Prediction of protein crystallization outcome using a hybrid method.

The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R(30), the ratio between a protein's DSF intensity at 30°C and at T(m). This hybrid model is shown to be more powerful than sequence-based prediction alone - and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories.

Use of thermal melt curves to assess the quality of enzyme preparations.

This study sought to determine whether the quality of enzyme preparations can be determined from their melting curves, which may easily be obtained using a fluorescent probe and a standard reverse transcription-polymerase chain reaction (RT-PCR) machine. Thermal melt data on 31 recombinant enzymes from Plasmodium parasites were acquired by incrementally heating them to 90 degrees C and measuring unfolding with a fluorescent dye. Activity assays specific to each enzyme were also performed. Four of the enzymes were denatured to varying degrees with heat and sodium dodecyl sulfate (SDS) prior to the thermal melt and activity assays. In general, melting curve quality was correlated with enzyme activity; enzymes with high-quality curves were found almost uniformly to be active, whereas those with lower quality curves were more varied in their catalytic performance. Inspection of melting curves of bovine xanthine oxidase and Entamoeba histolytica cysteine protease 1 allowed active stocks to be distinguished from inactive stocks, implying that a relationship between melting curve quality and activity persists over a wide range of experimental conditions and species. Our data suggest that melting curves can help to distinguish properly folded proteins from denatured ones and, therefore, may be useful in selecting stocks for further study and in optimizing purification procedures for specific proteins.

Buffer optimization of thermal melt assays of Plasmodium proteins for detection of small-molecule ligands.

In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of Tm for 58 of 61 Plasmodium proteins tested. However, with several proteins, Tm could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. The authors conclude that buffer optimization to minimize variability in Tm measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal.

The double-length tyrosyl-tRNA synthetase from the eukaryote Leishmania major forms an intrinsically asymmetric pseudo-dimer.

The single tyrosyl-tRNA synthetase (TyrRS) gene in trypanosomatid genomes codes for a protein that is twice the length of TyrRS from virtually all other organisms. Each half of the double-length TyrRS contains a catalytic domain and an anticodon-binding domain; however, the two halves retain only 17% sequence identity to each other. The structural and functional consequences of this duplication and divergence are unclear. TyrRS normally forms a homodimer in which the active site of one monomer pairs with the anticodon-binding domain from the other. However, crystal structures of Leishmania major TyrRS show that, instead, the two halves of a single molecule form a pseudo-dimer resembling the canonical TyrRS dimer. Curiously, the C-terminal copy of the catalytic domain has lost the catalytically important HIGH and KMSKS motifs characteristic of class I aminoacyl-tRNA synthetases. Thus, the pseudo-dimer contains only one functional active site (contributed by the N-terminal half) and only one functional anticodon recognition site (contributed by the C-terminal half). Despite biochemical evidence for negative cooperativity between the two active sites of the usual TyrRS homodimer, previous structures have captured a crystallographically-imposed symmetric state. As the L. major TyrRS pseudo-dimer is inherently asymmetric, conformational variations observed near the active site may be relevant to understanding how the state of a single active site is communicated across the dimer interface. Furthermore, substantial differences between trypanosomal TyrRS and human homologs are promising for the design of inhibitors that selectively target the parasite enzyme.

Identification of inhibitors for putative malaria drug targets among novel antimalarial compounds.

The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25µM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology.

Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors.

New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.