High-resolution crystal structure and in vivo function of a kinesin-2 homologue in Giardia intestinalis.

A critical component of flagellar assembly, the kinesin-2 heterotrimeric complex powers the anterograde movement of proteinaceous rafts along the outer doublet of axonemes in intraflagellar transport (IFT). We present the first high-resolution structures of a kinesin-2 motor domain and an ATP hydrolysis-deficient motor domain mutant from the parasitic protist Giardia intestinalis. The high-resolution crystal structures of G. intestinalis wild-type kinesin-2 (GiKIN2a) motor domain, with its docked neck linker and the hydrolysis-deficient mutant GiKIN2aT104N were solved in a complex with ADP and Mg(2+) at 1.6 and 1.8 A resolutions, respectively. These high-resolution structures provide unique insight into the nucleotide coordination within the active site. G. intestinalis has eight flagella, and we demonstrate that both kinesin-2 homologues and IFT proteins localize to both cytoplasmic and membrane-bound regions of axonemes, with foci at cell body exit points and the distal flagellar tips. We demonstrate that the T104N mutation causes GiKIN2a to act as a rigor mutant in vitro. Overexpression of GiKIN2aT104N results in significant inhibition of flagellar assembly in the caudal, ventral, and posterolateral flagellar pairs. Thus we confirm the conserved evolutionary structure and functional role of kinesin-2 as the anterograde IFT motor in G. intestinalis.

Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5.

Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.

The cenH3 histone variant defines centromeres in Giardia intestinalis.

Histone H3 variants play critical roles in the functional specialization of chromatin by epigenetically marking centromeric chromatin and transcriptionally active or silent genes. Specifically, the cenH3 histone variant acts as the primary epigenetic determinant of the site of kinetochore assembly at centromeres. Although the function of histone variants is well studied in plants, animals, and fungi, there is little knowledge of the evolutionary conservation of histone variants and their function in most protists. We find that Giardia intestinalis--a diplomonad parasite with two equivalent nuclei--has two phylogenetically distinct histone H3 variants with N-terminal extensions and nonconserved promoters. To determine their role in chromatin dynamics, conventional H3 and the two H3 variants were GFP-tagged, and their subcellular location was monitored during interphase and mitosis. We demonstrate that one cenH3-like variant has a conserved function in epigenetically marking centromeres. The other H3 variant (H3B) has a punctate distribution on chromosomes, but does not colocalize with active transcriptional regions as indicated by H3K4 methylation. We suggest that H3B could instead mark noncentromeric heterochromatin. Giardia is a member of the Diplomonads and represents an ancient divergence from metazoans and fungi. We confirm the ancient role of histone H3 variants in modulating chromatin architecture, and suggest that monocentric chromosomes represent an ancestral chromosome morphology.