David M. Blow

An Automated System for Micro-Batch Protein Crystallization and Screening

An automatic sample dispenser has been constructed to aid with protein crystallization trials. This dispenser contains a bank of Hamilton syringes driven by stepper motors under computer control which is used to set up small samples (2 μl or less) for batch crystallization. Software has been written to create a series of trials which form a two-dimensional array of crystallization conditions. A specially designed fluoropolymer multibore microtip allows the very small volumes to be mixed and dispensed with great accuracy.

Structure of α-chymotrypsin refined at 1.68 Å resolution☆

Diffraction data for α-chymotrypsin crystals at −10 °C were measured at 1·68 A resolution and refined by restrained structure-factor least-squares refinement. The two independent chymotrypsin molecules in the crystallographic asymmetric unit were refined independently. The overall structure of α-chymotrypsin is little changed from published co-ordinates. The root-mean-square shift of Cα co-ordinates is 0·42 A, co-ordinates for the two molecules showing a root-mean-square difference of 0·19 A. Certain regions with high disorder (residues 9 to 14, 73 to 79) remain difficult to interpret and several side-chains are disordered. Some water molecule positions have been changed. The absence of the tosyl group has made a significant difference to the refined structure at the active site. This now agrees closely with other enzymes of the trypsin family that have been refined at high resolution. There is a strong hydrogen bond between Ne2 (His57) and Oγ (Ser195) in the free enzyme, in line with the published description of the charge relay system.Diffraction data for alpha-chymotrypsin crystals at -10 degrees C were measured at 1.68 A resolution and refined by restrained structure-factor least-squares refinement. The two independent chymotrypsin molecules in the crystallographic asymmetric unit were refined independently. The overall structure of alpha-chymotrypsin is little changed from published co-ordinates. The root-mean-square shift of C alpha co-ordinates is 0.42 A, co-ordinates for the two molecules showing a root-mean-square difference of 0.19 A. Certain regions with high disorder (residues 9 to 14, 73 to 79) remain difficult to interpret and several side-chains are disordered. Some water molecule positions have been changed. The absence of the tosyl group has made a significant difference to the refined structure at the active site. This now agrees closely with other enzymes of the trypsin family that have been refined at high resolution. There is a strong hydrogen bond between N epsilon 2 (His57) and O gamma (Ser195) in the free enzyme, in line with the published description of the charge relay system.

Microbatch crystallization under oil — a new technique allowing many small-volume crystallization trials

Abstract An approach to rapid protein crystallization using very small samples is described. A computer controlled microdispenser is used to make crystallization samples as microbatch droplets under oil. Samples of 1–2 μl are dispensed ready-mixed and with good precision. The samples are protected from evaporation, contamination and physical shock by the oil. When favourable conditions for crystallization have been found using one mode of the system, the size and quantity of crystals are optimized by a second program which generates a set of conditions throughout the area of interest. Crystals of diffraction size and quality have been grown in 1 μl drops.

Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy

BACKGROUND Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, carboxypeptidase G2 (CPG2), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. RESULTS We have determined the crystal structure of CPG2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. CONCLUSIONS We find that the CPG2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica, where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.

Tyrosyl-tRNA synthetase forms a mononucleotide-binding fold.

Abstract Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a dimeric molecule of approximately 90,000 M r . The crystal structure originally reported by Irwin et al. (1976) has been re-interpreted using a new density-modification technique. The reinterpretation is confirmed by the complete amino acid sequence (D. Barker & (G. Winter, personal communication). The structure consists of an amino-terminal α β domain, a domain containing five α-helices, and a region of 99 amino acids at the carboxyl terminus, which appears to be disordered. The re-interpretation reveals two new α-helices in the α β domain, and some changes in chain connections. The strands of the β-sheet are in the order A, F, E, B, C, D, with A antiparallel to the others. The arrangement of strands B to F is topologically identical to arrangements found in many other proteins, including the first five strands of the sheet in the NAD-binding domain of the dehydrogenases. Strands B, C, D form a mononucleotide-binding fold. In the complex with tyrosyl adenylate (Rubin & Blow, 1981), an intermediate in the reaction catalysed by the enzyme, the adenine lies near the carboxyl-terminal end of strand F of the β-sheet, with the ribose between the ends of strands B and E. This is similar to the nicotinamide position in dehydrogenases. The tyrosine moiety occupies a pocket at one side of the sheet, close to strands B and C. This tyrosine orientation is quite different from any part of the coenzyme in dehydrogenases. The ends of strands C and D of the sheet are buried, and binding of a nucleotide to the mononucleotide-binding fold formed by strands B, C, D would require a substantial structural change.

Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine☆

The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 A resolution using molecular replacement techniques. The genetically engineered molecule catalyses the activation of tyrosine with kinetic properties similar to those of the wild-type enzyme but no longer binds tRNATyr. It contains 319 residues corresponding to the region of the polypeptide chain for which interpretable electron density is present in crystals of the wild-type enzyme. The partly refined model of the wild-type enzyme was used as a starting point in determining the structure of the truncated mutant. The new crystals are of space group P2(1) and contain the molecular dimer within the asymmetric unit. The refined model has a crystallographic R-factor of 18.7% for all reflections between 8 and 2.5 A. Each subunit contains two structural domains: the alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet and the alpha-helical domain (residues 248 to 319) containing five helices. The alpha/beta domains are related by a non-crystallographic dyad while the alpha-helical domains are in slightly different orientations in the two subunits. The tyrosine substrate binds in a slot at the bottom of a deep active site cleft in the middle of the alpha/beta domain. It is surrounded by polar side-chains and water molecules that are involved in an intricate hydrogen bonding network. Both the alpha-amino and hydroxyl groups of the substrate make good hydrogen bonds with the protein. The amino group forms hydrogen bonds with Tyr169-OH, Asp78-OD1 and Gln173-OE1. The phenolic hydroxyl group forms hydrogen bonds with Asp76-OD1 and Tyr34-OH. In contrast, the substrate carboxyl group makes no direct interactions with the enzyme. The results of both substrate inhibition studies and site-directed mutagenesis experiments have been examined in the light of the refined structure.

Controlled nucleation of protein crystals

Control of nucleation may be needed to obtain a reliable supply of large protein crystals, when standard techniques give many small or twinned crystals. Heterogeneous nucleation may be controlled by the use of fine filters, with the elimination of airborne contaminants by working under paraffin oil. The area of contact with the supporting vessel also has an important effect. A heterogeneous nucleant for lysozyme (identified earlier) has been shown to be effective for carboxypeptidase G2. Control of homogeneous nucleation (previously demonstrated by dilutions of a nucleating sample after various times of incubation) may also be achieved by incubating a sample at 1 temperature, where nucleation can occur, and changing the temperature to conditions where there is growth but no nucleation.

Structural homology in the amino-terminal domains of two aminoacyl-tRNA synthetases

The three-dimensional structures of two animoacyl-tRNA synthetases, the methionyl-tRNA synthetase from Escherichia coli (MetRS) and the tyrosyl-tRNA synthetase from Bacillus stearothermophilus (TyrRS), show a remarkable similarity over a span of about 140 amino acids. The region of homologous folding corresponds to a five-stranded parallel beta-sheet, including a mononucleotide-binding fold. One cysteine and two histidine residues that were found to be invariant in the amino acid sequences occupy similar places in the nucleotide-binding fold. In TyrRS, these residues are close to the adenylate binding site, and in MetRS to the Mg2+-ATP binding site.

So do we understand how enzymes work

Between 1930 and 1975 biochemical and structural analysis of enzymes led to a clear set of ideas that might form a basis for detailed understanding of enzyme action. Further development required energetic and thermodynamic analysis of enzymes in an aqueous medium, beyond the computational power then available. Structural enzymology advanced in other directions, but the fundamental questions of enzyme action must soon be re-opened.

Amino acid activation in crystalline tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

Abstract A crystalline complex of tyrosyl adenylate with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was prepared by the catalytic action of the crystalline enzyme, on soaking with saturated tyrosine, followed by an excess of ATP. Difference Fourier analysis shows that tyrosyl adenylate takes up a conformation similar to that previously observed for the inhibitor tyrosinyl adenylate ( Monteilhet & Blow, 1978 ). The tyrosyl adenylate density straddles the central β-sheet of the structure, the adenosine and tyrosine moieties lying on opposite sides of the sheet. Formation of the tyrosyl adenylate complex is accompanied by extensive changes in enzyme structure, which are not observed in the tyrosinyl adenylate complex. The trinucleoside diphosphate CpCpA, soaked into crystals at the same time as ATP, did not appear to bind at any localized site. When puromycin is soaked into tyrosyl-tRNA synthetase crystals, strong positive electron density differences are found in the tyrosine binding site, but the changes elsewhere are weak and disconnected. Several small oligonucleotides were soaked into crystals and studied in projection. All indicate binding at, or near, the tyrosine binding site. Difference density for arsenate substitution of the mother liquor was also calculated, showing peaks of density in the binding site and over the surface of the molecule.