Initiation factor IF1, the smallest of the three factors, (Cummings and Hershey, 1994), indicating that one or more of the activities of IF1 must The structure of the translational initiation factor be essential for the cell. Maurizio IF1 from Escherichia coli has been determined withSeveral functions have been reported for IF1. These multidimensional NMR spectroscopy. Using 1041 disinclude: (i) the enhancement of the rate of 70S ribosome tance and 78 dihedral constraints, 40 distance geometry dissociation and subunit association (Godefroy-Colburn structures were calculated, which were refined by et al., 1975); (ii) the stimulation of the activity of IF2 restrained molecular dynamics. From this set, 19 strucand IF3 in the formation of the 30S initiation complex tures were selected, having low constraint energy and (Wintermeyer and Gualerzi, 1983; Pon and Gualerzi, few constraint violations. The ensemble of 19 structures 1984); and (iii) the modulation of the interaction of IF2 displays a root-mean-square deviation versus the averwith the ribosome, increasing its affinity for the 30S age of 0.49 Å for the backbone atoms and 1.12 Å for ribosomal subunit when IF1 is bound and indirectly all atoms for residues 6-36 and 46-67. The structure favouring its release when IF1 is ejected (Stringer et al., of IF1 is characterized by a five-stranded β-barrel.1977; Celano et al., 1988). In addition, by binding to the The loop connecting strands three and four contains a A-site of the 30S ribosomal subunit, IF1 may contribute short 3 10 helix but this region shows considerably to the fidelity of the selection of the initiation site of the higher flexibility than the β-barrel. The fold of IF1 is mRNA (Moazed et al., 1995). very similar to that found in the bacterial cold shock Equilibrium binding studies have shown that IF1 binds proteins CspA and CspB, the N-terminal domain of to the 30S ribosomal subunit in a 1:1 ratio (Zucker and aspartyl-tRNA synthetase and the staphylococcal Hershey, 1986;Celano et al., 1988). The binding affinity nuclease, and can be identified as the oligomer-binding depends strongly upon the ionic strength and upon the motif. Several proteins of this family are nucleic acidpresence of IF2 and IF3 which increase its affinity for the binding proteins. This suggests that IF1 plays its role ribosome; the K a ranging from 5ϫ10 5 to 2.5ϫ10 8 M -1 . in the initiation of protein synthesis by nucleic acid Interaction with 50S ribosomal subunits was also observed, interactions. Specific changes of NMR signals of IF1 but the affinity is considerably lower than for the 30S upon titration with 30S ribosomal subunit identifies ribosomal subunits. Stable interaction with 70S ribosomes several residues that are involved in the interaction has never been observed; in fact, the addition of 50S to with ribosomes.

The salivary antimicrobial peptide histatin-5 is able to aggregate and fuse negatively charged small unilamellar vesicles, and this fusogenic activity is selectively induced by the presence of zinc ions. Circular dichroism spectroscopy shows that histatin-5, in the presence of negatively charged vesicles and zinc ions, undergoes a conformational change leading to the stabilization of an alpha-helical secondary structure. We attribute the specific action of the zinc ions to the presence of a consensus sequence, HEXXH, located in the C-terminal functional domain of histatin-5, a recognized zinc-binding motif in many proteins. Two-dimensional proton NMR spectroscopy of histatin-5 in a trifluoroethanol/water mixture (a membrane mimetic environment) has been performed and the results analyzed by means of distance geometry and restrained molecular dynamics simulations. Our results reveal that the peptide chain, including the Zn-binding consensus sequence corresponding to residues 15-19, is in a helicoidal conformation. Comparison of the chemical shifts of the individual amino acids in histatin-5 with those recently reported in other solvents indicates that trifluoroethanol/water has a structuring capability somewhere between water and dimethyl sulfoxide. The mechanism of action of this antimicrobial peptide is discussed on the basis of its structural characteristics with particular attention to the Zn-binding motif.

