Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Lagging strand replication of rolling-circle plasmids: Specific recognition of the ssoA-type origins in different gram-positive bacteria

Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed ssoA, which have conserved sequence and structural features, function efficiently only in their natural hosts in vivo. To study the host specificity of sso sequences, we have analyzed the functions of two closely related ssoAs belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in Staphylococcus aureus. The pLS1 ssoA functioned poorly in vivo in S. aureus as evidenced by accumulation of high levels of ssDNA but supported efficient replication in vitro in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in S. aureus cell-free extracts may be limiting in vivo. Mapping of the initiation points of lagging strand synthesis in vivo and in vitro showed that DNA synthesis initiates from specific sites within the pLS1 ssoA. These results demonstrate that specific initiation of replication can occur from the pLS1 ssoA in S. aureus although it plays a minimal role in lagging strand synthesis in vivo. Therefore, the poor functionality of the pLS1 in vivo in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the S. aureus and Bacillus subtilis RNA polymerases from the pE194 and pLS1 ssoAs. The S. aureus RNA polymerase bound more efficiently to the native pE194 ssoA as compared with the pLS1 ssoA, suggesting that the strength of RNA polymerase–ssoA interaction may play a major role in the functionality of the ssoA sequences in Gram-positive bacteria.

Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities.

Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits.

Cytotoxic T-lymphocyte clones from different patients display limited T-cell-receptor variable-region gene usage in HLA-A2-restricted recognition of the melanoma antigen Melan-A/MART-1.

To determine whether T-cell-receptor (TCR) usage by T cells recognizing a defined human tumor antigen in the context of the same HLA molecule is conserved, we analyzed the TCR diversity of autologous HLA-A2-restricted cytotoxic T-lymphocyte (CTL) clones derived from five patients with metastatic melanoma and specific for the common melanoma antigen Melan-A/MART-1. These clones were first identified among HLA-A2-restricted anti-melanoma CTL clones by their ability to specifically release tumor necrosis factor in response to HLA-A2.1+ COS-7 cells expressing this tumor antigen. A PCR with variable (V)-region gene subfamily-specific primers was performed on cDNA from each clone followed by DNA sequencing. TCRAV2S1 was the predominant alpha-chain V region, being transcribed in 6 out of 9 Melan-A/MART-1-specific CTL clones obtained from the five patients. beta-chain V-region usage was also restricted, with either TCRBV14 or TCRBV7 expressed by all but one clone. In addition, a conserved TCRAV2S1/TCRBV14 combination was expressed in four CTL clones from three patients. None of these V-region genes was found in a group of four HLA-A2-restricted CTL clones recognizing different antigens (e.g., tyrosinase) on the autologous tumor. TCR joining regions were heterogeneous, although conserved structural features were observed in the complementarity-determining region 3 sequences. These results indicate that a selective repertoire of TCR genes is used in anti-melanoma responses when the response is narrowed to major histocompatibility complex-restricted antigen-specific interactions.

Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor

2C is a typical alloreactive cytotoxic T lymphocyte clone that recognizes two different ligands. These ligands are adducts of the allo-major histocompatibility complex (MHC) molecule H-2Ld and an endogenous octapeptide, and of the self-MHC molecule H-2Kb and another peptide. MHC-binding and T-cell assays with synthetic peptides in combination with molecular modeling studies were employed to analyze the structural basis for this crossreactivity. The molecular surfaces of the two complexes differ greatly in densities and distributions of positive and negative charges. However, modifications of the peptides that increase similarity decrease the capacities of the resulting MHC peptide complexes to induce T-cell responses. Moreover, the roles of the peptides in ligand recognition are different for self- and allo-MHC-restricted T-cell responses. The self-MHC-restricted T-cell responses were finely tuned to recognition of the peptide. The allo-MHC-restricted responses, on the other hand, largely ignore modifications of the peptide. The results strongly suggest that adaptation of the T-cell receptor to the different ligand structures, rather than molecular mimicry by the ligands, is the basis for the crossreactivity of 2C. This conclusion has important implications for T-cell immunology and for the understanding of immunological disorders.

