Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To that end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone-mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance-conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side-chains may determine atovaquone-binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.     

Structure of B-MLV capsid amino-terminal domain reveals key features of viral tropism, gag assembly and core formation

The Gag polyprotein is the major structural protein found in all classes of retroviruses. Interactions between Gag molecules control key events at several stages in the cycle of infection. In particular, the capsid (CA) domain of Gag mediates many of the protein-protein interactions that drive retrovirus assembly, maturation and disassembly. Moreover, in murine leukaemia virus (MLV), sequence variation in CA confers N and B tropism that determines susceptibility to the intracellular restriction factors Fv1n and Fv1b. We have determined the structure of the N-terminal domain (NtD) of CA from B-tropic MLV. A comparison of this structure with that of the NtD of CA from N-tropic MLV reveals that although the crystals belong to different space groups, CA monomers are packed with the same P6 hexagonal arrangement. Moreover, interhexamer crystal contacts between residues located at the periphery of the discs are conserved, indicating that switching of tropism does not result in large differences in the backbone conformation, nor does it alter the quaternary arrangement of the disc. We have also examined crystals of the N-tropic MLV CA containing both N- and C-terminal domains. In this case, the NtD hexamer is still present; however, the interhexamer spacing is increased and the conserved interhexamer contacts are absent. Investigation into the effects of mutation of residues that mediate interhexamer contacts reveals that amino acid substitutions at these positions cause severe defects in viral assembly, budding and Gag processing. Based on our crystal structures and mutational analysis, we propose that in MLV, interactions between the NtDs of CA are required for packing of Gag molecules in the early part of immature particle assembly. Moreover, we present a model where proteolytic cleavage at maturation results in migration of CA C-terminal domains into interstitial spaces between NtD hexamers. As a result, NtD-mediated interhexamer contacts present in the immature particle are displaced and the less densely packed lattice with increased hexamer-hexamer spacing characteristic of the viral core is produced.     

Cysteine substitutions reveal dual functions of the amino-terminal tail in cystic fibrosis transmembrane conductance regulator channel gating

Previously, we observed that the cystic fibrosis transmembrane conductance regulator (CFTR) channel openings are destabilized by replacing several acidic residues in the amino-terminal tail with alanines (Naren, A. P., Cormet-Boyaka, E., Fu, J., Villain, M., Blalock, J. E., Quick, M. W., and Kirk, K. L. (1999) Science 286, 544-548). Here we determined whether this effect is due to the loss of negative charge at these sites and whether the amino-terminal tail also modulates other aspects of channel gating. We introduced cysteines at two of these positions (E54C/D58C) and tested a series of methanethiosulfonate (MTS) reagents for their effects on the gating properties of these cysteine mutants in intact Xenopus oocytes and excised membrane patches. Covalent modification of these sites with either neutral (MMTS) or charged (2-carboxyethylmethanethiosulfonate (MTSCE) and 2-(trimethylammonium)ethylmethanethiosulfonate (MTSET)) reagents markedly inhibited channel open probability primarily by reducing the rate of channel opening. The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions. The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could be reversed by the reducing agent dithiothreitol (200 microm) or by elevating the bath ATP concentration above that required to activate maximally the wild type channel (>1 mm). Interestingly, the three MTS reagents had qualitatively different effects on the duration of channel openings (i.e. channel closing rate), namely the duration of openings was negligibly changed by the neutral MMTS, decreased by the positively charged MTSET, and increased by the negatively charged MTSCE. Our results indicate that the CFTR amino tail modulates both the rates of channel opening and channel closing and that the negative charges at residues 54 and 58 are important for controlling the duration of channel openings.     

Transposition of a Ty3 GAG3-POL3 fusion mutant is limited by availability of capsid protein.

