A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch

The regulation of cation content is critical for cell growth. However, the molecular mechanisms that gate the systems that control K+ movements remain unclear. KTN is a highly conserved cytoplasmic domain present ubiquitously in a variety of prokaryotic and eukaryotic K+ channels and transporters. Here we report crystal structures for two representative KTN domains that reveal a dimeric hinged assembly. Alternative ligands NAD+ and NADH block or vacate, respectively, the hinge region affecting the dimer's conformational flexibility. Conserved, surface-exposed hydrophobic patches that become coplanar upon hinge closure provide an assembly interface for KTN tetramerization. Mutational analysis using the KefC system demonstrates that this domain directly interacts with its respective transmembrane constituent, coupling ligand-mediated KTN conformational changes to the permease's activity.     

A geometric approach to cranial sexual dimorphism in the orang-utan

Adult craniofacial morphology is quantified and compared using Euclidean distance matrix analysis (EDMA), a three-dimensional morphometric method for the comparison of forms, which localizes form differences between comparative groups. Results indicate that the number and magnitude of differences between male and female crania are striking. The face, basicranium and neurocranium exhibit the most dimorphism, while the palate shows the least. Significant differences also exist between young adult and fully adult individuals, especially males, supporting the delayed onset of sexual maturity and secondary sex characteristics in males. As one of the many new morphometric techniques available, EDMA was useful for identifying local form difference and provides insights into the understanding of sexual dimorphism in this species beyond that obtained from traditional statistical methods based on linear caliper measurements.     

Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene

Many mutations in the Human Ether-à-go-go-Related Gene (HERG) cause type 2 congenital long QT syndrome (LQT2) by disrupting trafficking of the HERG-encoded potassium channel. Beyond observations that some mutations trap channels in the endoplasmic reticulum, little is known about how trafficking fails. Even less is known about what checkpoints are encountered in normal trafficking. To identify protein partners encountered as HERG channels are transported among subcellular compartments, we screened a human heart library with the C terminus of HERG using yeast two-hybrid technology. Among the proteins isolated was GM130, a Golgi-associated protein involved in vesicular transport. The interaction mapped to two non-contiguous regions of HERG and to a region just upstream of the GRASP-65 interaction domain of GM130. GM130 did not interact with the N or C terminus of either KvLQT1 or Shaker channels. LQT2-causing mutations in the HERG C terminus selectively disrupted interactions with GM130 but not Tara, another HERG-interacting protein. Native GM130 and stably expressed HERG were co-immunoprecipitated from HEK-293 cells using GM130 antibodies. In rat cardiac myocytes and HEK-293 cells, confocal immunocytochemistry showed co-localization of GM130 and HERG to the Golgi apparatus. Overexpression of GM130 suppressed HERG current amplitude in Xenopus oocytes, as if by providing an excess of substrate at the Golgi checkpoint. These findings indicate that GM130 plays a previously undefined role in cargo transport. We propose that the cytoplasmic C terminus of HERG participates in the tethering or possibly targeting of HERG-containing vesicles within the Golgi via its interaction with GM130.     

Structural determinants of aromatase cytochrome p450 inhibition in substrate recognition site-1

The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I(133)M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B'-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I(133)M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile(133) to either alanine (I(133)A) or tyrosine (I(133)Y) decreased apparent enzyme activity, but the I(133)A mutant was sensitive to etomidate inhibition, suggesting that it is Ile(133) that decreases etomidate binding rather than Met(133) increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I(133)M mutant and the native placental isozyme. These data suggest that Ile(133) is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees.     

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.     

Structural insights into the avian AICAR transformylase mechanism

ATIC encompasses both AICAR transformylase and IMP cyclohydrolase activities that are responsible for the catalysis of the penultimate and final steps of the purine de novo synthesis pathway. The formyl transfer reaction catalyzed by the AICAR Tfase domain is substantially more demanding than that catalyzed by the other folate-dependent enzyme of the purine biosynthesis pathway, GAR transformylase. Identification of the AICAR Tfase active site and key catalytic residues is essential to elucidate how the non-nucleophilic AICAR amino group is activated for formyl transfer. Hence, the crystal structure of dimeric avian ATIC was determined as a complex with the AICAR Tfase substrate AICAR, as well as with an IMP cyclohydrolase inhibitor, XMP, to 1.93 A resolution. AICAR is bound at the dimer interface of the transformylase domains and forms an extensive hydrogen bonding network with a multitude of active site residues. The crystal structure suggests that the conformation of the 4-carboxamide of AICAR is poised to increase the nucleophilicity of the C5 amine, while proton abstraction occurs via His(268) concomitant with formyl transfer. Lys(267) is likely to be involved in the stabilization of the anionic formyl transfer transition state and in subsequent protonation of the THF leaving group.     

Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition

This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.     

Crystal structures of human GAR Tfase at low and high pH and with substrate beta-GAR

Glycinamide ribonucleotide transformylase (GAR Tfase) is a key folate-dependent enzyme in the de novo purine biosynthesis pathway and, as such, has been the target for antitumor drug design. Here, we describe the crystal structures of the human GAR Tfase (purN) component of the human trifunctional protein (purD-purM-purN) at various pH values and in complex with its substrate. Human GAR Tfase exhibits pH-dependent enzyme activity with its maximum around pH 7.5-8. Comparison of unliganded human GAR Tfase structures at pH 4.2 and pH 8.5 reveals conformational differences in the substrate binding loop, which at pH 4.2 occupies the binding cleft and prohibits substrate binding, while at pH 8.5 is permissive for substrate binding. The crystal structure of GAR Tfase with its natural substrate, beta-glycinamide ribonucleotide (beta-GAR), at pH 8.5 confirms this conformational isomerism. Surprisingly, several important structural differences are found between human GAR Tfase and previously reported E. coli GAR Tfase structures, which have been used as the primary template for drug design studies. While the E. coli structure gave valuable insights into the active site and formyl transfer mechanism, differences in structure and inhibition between the bacterial and mammalian enzymes suggest that the human GAR Tfase structure is now the appropriate template for the design of anti-cancer agents.     

Natural variation for seed oil composition in Arabidopsis thaliana

The biochemical pathways involved in the biosynthesis and accumulation of storage lipids in seeds have been extensively studied. However, the regulatory mechanisms of those pathways, their environmental interactions and the ecological implications of variation are poorly understood. We have initiated a new approach: the analysis of natural variation in Arabidopsis thaliana. Three hundred and sixty accessions were surveyed for content of oil, very long chain fatty acids (VLCFAs) and polyunsaturated fatty acids (PUFAs) in their seeds. The results revealed extensive natural variation. A core set of accessions, the seeds of which reproducibly contain extreme amounts of oil, VLCFAs and PUFAs have been identified. Reproducible oil content ranged from 34.6 to 46.0% of seed dry weight. VLCFA content ranged from 13.0 to 21.2% of total fatty acids. PUFA content, ranged from 53.3 to 66.1% of total fatty acids. Interactions were also identified for PUFA and VLCFA content of seeds with vernalisation of plants. Mapping of the regions of the genome involved in controlling the traits was conducted in an F(2) population and indicated that natural variation at the loci FAE1 and FAD3 might be involved in the regulation of VLCFA and PUFA content, respectively. A set of accessions, which capture a broad range of the natural variation for these traits available in A. thaliana, has been selected to form a core set which can be used to further dissect the genetics of the regulation of seed lipid traits and to identify the genes involved.     

Transmissible and nontransmissible complex chromosome aberrations characterized by three-color and mFISH define a biomarker of exposure to high-LET alpha particles

Insertions have been proposed as potential stable biomarkers of chronic high-LET radiation exposure. To examine this in vitro, we irradiated human peripheral blood lymphocytes in G(0) with either 50 cGy (238)Pu alpha particles (LET 121.4 keV/microm) or 3 Gy 250 kV X rays and stimulated their long-term culture up to approximately 22 population doublings postirradiation. Mitotic cells were harvested at regular intervals throughout this culture period and were assayed for chromosome aberrations using the techniques of three-color and 24-color mFISH. We observed the stable persistence of transmissible-type complex rearrangements, all involving at least one insertion. This supports the hypothesis that insertions are relevant indicators of exposure to high-LET radiation. However, one practical caveat of insertions being effective biomarkers is that their frequency is low due to the complexity and cell lethality of the majority of alpha-particle-induced complexes. Therefore, we propose a "profile of damage" that relies on the presence of insertions, a low frequency of stable simple reciprocal translocations (2B), and, significantly, the complexity of the damage initially induced. We suggest that the complexity of first- and second-division alpha-particle-induced nontransmissible complex aberrations reflects the structure of the alpha-particle track and as a consequence adds radiation-quality specificity to the biomarker, increasing the signal:noise ratio of the characteristic 2B:insertion ratio.