Differentiation between homoeologous chromosomes 1A of wheat and 1Am of Triticum monococcum and its recognition by the wheat Ph1 locus.

In most allopolyploid plants, only homogenetic chromosome pairing occurs in meiosis, as a result of the recognition of genome differentiation by the genetic system regulating meiotic chromosome pairing. The nature of differentiation between chromosomes of closely related genomes is examined here by investigating recombination between wheat chromosome 1A and the closely related homoeologous chromosome 1Am of Triticum monococcum. The recognition of the differentiation between these chromosomes by the Ph1 locus, which prevents heterogenetic chromosome pairing in wheat, is also investigated. Chromosomes 1A and 1Am are shown to be colinear, and it is concluded that they are differentiated "substructurally." This substructural differentiation is argued to be recognized by the Ph1 locus. In the absence of Ph1, the distribution and frequencies of crossing over between the 1A and 1Am homoeologues were similar to the distribution and frequencies of crossing over between 1A homologues. The cytogenetic and evolutionary significance of these findings is discussed.

Geometry of circular vectors and pattern recognition of shape of a boundary

This paper deals with pattern recognition of the shape of the boundary of closed figures on the basis of a circular sequence of measurements taken on the boundary at equal intervals of a suitably chosen argument with an arbitrary starting point. A distance measure between two boundaries is defined in such a way that it has zero value when the associated sequences of measurements coincide by shifting the starting point of one of the sequences. Such a distance measure, which is invariant to the starting point of the sequence of measurements, is used in identification or discrimination by the shape of the boundary of a closed figure. The mean shape of a given set of closed figures is defined, and tests of significance of differences in mean shape between populations are proposed.

Orientation of the transcription preinitiation complex in Archaea

The basal transcription machinery of Archaea corresponds to the minimal subset of factors required for RNA polymerase II transcription in eukaryotes. Using just two factors, Archaea recruit the RNA polymerase to promoters and define the direction of transcription. Notably, the principal determinant for the orientation of transcription is not the recognition of the TATA box by the TATA-box-binding protein. Instead, transcriptional polarity is governed by the interaction of the archaeal TFIIB homologue with a conserved motif immediately upstream of the TATA box. This interaction yields an archaeal preinitiation complex with the same orientation as the analogous eukaryal complex.

Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors

Deamination of 5-methylcytosine residues in DNA gives rise to the G/T mismatched base pair. In humans this lesion is repaired by a mismatch-specific thymine DNA glycosylase (TDG or G/T glycosylase), which catalyzes specific excision of the thymine base through N-glycosidic bond hydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant pairing of two normal bases rather than a damaged base per se. An important structural issue is thus to understand how the enzyme specifically targets the T (or U) residue of the mismatched base pair. Our approach toward the study of substrate recognition and processing by catalytic DNA binding proteins has been to modify the substrate so as to preserve recognition of the base but to prevent its excision. Here we report that replacement of 2′-hydrogen atoms with fluorine in the substrate 2′-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, thereby leading to the formation of a tight, specific glycosylase–DNA complex. Biochemical characterization of these complexes reveals that the enzyme protects an ≈20-bp stretch of the substrate from DNase I cleavage, and directly contacts a G residue on the 3′ side of the mismatched U derivative. These studies provide a mechanistic rationale for the preferential repair of deaminated CpG sites and pave the way for future high-resolution studies of TDG bound to DNA.

Crystal structure of the ribosomal RNA domain essential for binding elongation factors

The structure of a 29-nucleotide RNA containing the sarcin/ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 Å resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5′-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be flexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the “flexible” region and to a lesser extent in the G-bulged cross-strand A stack.

Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1–Stat1 interaction

STATs are activated by tyrosine phosphorylation on cytokine stimulation. A tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src homology 2 domain (SH2)–phosphotyrosyl peptide interactions. IFN treatment induces the association of PIAS1 and Stat1, which results in the inhibition of Stat1-mediated gene activation. The molecular basis of the cytokine-dependent PIAS1–Stat1 interaction has not been understood. We report here that a region near the COOH terminus of PIAS1 (amino acids 392–541) directly interacts with the NH2-terminal domain of Stat1 (amino acids 1–191). A mutant PIAS1 lacking the Stat1-interacting domain failed to inhibit Stat1-mediated gene activation. By using a modified yeast two-hybrid assay, we demonstrated that PIAS1 specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or -unphosphorylated Stat1 monomer. In addition, whereas the NH2-terminal region of PIAS1 does not interact with Stat1, it serves as a modulatory domain by preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1 monomer. Thus, the cytokine-induced PIAS1–Stat1 interaction is mediated through the specific recognition of the dimeric form of Stat1 by PIAS1.

Dual HLA class I and class II restricted recognition of alloreactive T lymphocytes mediated by a single T cell receptor complex

The alloreactive human T cell clone MBM15 was found to exhibit dual specificity recognizing both an antigen in the context of the HLA class I A2 molecule and an antigen in the context of the HLA class II DR1. We demonstrated that the dual reactivity that was mediated via a single clonal T cell population depended on specific peptide binding. For complete recognition of the HLA-A2-restricted specificity the interaction of CD8 with HLA class I is essential. Interestingly, interaction of the CD8 molecule with HLA class I contributed to the HLA-DR1-restricted specificity. T cell clone MBM15 expressed two in-frame T cell receptor (TCR) Vα transcripts (Vα1 and Vα2) and one TCR Vβ transcript (Vβ13). To elucidate whether two TCR complexes were responsible for the dual recognition or one complex, cytotoxic T cells were transduced with retroviral vectors encoding the different TCR chains. Only T cells transduced with the TCR Vα1Vβ13 combination specifically recognized both the HLA-A2+ and HLA-DR1+ target cells, whereas the Vα2Vβ13 combination did not result in a TCR on the cell surface. Thus a single TCRαβ complex can have dual specificity, recognizing both a peptide in the context of HLA class I as well as a peptide in the context of HLA class II. Transactivation of T cells by an unrelated antigen in the context of HLA class II may evoke an HLA class I-specific T cell response. We propose that this finding may have major implications for immunotherapeutic interventions and insight into the development of autoimmune diseases.

Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus.

We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.

Mimics of the binding sites of opioid receptors obtained by molecular imprinting of enkephalin and morphine.

Molecular imprinting of morphine and the endogenous neuropeptide [Leu5]enkephalin (Leu-enkephalin) in methacrylic acid-ethylene glycol dimethacrylate copolymers is described. Such molecular imprints possess the capacity to mimic the binding activity of opioid receptors. The recognition properties of the resultant imprints were analyzed by radioactive ligand binding analysis. We demonstrate that imprinted polymers also show high binding affinity and selectivity in aqueous buffers. This is a major breakthrough for molecular imprinting technology, since the binding reaction occurs under conditions relevant to biological systems. The antimorphine imprints showed high binding affinity for morphine, with Kd values as low as 10(-7) M, and levels of selectivity similar to those of antibodies. Preparation of imprints against Leu-enkephalin was greatly facilitated by the use of the anilide derivative rather than the free peptide as the print molecule, due to improved solubility in the polymerization mixture. Free Leu-enkephalin was efficiently recognized by this polymer (Kd values as low as 10(-7) M were observed). Four tetra- and pentapeptides, with unrelated amino acid sequences, were not bound. The imprints showed only weak affinity for two D-amino acid-containing analogues of Leu-enkephalin. Enantioselective recognition of the L-enantiomer of phenylalanylglycine anilide, a truncated analogue of the N-terminal end of enkephalin, was observed.