Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.
The consensus motifs of HLA-Cw3, -Cw4, -Cw6, and -Cw7 ligands were determined by pool sequencing. Together with information obtained by sequencing of some prominent individual peptides, the results indicate the following: (t) all four HLA-C molecules are associated with peptides. (i) These peptides adhere to allele-specific motifs that are similar to those of to HLA-A or -B molecules; they have a preferred length of nine amino acids and an anchor residue at the C terminus. (Mi) AU four HLA-C molecules analyzed exhibit related peptide motifs, although each ailelic product shows individual characteristics in fine specificity. (iv) Processing and origin of peptides appear not to be different from that of other class I molecules. (v) No obvious difference at C-terminal position 9 was present in the peptides isolated from the two dimorphic variants of HLA-C that determine dominant resistance to natural killer NKl-speciflc cells (HLA-Cw4, -Cw6) or to NK2-specific cells (HLA-Cw3, -Cw7) and that differ in two residues in or near the pocket at position 9.
The peptide motifs of two HLA molecules, B8 and DR3(17), which are associated with autoimmune diseases including myasthenia gravis, were determined from natural peptide pools using Edman degradation. The majority of HLA-B8 ligands are nonamers preferentially terminated by leucine. As a characteristic feature of the HLA-B8 motif, there is a high degree of conservation of positively charged amino acids at position 3 and 5, exclusively lysine at position 3, and lysine or arginine at position 5. Additional evidence for this allele-specific motif is the presence of these features in several viral peptides recognized by HLA-B8 restricted T cells. The DR3(17) motif is characterized by four conserved anchor-like positions ordered in an almost symmetrical arrangement, as has been found for DR1 and DR5 motifs. A first hydrophobic/aromatic anchor three to four residues apart from the N-terminus (at relative position 1) appears to be a common feature of DR ligands. The second anchor is an aspartate at relative position 4, which is likely to be the DR3(17)-specific contact site in the groove. Two additional conserved positions closer to the C-terminus are occupied by charged amino acids at relative position 6 and by hydrophobic/aromatic residues at positions 8, 9, or 10. Eight individual naturally processed DR17 ligands were sequenced and were found to be derived from exogenous proteins and cytoplasmic membrane receptors. These natural peptides conform well to the determined motif. A single exchange of the anchor-like positions in a model peptide abrogated binding to DR17+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)
SummaryClass II-associated invariant chain peptides (CLIPs) compete with natural allele-specific ligands for binding to several purified HLA-DR molecules. Truncation and substitution analysis showed that a minimal sequence of 13 amino acids is sufficient for excellent binding to DR17 and DR1. Hydrophobic residues at relative positions 1 and 9 (P1 and P9) which are shared among these DK-ligands, and are found to be anchored in complementary pockets by x-ray crystallography allow specific binding. Two flanking residues at either end next to the specific contact sites Metl0v and Metlts contribute to binding irrespective of their side chains, suggesting H-bonds to the major histocompatibility complex (MHC) molecule. Thus, CLIPs behave like conventional ligands, however, lack their allele-specific contact sites. Introduction of the DK17-specific contact site aspartate at P4 dramatically improves invariant chain-peptide binding to DR.17, but reduces DK1 binding. By contrast, binding to DR1, but not DK17 is strongly improved after introduction of the DKl-specific contact site alanine at P6. In addition, analyzing the fine specificity of the hydrophobic contact sites at P1 and P9, CLIP variants reflected the allele-specific preferences of DR17-or DKl-ligands, respectively, for aliphatic or aromatic residues. Alignment studies suggest that CLIPs are designed for promiscuous binding in the groove of many MHC class II molecules by taking advantage of one or more supermotifs. One such supermotif, for example, does not include the DK17-specific contact site aspartate at P4, which in conventional natural ligands like Apolipoprotein (2877-94) is necessary to confer a stable conformation. Introduction of aspartate at P4 generates a CLIP variant that is stable in the presence of sodium dodecyl sulfate, such as allele-specific ligands. Studying the stability of class II-CLIP complexes at pH 5, we found that CLIPs, similar to anchor-amputated ligands, can be released from class II molecules, in contrast to conventional natural ligands, which were irreversibly bound. Taken together, our data provide compelling evidence that CLIP peptides bind into the class II groove.
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