Crystal structure of a bicupin protein HutD involved in histidine utilization in Pseudomonas.

Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc.

High throughput crystallography of TB drug targets.

Tuberculosis (TB) infects one-third of the world population. Despite 50 years of available drug treatments, TB continues to increase at a significant rate. The failure to control TB stems in part from the expense of delivering treatment to infected individuals and from complex treatment regimens. Incomplete treatment has fueled the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Reducing non-compliance by reducing the duration of chemotherapy will have a great impact on TB control. The development of new drugs that either kill persisting organisms, inhibit bacilli from entering the persistent phase, or convert the persistent bacilli into actively growing cells susceptible to our current drugs will have a positive effect. We are taking a multidisciplinary approach that will identify and characterize new drug targets that are essential for persistent Mtb. Targets are exposed to a battery of analyses including microarray experiments, bioinformatics, and genetic techniques to prioritize potential drug targets from Mtb for structural analysis. Our core structural genomics pipeline works with the individual laboratories to produce diffraction quality crystals of targeted proteins, and structural analysis will be completed by the individual laboratories. We also have capabilities for functional analysis and the virtual ligand screening to identify novel inhibitors for target validation. Our overarching goals are to increase the knowledge of Mtb pathogenesis using the TB research community to drive structural genomics, particularly related to persistence, develop a central repository for TB research reagents, and discover chemical inhibitors of drug targets for future development of lead compounds.

Structure of HisF, a histidine biosynthetic protein from Pyrobaculum aerophilum.

HisF (imidazole glycerol phosphate synthase) is an important branch-point enzyme in the histidine biosynthetic pathway of microorganisms. Because of its potential relevance for structure-based drug design, the crystal structure of HisF from the hyperthermophilic archaeon Pyrobaculum aerophilum has been determined. The structure was determined by molecular replacement and refined at 2.0 A resolution to a crystallographic R factor of 20.6% and a free R of 22.7%. The structure adopts a classic (beta/alpha)(8) barrel fold and has networks of surface salt bridges that may contribute to thermostability. The active site is marked out by the presence of two bound phosphate ions and two glycerol molecules that delineate a long groove at one end of the (beta/alpha)(8) barrel. The two phosphate ions, 17 A apart, are bound to sequence-conserved structural motifs that seem likely to provide much of the specificity for the two phosphate groups of the HisF substrate. The two glycerol molecules bind in the vicinity of other sequence-conserved residues that are likely to be involved in binding and/or catalysis. Comparisons with the homologous HisF from Thermatoga maritima reveal a displaced loop that may serve as a lid over the active site.

Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation.

N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We have determined the crystal structure of the NAD(+) complex of a truncated form of the enzyme, GFORDelta, in which the first 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, refined at 2.7 A resolution (R(cryst)=24.1%, R(free)=28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of the N-terminal arm also greatly increases the solvent exposure of the cofactor; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORDelta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity.

The ordered visual transduction complex of the squid photoreceptor membrane.

The study of visual transduction has given invaluable insight into the mechanisms of signal transduction by heptahelical receptors that act via guanine nucleotide binding proteins (G-proteins). However, the cyclic-GMP second messenger system seen in vertebrate photoreceptor cells is not widely used in other cell types. In contrast, the retina of higher invertebrates, such as squid, offers an equally accessible transduction system, which uses the widespread second messenger chemistry of an increase in cytosolic calcium caused by the production of inositol-(1,4,5)-trisphosphate (InsP3) by the enzyme phospholipase C, and which may be a model for store-operated calcium influx. In this article, we highlight some key aspects of invertebrate visual transduction as elucidated from the combination of biochemical techniques applied to cephalopods, genetic techniques applied to flies, and electrophysiology applied to the horseshoe crab. We discuss the importance and applicability of ideas drawn from these model systems to the understanding of some general processes in signal transduction, such as the integration of the cytoskeleton into the signal transduction process and the possible modes of regulation of store-operated calcium influx.

The cloning, expression and crystallisation of a thermostable arginase.

The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox' has been cloned and sequenced. Expression of recombinant arginase at high levels has been achieved in E. coli using an inducible T7 RNA polymerase-based system. A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture. The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme. The recombinant protein has been crystallised and one crystal form is isomorphous to crystals of the native protein.

The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.

Malignant lymphoma of the thyroid gland.

Malignant lymphoma of the thyroid gland is a rare entity; only 200 cases have been reported to date since 1960. Four patients with this disease presented at the Kingston clinic of the Ontario Cancer Treatment and Research Foundation. Those with localized malignant lymphoma, particularly the histiocytic types, responded favourably to resection of as much of the tumour as possible and subsequent local radiation with cobalt-60 telecurietherapy (3000 to 4000 rads in 3 to 4 weeks). The results of local radiation alone after a biopsy in patients with inoperable localized diseases are encouraging; it is possible, but not yet established, that some of these patients are cured. It has been suggested that the tumour is dependent on thyroid-stimulating hormone but conclusive evidence is not yet available.