystallin IIIb belongs to space group P 2 1 2 1 2 1 with unit cell dimensions a =58·6 A, b =69·8 A, c =117·6 A. The assumed number of protein molecules per asymmetric part of the unit cell is two. In both cases the packing of molecules inside the unit cell seems to be similar.

Crystallization and preliminary X-ray diffraction analysis of shikimate kinase from Mycobacterium tuberculosis in complex with MgADP.

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.

Crystallization and preliminary X-ray diffraction studies of NusG, a protein shared by the transcription and translation machines.

N-utilization factor G (NusG) from Aquifex aeolicus (Aa) was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion technique. The drops consisted of 2.5 microl protein solution (approximately 30 mg ml(-1) in 20 mM Tris-HCl pH 8.0, 200 mM NaCl, 2 mM EDTA and 10 mM DTT) and 2.5 microl reservoir solution (0.085 M Na HEPES pH 7.5, 15% glycerol, 11% 2-propanol and 20% PEG 4000) derived from condition number 41 of the Hampton Cryo Screen. The crystals grew at 291 +/- 1 K and reached dimensions of 0.2 x 0.1 x 0.05 mm in 5-7 d. The crystals, which diffracted to 2.45 A resolution, belonged to space group C222(1), with unit-cell parameters a = 65.95, b = 124.58, c = 83.60 A. One AaNusG molecule is present in the asymmetric unit, corresponding to a solvent content of 59.80% (Matthews coefficient = 3.06 A(3) Da(-1)). Crystal structure determination is in progress.

Crystallographic study of the large tryptic fragments of elongation factor G from Escherichia coli

Crystals of N- and C-terminal fragments of elongation factor G (EF-G) from Escherichia coli have been grown from the preparations obtained by limited tryptic hydrolysis. Molecular masses of these fragments are equal to about 49,000 and 25,000, respectively. In the form of an additive complex they appear to be a major part of the native spatial structure of EF-G (Alakhov et al., 1979). Crystals of N-terminal fragment belong to space group P41212 or P43212 with unit cell dimensions a = b = 76.6 A and c = 191.6 A. Crystals of C-terminal fragment belong to space group P4122 or P4322 with unit cell dimensions a = b = 77.1 A and c = 75.0 A. In both cases the assumed number of protein molecules per asymmetric part of the unit cell is one.