Ty3 encodes structural proteins in its upstream open reading frame (GAG3) and catalytic proteins in an overlapping open reading frame (POL3). As is the case for retroviruses, high levels of structural protein versus catalytic proteins are synthesized and we show here that catalytic proteins are derived from a GAG3-POL3 fusion polyprotein. To evaluate the relative contributions of structural and catalytic components of the Ty3 particle, we perturbed the balance of these proteins by fusing the GAG3 and POL3 frames. This fusion Ty3 was capable of complementing low levels of transposition of a donor Ty3 which contained only cis-acting sequences required for transposition. Examination of extracts of cells expressing the GAG3-POL3 fusion mutant showed that particle formation differed qualitatively and quantitatively from viruslike particle formation by wild-type Ty3. Suprisingly, expression of 238 codons of GAG3, encoding only capsid protein, complemented transposition and particle formation defects of the fusion mutant, showing that the limiting deficiency was in capsid, and not in nucleocapsid, function. In addition, protein containing the capsid domain expressed alone accumulated in the same particulate fraction as viruslike particles, showing that it was sufficient for particle formation. The activity of the Ty3 fusion mutant contrasts with the inviability of mutant retroviruses in which gag and pol frames were fused and argues that retrotransposons tolerate considerable variation in the nucleoprotein complexes that permit replication and integration.     

Second-site compensatory mutations of HIV-1 capsid mutations

The human immunodeficiency virus (HIV) capsid (CA) protein assembles into a hexameric lattice that forms the mature virus core. Contacts between the CA N-terminal domain (NTD) of one monomer and the C-terminal domain (CTD) of the adjacent monomer are important for the assembly of this core. In this study, we have examined the effects of mutations in the NTD region associated with this interaction. We have found that such mutations yielded modest reductions of virus release but major effects on viral infectivity. Cell culture and in vitro assays indicate that the infectivity defects relate to abnormalities in the viral cores. We have selected second-site compensatory mutations that partially restored HIV infectivity. These mutations map to the CA CTD and to spacer peptide 1 (SP1), the portion of the precursor Gag protein immediately C terminal to the CTD. The compensatory mutations do not locate to the molecularly modeled intermolecular NTD-CTD interface. Rather, the compensatory mutations appear to act indirectly, possibly by realignment of the C-terminal helix of the CA CTD, which participates in the NTD-CTD interface and has been shown to serve an important role in the assembly of infectious virus.     

High-resolution 3D structures reveal the biological functions of reoviruses

Viruses in the family Reoviridae are non-enveloped particles comprising a segmented double-stranded RNA genome surrounded by a two-layered or multi-layered icosahedral protein capsid. These viruses are classified into two sub-families based on their particle structural organization. Recent studies have focused on high-resolution three-dimensional structures of reovirus particles by using cryo-electron microscopy （cryo-EM） to approach the resolutions seen in X-ray crystallographic structures. The results of cryo-EM image reconstructions allow tracing of most of the protein side chains, and thus permit integration of structural and functional information into a coherent mechanism for reovirus assembly and entry.     

Targeted mutations in beta1,4-galactosyltransferase I reveal its multiple cellular functions

Beta1,4-galactosyltransferase I (GalT I) is one of the most extensively studied glycosyltransferases. It is localized in the trans-Golgi compartment of most eukaryotic cells, where it participates in the elongation of oligosaccharide chains on glycoproteins and glycolipids. GalT I has also been reported in non-Golgi locations, most notably the cell surface, where it has been suggested to function non-biosynthetically as a receptor for extracellular glycoside substrates. Cloning of the GalT I cDNAs revealed that the gene encodes two similar proteins that differ only in the length of their cytoplasmic domains. Whether these different GalT I proteins, or isoforms, have similar or different biological roles is a matter of active investigation. The functions of the GalT I proteins have been addressed by targeted mutations that eliminate either both GalT I isoforms or just the long GalT I isoform. Eliminating both GalT I proteins abolishes most, but not all, GalT activity, an observation that led to the realization that other GalT family members must exist. The loss of both GalT I isoforms leads to neonatal lethality due to a wide range of phenotypic abnormalities that are most likely the result of decreased galactosylation. When the long isoform of GalT I is eliminated, galactosylation proceeds grossly normal via the short GalT I isoform, but specific defects in cell interactions occur that are thought to depend upon a non-biosynthetic function of the long GalT I isoform.     

