David N. Garboczi

Dissection of Merozoite Surface Protein 3, a Representative of a Family of Plasmodium falciparum Surface Proteins, Reveals an Oligomeric and Highly Elongated Molecule

Vaccination with the merozoite surface protein 3 (MSP3) of Plasmodium falciparum protects against infection in primates and is under development as a vaccine against malaria in humans. MSP3 is secreted and associates with the parasite membrane but lacks a predicted transmembrane domain or a glycosylphosphatidylinositol anchor. Its role in the invasion of red blood cells is unclear. To study MSP3, we produced recombinant full-length protein and found by size exclusion chromatography that the apparent size of MSP3 was much larger than predicted from its sequence. To investigate this, we used several biophysical techniques to characterize the full-length molecule and four smaller polypeptides. The MSP3 polypeptides contain a large amount of α-helix and random coil secondary structure as measured by circular dichroism spectroscopy. The full-length MSP3 forms highly elongated dimers and tetramers as revealed by chemical cross-linking and analytical ultracentrifugation. The dimer is formed through a leucine zipper-like domain located between residues 306 and 362 at the C terminus. Two dimers interact through their C termini to form a tetramer with an apparent association constant of 3 μm. Sedimentation velocity experiments determined that the MSP3 molecules are highly extended in solution (some with f/f0 > 2). These data, in light of the recent discoveries of three other Plasmodium proteins containing very similar C-terminal sequences, suggest that the members of this newly identified family may adopt highly extended and oligomeric novel structures capable of interacting with a red blood cell at relatively long distances.

Subdomain 3 of Plasmodium falciparum VAR2CSA DBL3x Is Identified as a Minimal Chondroitin Sulfate A-binding Region

Molecular interactions between the VAR2CSA protein, expressed on the surface of Plasmodium falciparum-infected erythrocytes, and placental chondroitin sulfate A (CSA) are primarily responsible for pregnancy-associated malaria (PAM). Interrupting these interactions may prevent or ameliorate the severity of PAM. Several of the Duffy binding-like (DBL) domains of VAR2CSA, including the DBL3x domain, have been shown to bind CSA in vitro, but a more detailed understanding of how DBL domains bind CSA is needed. In this study, we demonstrate that subdomain 3 (S3), one of the three subdomains of VAR2CSA DBL3x by itself, is the major contributor toward CSA binding. NMR spectroscopy and flow cytometry analyses show that S3 and the intact DBL3x domain bind CSA similarly. Mutations within the S3 portion of DBL3x markedly affect CSA binding. Both recombinant molecules, S3 and DBL3x, are recognized by antibodies in the plasma of previously pregnant women living in malaria-endemic regions of Mali, but much less so by plasma from men of the same regions. As the S3 sequence is highly conserved in all known VAR2CSA proteins expressed by different parasite isolates obtained from various malaria endemic areas of the world, the identification of S3 as an independent CSA-binding region provides a compelling molecular basis for designing interventions against PAM.

Plasmodium P25 and P28 Surface Proteins: Potential Transmission-Blocking Vaccines

Malaria arises from the infection of red blood cells by protozoan parasites of the genus Plasmodium that are transmitted by anopheline mosquitoes. More than 400 species of Anopheles mosquitoes are known, of which about 40 species are characterized as important disease vectors for human malaria transmission (29). It is estimated that 300 to 500 million cases of malaria and over 1 million deaths from the disease occur each year (49). The Plasmodium parasite must complete its development in the mosquito before it can be transmitted to the vertebrate host and cause malaria. Each stage of parasite development in the mosquito offers potential targets to interfere with malaria transmission. Development of the malaria parasite in the mosquito begins when the gametocyte forms of the parasite are picked up by the mosquito in the blood meal from an infected human and quickly develop into extracellular gametes in the mosquito midgut. After fertilization, round zygotes form and transform into banana-shaped ookinetes. The ookinetes are motile and must exit the gut by crossing the peritrophic membrane and midgut epithelium. On the basal side of the epithelium, surviving ookinetes lodge against the basal lamina and transform into spherical oocysts. In the oocyst, the parasite develops into several thousand sporozoites, which then exit the oocyst and are carried by the hemolymph to the mosquito’s salivary glands to infect another host (22). There is ongoing research to develop antiparasite vaccines against each stage of the complicated life cycle of Plasmodium (17, 24). Liver-stage vaccines are intended to reduce infection rates, and asexual-blood-stage vaccines will reduce disease severity and the risk of death during infection. Transmissionblocking vaccines would prevent the spread of disease by targeting antigens expressed in the mosquito stage on the surfaces of the gametocyte, gamete, zygote, and ookinete forms of the parasite (6, 61). These vaccines induce antibodies in the human host that inhibit parasite development in the mosquito midgut and thereby block parasite transmission to another person. This article reviews the biology and structural knowledge of the Plasmodium P25 and P28 proteins and their contributions to transmission-blocking vaccine development.

