Structure of the essential Plasmodium host cell traversal protein SPECT1.

Host cell traversal by Plasmodium, the protozoan cause of malaria, is an essential part of this parasite's virulence. In this process, the parasite enters a host cell through a parasite-induced pore, traverses the host cell, and then exits the host cell. Two P. berghei proteins, SPECT1 and SPECT2, are required for host cell traversal by the sporozoite form of the parasite. In the absence of either, no pore formation is observed. While SPECT2 has sequence homology to pore-forming proteins, SPECT1 has no homology to proteins of known structure or function. Here we present the 2.75 Å resolution structure of a slightly truncated version of P. berghei SPECT1. The structure reveals that the protein forms a four-helix bundle, with the rare feature of having all of these helices in parallel or antiparallel alignment. Also notable is the presence of a large, conserved, hydrophobic internal cavity in the protein, which may constitute a ligand-binding site or be indicative of partial instability in SPECT1, or both. The structure of SPECT1 will make possible targeted mutagenesis experiments aimed at understanding its mechanism of action in host cell traversal.

Two translation products of Yersinia yscQ assemble to form a complex essential to type III secretion.

The bacterial flagellar C-ring is composed of two essential proteins, FliM and FliN. The smaller protein, FliN, is similar to the C-terminus of the larger protein, FliM, both being composed of SpoA domains. While bacterial type III secretion (T3S) systems encode many proteins in common with the flagellum, they mostly have a single protein in place of FliM and FliN. This protein resembles FliM at its N-terminus and is as large as FliM but is more like FliN at its C-terminal SpoA domain. We have discovered that a FliN-sized cognate indeed exists in the Yersinia T3S system to accompany the FliM-sized cognate. The FliN-sized cognate, YscQ-C, is the product of an internal translation initiation site within the locus encoding the FliM-sized cognate YscQ. Both intact YscQ and YscQ-C were found to be required for T3S, indicating that the internal translation initiation site, which is conserved in some but not all YscQ orthologs, is crucial for function. The crystal structure of YscQ-C revealed a SpoA domain that forms a highly intertwined, domain-swapped homodimer, similar to those observed in FliN and the YscQ ortholog HrcQ(B). A single YscQ-C homodimer associated reversibly with a single molecule of intact YscQ, indicating conformational differences between the SpoA domains of intact YscQ and YscQ-C. A "snap-back" mechanism suggested by the structure can account for this. The 1:2 YscQ-YscQ-C complex is a close mimic of the 1:4 FliM-FliN complex and the likely building block of the putative Yersinia T3S system C-ring.

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling.

Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

The crystal structures of EAP domains from Staphylococcus aureus reveal an unexpected homology to bacterial superantigens.

The Eap (extracellular adherence protein) of Staphylococcus aureus functions as a secreted virulence factor by mediating interactions between the bacterial cell surface and several extracellular host proteins. Eap proteins from different Staphylococcal strains consist of four to six tandem repeats of a structurally uncharacterized domain (EAP domain). We have determined the three-dimensional structures of three different EAP domains to 1.8, 2.2, and 1.35 A resolution, respectively. These structures reveal a core fold that is comprised of an alpha-helix lying diagonally across a five-stranded, mixed beta-sheet. Comparison of EAP domains with known structures reveals an unexpected homology with the C-terminal domain of bacterial superantigens. Examination of the structure of the superantigen SEC2 bound to the beta-chain of a T-cell receptor suggests a possible ligand-binding site within the EAP domain (Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., and Mariuzza, R. (1996) Nature 384, 188-192). These results provide the first structural characterization of EAP domains, relate EAP domains to a large class of bacterial toxins, and will guide the design of future experiments to analyze EAP domain structure/function relationships.

Crystal structure of Caenorhabditis elegans HER-1 and characterization of the interaction between HER-1 and TRA-2A.

HER-1 is a secreted protein that promotes male development in the nematode Caenorhabditis elegans. HER-1 inhibits the function of TRA-2A, a multipass integral membrane protein thought to serve as its receptor. We report here the 1.5-A crystal structure of HER-1. The structure was solved by the multiwavelength anomalous diffraction method by using selenomethionyl-substituted HER-1 produced in Chinese hamster ovary cells. The HER-1 structure consists of two all-helical domains and is not closely homologous to any known structure. Sites of amino acid substitutions known to impair HER-1 function were mapped on the HER-1 structure and classified according to the likely mechanism by which they affect HER-1 activity. A subset of these and other amino acid substitutions on the HER-1 surface were assayed for their ability to disrupt interactions between HER-1 and TRA-2A-expressing cells, and a localized region on the HER-1 surface important for mediating this interaction was identified.

Agonist-induced transitions of the acetylcholine receptor.

Anti-acetylcholine receptor (AChR) monoclonal antibody 383C binds to the beta-hairpin loop alpha(187-199) of only one of the two Torpedo AChR alpha subunits. The loop recognized is associated with the alpha subunit corresponding to the high-affinity d-tubocurarine (dTC) binding site. Desensitization of the receptor with carbamylcholine completely blocks the binding of 383C. Mild reduction of AChR alpha subunit cys 192-193 disulfide with DTT and subsequent reaction with 5-iodoacetamidofluorescein label only the high-affinity dTC alpha subunit. Rhodamine-labeled alpha-bungarotoxin (R-Btx) binds to the unlabeled AChR alpha subunit as monitored by fluorescence resonance energy transfer between the fluorescein and rhodamine dyes. A 10-A contraction of the distance between the dyes is observed following the addition of carbamylcholine. In a small angle X-ray diffraction experiment exploiting anomalous X-ray scattering from Tb(III) ions titrated into AChR Ca(II) binding sites, we find evidence for a change in the Tb(III) ion distribution in the region of the ion channel following addition of carbamylcholine to the AChR. The carbamylcholine-induced loss of the 383C epitope, the 10-A contraction of the beta-hairpin loop, and the loss of multivalent cations from the channel likely represent the first molecular transitions leading to AChR channel opening.