The amino-terminal domain of chloroplast Hsp93 is important for its membrane association and functions in vivo

Chloroplast 93-kD heat shock protein (Hsp93/ClpC), an Hsp100 family member, is suggested to have various functions in chloroplasts, including serving as the regulatory chaperone for the ClpP protease in the stroma and acting as a motor component of the protein translocon at the envelope. Indeed, although Hsp93 is a soluble stromal protein, a portion of it is associated with the inner envelope membrane. The mechanism and functional significance of this Hsp93 membrane association have not been determined. Here, we mapped the region important for Hsp93 membrane association by creating various deletion constructs and found that only the construct with the amino-terminal domain deleted, Hsp93-ΔN, had reduced membrane association. When transformed into Arabidopsis (Arabidopsis thaliana), most atHsp93V-ΔN proteins did not associate with membranes and atHsp93V-ΔΝ failed to complement the pale-green and protein import-defective phenotypes of an hsp93V knockout mutant. The residual atHsp93V-ΔN at the membranes had further reduced association with the central protein translocon component Tic110. However, the degradation of chloroplast glutamine synthetase, a potential substrate for the ClpP protease, was not affected in the hsp93V mutant or in the atHSP93V-ΔN transgenic plants. Hsp93-ΔN also had the same ATPase activity as that of full-length Hsp93. These data suggest that the association of Hsp93 with the inner envelope membrane through its amino-terminal domain is important for the functions of Hsp93 in vivo.     

Species-specific epitopes exist on the cytoplasmic amino-terminal domain of erythrocyte band 3 protein.

Monoclonal antibodies (P3-9H, P3-1F, P3-2H, P3-4A, and P3-4C) to human erythrocyte band 3 were produced using human erythrocyte membranes as the immunogen. All epitopes defined by these antibodies were found on the amino-terminal cytoplasmic domain of erythrocyte band 3. The antibodies crossreacted variously with erythrocyte band 3 of primates (chimpanzee, orangutan, Rhesus monkey, Japanese monkey, spider monkey, and capuchin monkey) in enzyme-linked immunosorbent assay. P3-9H did not crossreact with erythrocyte band 3 of any primate examined; P3-1F crossreacted only with that of chimpanzee; P3-2H crossreacted with erythrocyte band 3 of chimpanzee, spider monkey, and capuchin monkey; and P3-4A and P3-4C crossreacted with erythrocyte band 3 of all primates examined. These results suggest that evolutional changes in primates are accumulated in the amino-terminal cytoplasmic domain of band 3 and that species-specific epitopes exist on this domain.     

Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility

The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes.     

Modular evolution of the integrase domain in the Ty3/Gypsy class of LTR retrotransposons

A phylogenetic analysis of the Ty3/Gypsy group of retrotransposons identified a conserved domain (GPY/F) present in the integrases of several members of this group as well as of certain vertebrate retroviruses. The analysis suggested an evolutionary scheme for the acquisition and loss of the GPY/F domain as well as the acquisition of a chromodomain module in the integrase encoded by this group of elements that may direct targeting specificity in the host genome.     

The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain.

The retroviral oncogene v-myb encodes a 45,000 Mr nuclear protein (p45v-myb) that is predominantly associated with the chromatin of transformed cells. It has previously been shown that p45v-myb, when released from chromatin by salt-treatment, binds to DNA. To analyse the biochemical properties of p45v-myb in more detail we have expressed the v-myb coding region in Escherichia coli. Our results demonstrate that bacterially expressed myb protein has an intrinsic DNA-binding activity. Using two alternative strategies, (i) inhibition of DNA-binding by monoclonal antibodies and (ii) analysis of DNA-binding activities of partially deleted forms of the bacterial myb protein, we show that the DNA-binding domain is located in the amino-terminal region of the v-myb protein. This region has been highly conserved between myb genes of different species. Our results are therefore consistent with the hypothesis that DNA-binding is an important aspect of myb protein function.     

Distinct domains mediate the early and late functions of the Drosophila ovarian tumor proteins

The Drosophila melanogaster ovarian tumor (otu) gene encodes two novel protein isoforms that are required at multiple stages of oogenesis. We have examined the activity of a set of C-terminal truncation Otu proteins as well as a GFP-tagged Otu (Otu-GFP). These experiments have shown that a putative Tudor domain in the central region of the large Otu isoform and a separate domain in the C-terminal region are required for regulation of cyst formation and oocyte maturation, respectively. We also present evidence that a portion of Otu co-fractionates with mRNA/protein complexes (mRNPs) and show that Otu-GFP associates with cytoplasmic aggregates at periphery of the nucleus at an intermediate stage of oogenesis. This study substantially clarifies the relationship between Otu structure and function and reveals new clues about interacting components.