Preparation, crystallization and preliminary X-ray analysis of a complex between the Plasmodium vivax sexual stage 25 kDa protein Pvs25 and a malaria transmission-blocking antibody Fab fragment

The Plasmodium vivax sexual stage 25 kDa protein Pvs25, located on the surface of the ookinete form of the parasite, is a vaccine candidate designed to elicit immunity that blocks the transmission of malaria by mosquitoes. The 2A8 murine monoclonal antibody directed against recombinant Pvs25 prevents the formation of P. vivax oocysts in mosquitoes fed in the laboratory. The complex between recombinant Pvs25 and the Fab fragment of 2A8 forms crystals that diffract X-rays to 3.5 A. Two native data sets, A and B, have been collected from crystals of the Pvs25-Fab complex. Both crystals belong to space group P2(1), with unit-cell parameters of a = 86.3, b = 61.7, c = 142.7 A, beta = 101.7 degrees for data set A and a = 86.8, b = 61.0, c = 149.3 A, beta = 104.3 degrees for data set B, and contain two complex molecules per asymmetric unit. Efforts are under way to reveal the structure of the Pvs25-Fab complex by molecular replacement. The three-dimensional structure of the Pvs25-Fab complex will provide an understanding of the interaction between Pvs25 and the 2A8 antibody that inhibits ookinete development in the mosquito and should aid in the development of transmission-blocking vaccines against P. vivax malaria.

Biochemical and biophysical characterization of cytokine-like protein 1 (CYTL1)

HighlightsSecondary structure content of CYTL1 is consistent with chemokines.Secondary structure content of CYTL1 is not consistent with 4‐helical cytokines.CYTL1 induces calcium flux in human chondrocytes.CYTL1 appears to be an atypical chemokine receptor ligand. Abstract Cytokine‐like protein 1 (CYTL1) is a small widely expressed secreted protein lacking significant primary sequence homology to any other known protein. CYTL1 expression appears to be highest in the hematopoietic system and in chondrocytes; however, maintenance of cartilage in mouse models of arthritis is its only reported function in vivo. Despite lacking sequence homology to chemokines, CYTL1 is predicted by computational methods to fold like a chemokine, and has been reported to function as a chemotactic agonist at the chemokine receptor CCR2 in mouse monocyte/macrophages. Nevertheless, since chemokines are defined by structure and chemokine receptors are able to bind many non‐chemokine ligands, direct determination of the CYTL1 tertiary structure will ultimately be required to know whether it actually folds as a chemokine and therefore is a chemokine. Towards this goal, we have developed a method for producing functional recombinant human CYTL1 in bacteria, and we provide new evidence about the biophysical and biochemical properties of recombinant CYTL1. Circular dichroism analysis showed that, like chemokines, CYTL1 has a higher content of beta‐sheet than alpha‐helix secondary structure. Furthermore, recombinant CYTL1 promoted calcium flux in chondrocytes. Nevertheless, unlike chemokines, CYTL1 had limited affinity to proteoglycans. Together, these properties further support cytokine‐like properties for CYTL1 with some overlap with the chemokines.

F-type ATPases: Are nucleotide domains in adenylate kinase appropriate models for nucleotide domains in ATP synthase/ATPase complexes?

There are three classes of ATPases that have received considerable atten tion in recent years. These are the P, V, and F types.’ Of these ATPases, only the F type function physiologically in eukaryotes in the direction of ATP synthesis. Hence, these ATPases are referred to also as “ATP synthases.” ATP synthase molecules are comprised of two major units (FIG. l), Fo and FI. (For reviews see refs. 2-7). The major function of Fo is to direct protons from an electrochemical proton gradient to the FI unit. Currently it is believed that ATP synthesis takes place on Fl without the input of energy and that the role of the energy derived from the electrochemical proton gradient is to release bound ATP. As the F, unit consists of three a@ pairs, each pair “in turn” is postulated to participate in ATP synthesis. This “rotational catalysis” is thought to be possible as Fl consists also of small subunits y, 6, and E, one or all three of which may change position (or “rotate”) in response to the electrochemical gradient, thus specifying in sequence each a@ pair for catalysis. In this scheme the principal location of the catalytic sites is thought to be on the @ subunits, although some sharing may take place with a subunits. Sequence analysis of the a and p subunits of the FI moiety of ATP synthases reveals regions of homology with the enzyme adenylate k i n a ~ e , ~ , ~ an enzyme that catalyzes the reaction 2ADP ATP + AMP. These regions are referred to as 4, EJ and _C, and the consensus amino acid sequences are, respectively, GX,GKT, RX7h4D, and VXADX3DX8HLDA. The A and S consensus sequences are those usually discussed, but particularly in the p subunit the C consensus is quite striking. Significantly, the three-dimensional structure of adenylate kinase is known from the work of Schultz and coworkers.’@-*-’ In addition, the NMR data of Mildvan and coworkers14 has resulted in the assignment of the ATP site (FIG. 2) which involves in part the 4 consensusiglycine flexible loop region in binding the triphosphate moiety of ATP. The region is suggested to participate in binding the remainder of the