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

Abstract 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 Å resolution, respectively. These structures reveal a core fold that is comprised of an -helix lying diagonally across a five-stranded, mixedsheet. 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 -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

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

Surface of <i>Pb</i>SPECT1Δ41.

<p><b>a</b>. Residues conserved among homologs of <i>Pb</i>SPECT1 were mapped to the molecular surface of <i>Pb</i>SPECT1Δ41. Two views of <i>Pb</i>SPECT1 related by a 180° rotation around the indicated axis are shown. Color scale for conservation is shown at bottom. <b>b</b>. The electrostatic surface mapped to the van der Waals surface of <i>Pb</i>SPECT1Δ41 is shown. As in panel a, two views and a color scale are shown.</p

Monomeric and Dimeric Forms of <i>Pb</i>SPECT1Δ41.

<p><b>a</b>. Schematic to scale of <i>Pb</i>SPECT1. Yellow denotes the putative signal sequence, purple the portion of the mature protein not included in <i>Pb</i>SPECT1Δ41, and blue <i>Pb</i>SPECT1Δ41. <b>b</b>. Size exclusion chromatogram of <i>Pb</i>SPECT1Δ41 showing the appearance of monomeric and dimeric species. <b>c</b>. “V”-shaped association between two <i>Pb</i>SPECT1Δ41 molecules (red and blue) observed in the asymmetric unit of the crystal.</p

Structure and Conservation of <i>Pb</i>SPECT1Δ41.

<p><b>a</b>. Ribbon representation of the four-helix bundle structure of <i>Pb</i>SPECT1Δ41 (4U5A), with individual α-helices in different colors. <b>b</b>. The structure of <i>Pb</i>SPECT1Δ41 viewed down the axis of the four-helix bundle. Coloring of individual α-helices is as in panel a. <b>c</b>. Structure-based sequence alignment of <i>Pb</i>SPECT1Δ41 (Genbank BAD08209.1, PlasmoDB PBANKA_135560) with <i>Plasmodium</i> homologs: <i>P. cynomolgi</i> (XP_004223591.1, PCYB_122110), <i>P. vivax</i> (PVX_083025), <i>P. knowlesi</i> (CAQ41197.1, PKH_121200), <i>P. inui</i> (EUD67722.1), <i>P. vinckei petteri</i> (EUD71736.1), <i>P. chabaudi chabaud</i> (PCHAS_136020), <i>P. yoelii</i> YM (PYYM_1357700), <i>P. falciparum</i> 3D7 (PF3D7_1342500), and <i>P. reichenowi</i> (CDO66209.1). Putative secretion signals were excluded. The secondary structure of <i>Pb</i>SPECT1Δ41 is shown above the sequence. Absolutely conserved residues are in white on a red background, and similar residues are in red; both types are in blue boxes.</p

The internal cavity of <i>Pb</i>SPECT1Δ41.

<p><b>a</b>. Stereoview of the interior cavity in <i>Pb</i>SPECT1Δ41 depicted as a surface, and the backbone of <i>Pb</i>SPECT1Δ41 as sticks. The positions of the cavity, pocket, and hook are indicated. <b>b</b>. A molecular surface representation of <i>Pb</i>SPECT1Δ41 with its deep pocket is shown. Residues at the entrance of the pocket are indicated.</p

Crystallographic Data Collection and Refinement.

<p>Crystallographic Data Collection and Refinement.</p

Two Translation Products of <i>Yersinia yscQ</i> 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 <i>Yersinia</i> 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 <i>Yersinia</i> 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