Key residues revealed in a major conformational epitope of the U1-70K protein

Epitopes depending on three-dimensional folding of proteins have during recent years been acknowledged to be main targets for many autoantibodies. However, a detailed resolution of conformation-dependent epitopes has to date not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of immunodominant epitopes of the U1-70K protein, the major autoantigen recognized by human ribonucleoprotein (RNP)-positive sera, we have used diversely mutated recombinant Drosophila melanogaster 70K proteins as antigens in assays for human anti-RNP antibodies. Thus, the contribution of individual amino acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be reconstituted rather than obliterated was enabled. Our results reveal that amino acid residue 125 is situated at a crucial position for recognition by human anti-RNP autoantibodies and that flanking residues at positions 119–126 also appear to be of utmost importance for recognition. These results are discussed in relation to structural models of RNA-binding domains, and tertiary structure modeling indicates that the residues 119–126 are situated at easily accessible positions in the end of an α-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, we believe it will be possible to analyze other immunodominant regions in which protein conformation has a strong impact.

A Transmembrane Form of the Prion Protein Contains an Uncleaved Signal Peptide and Is Retained in the Endoplasmic Reticululm

Although there is considerable evidence that PrPSc is the infectious form of the prion protein, it has recently been proposed that a transmembrane variant called CtmPrP is the direct cause of prion-associated neurodegeneration. We report here, using a mutant form of PrP that is synthesized exclusively with the CtmPrP topology, that CtmPrP is retained in the endoplasmic reticulum and is degraded by the proteasome. We also demonstrate that CtmPrP contains an uncleaved, N-terminal signal peptide as well as a C-terminal glycolipid anchor. These results provide insight into general mechanisms that control the topology of membrane proteins during their synthesis in the endoplasmic reticulum, and they also suggest possible cellular pathways by which CtmPrP may cause disease.

Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

Methionine residues as endogenous antioxidants in proteins

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they can form disulfide bonds that contribute to protein structure. In contrast, the specific functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, available as an efficient oxidant scavenger. Reduction back to methionine by methionine sulfoxide reductases would allow the antioxidant system to function catalytically. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the 16 methionine residues could be oxidized with little effect on catalytic activity of the enzyme. The oxidizable methionine residues were found to be relatively surface exposed, whereas the intact residues were generally buried within the core of the protein. Furthermore, the susceptible residues were physically arranged in an array that guarded the entrance to the active site.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492–742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Ecological intensification of cereal production systems: Yield potential, soil quality, and precision agriculture

Wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) provide about two-thirds of all energy in human diets, and four major cropping systems in which these cereals are grown represent the foundation of human food supply. Yield per unit time and land has increased markedly during the past 30 years in these systems, a result of intensified crop management involving improved germplasm, greater inputs of fertilizer, production of two or more crops per year on the same piece of land, and irrigation. Meeting future food demand while minimizing expansion of cultivated area primarily will depend on continued intensification of these same four systems. The manner in which further intensification is achieved, however, will differ markedly from the past because the exploitable gap between average farm yields and genetic yield potential is closing. At present, the rate of increase in yield potential is much less than the expected increase in demand. Hence, average farm yields must reach 70–80% of the yield potential ceiling within 30 years in each of these major cereal systems. Achieving consistent production at these high levels without causing environmental damage requires improvements in soil quality and precise management of all production factors in time and space. The scope of the scientific challenge related to these objectives is discussed. It is concluded that major scientific breakthroughs must occur in basic plant physiology, ecophysiology, agroecology, and soil science to achieve the ecological intensification that is needed to meet the expected increase in food demand.

The effect of cultivation on the size, shape, and persistence of disease patches in fields

Epidemics of soil-borne plant disease are characterized by patchiness because of restricted dispersal of inoculum. The density of inoculum within disease patches depends on a sequence comprising local amplification during the parasitic phase followed by dispersal of inoculum by cultivation during the intercrop period. The mechanisms that control size, shape, and persistence have received very little rigorous attention in epidemiological theory. Here we derive a model for dispersal of inoculum in soil by cultivation that takes account into the discrete stochastic nature of the system in time and space. Two parameters, probability of movement and mean dispersal distance, characterize lateral dispersal of inoculum by cultivation. The dispersal parameters are used in combination with the characteristic area and dimensions of host plants to identify criteria that control the shape and size of disease patches. We derive a critical value for the probability of movement for the formation of cross-shaped patches and show that this is independent of the amount of inoculum. We examine the interaction between local amplification of inoculum by parasitic activity and subsequent dilution by dispersal and identify criteria whereby asymptomatic patches may persist as inoculum falls below a threshold necessary for symptoms to appear in the subsequent crop. The model is motivated by the spread of rhizomania, an economically important soil-borne disease of sugar beet. However, the results have broad applicability to a very wide range of diseases that survive as discrete units of inoculum. The application of the model to patch dynamics of weed seeds and local introductions of genetically modified seeds is also discussed.

The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin.

In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction.

Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by site-directed mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of the [Fe4S4]3+ core.