The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudomonas syringae pv. syringae has been determined by a combination of chemical methods, 'H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin-producingwith Tyr acylating aThr to form a macrolactone ring, and smaller amounts of the 3-hydroxydodccanoyl homologue. Evidence was obtained that a third syringomycin-producing strain and a syringotoxin-producing strain synthesize 3-hydroxydecanoyl-Dhb-Pro-Val-Ala-AlaVal-Leu-Ala-Ala-Dhb-Val-Dhb-Ala-Val-Ala-Ala-Dhb-aThr-Ser-Ala-Vnl~~Ala-Dab-Dab-Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3-hydroxydodecanoyl homologue.Phytotoxin; Lipodepsipeptide; Syringopeptin; Pseudomonas syringae pv. syringae

Among the members of the ubiquitin‐like (Ubl) protein family, neural precursor cell expressed developmentally down‐regulated protein 8 (NEDD8) is the closest in sequence to ubiquitin (57% identity). The two modification mechanisms and their functions, however, are highly distinct and the two Ubls are not interchangeable. A complex network of interactions between modifying enzymes and adaptors, most of which are specific while others are promiscuous, ensures selectivity. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that specifically binds NEDD8 has yet been described. Here, we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their specificity/promiscuity. Many ubiquitin‐binding domains bind ubiquitin preferentially and, to a lesser extent, NEDD8. In a few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We have also identified a unique domain, mapping to the carboxyl end of the protein KHNYN, which has a stark preference for NEDD8. Given its ability to bind neddylated cullins, we have named this domain CUBAN (Cullin‐Binding domain Associating with NEDD8). We present here the solution structure of the CUBAN domain both in the isolated form and in complex with NEDD8. The results contribute to the understanding of the discrimination mechanism between ubiquitin and the Ubl. They also provide new insights on the biological role of a ill‐defined protein, whose function is hitherto only predicted.

Bile acids are physiological detergents facilitating absorption, transport, and distribution of lipid-soluble vitamins and dietary fats;they also play a role as signaling molecules that activate nuclear receptors and regulate cholesterol metabolism. Bile acid circulation is mediated by bile acid binding proteins (BABPs), and a detailed structural study of the complex of BABPs with bile salts has become a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. The solution structure here reported describes, at variance with previously determined singly ligated structures, a BABP in a ternary complex with two bile acid molecules, obtained by employing a variety of NMR experiments. Exchange processes between the two bound chenodeoxycholate molecules as well as the more superficial ligand and the free pool have been detected through ROESY and diffusion experiments. Significant backbone flexibility has been observed in regions located at the protein open end, facilitating bile salts exchange. A detailed description of the protonation states and tautomeric forms of histidines strongly supports the view that histidine protonation modulates backbone flexibility and regulates ligand binding. This structure opens the way to targeted site-directed mutagenesis and interaction studies to investigate both binding and nuclear localization mechanisms.

Proteasomal-mediated rapid turnover of proteins is often modulated by phosphorylation of PEST sequences. The E2 protein from papillomavirus participates in gene transcription, DNA replication, and episomal genome maintenance. Phosphorylation of a PEST sequence located in a flexible region accelerates its degradation. NMR analysis of a 29 amino acid peptide fragment derived from this region shows pH-dependent polyproline II and alpha helix structures, connected by a turn. Phosphorylation, in particular that at serine 301, disrupts the overall structure, and point mutations have either stabilizing or destabilizing effects. There is an excellent correlation between the thermodynamic stability of different peptides and the half-life of E2 proteins containing the same mutations in vivo. The structure around the PEST region appears to have evolved a marginal stability that is finely tunable by phosphorylation. Thus, conformational stability, rather than recognition of a phosphate modification, modulates the degradation of this PEST sequence by the proteasome machinery.

Strain 16 of the human papillomavirus is responsible for the largest number of cases of cervical cancers linked to this virus, and the E2 protein is the transcriptional regulator of all viral genes. We present the first structure for the DNA binding domain of HPV16 E2 bound to DNA, and in particular, to a natural cognate sequence. The NMR structure of the protein backbone reveals that the overall conformation remains virtually unchanged, and chemical shift analysis of the protein bound to a shorter DNA duplex uncovered a contact out of the minimal E2 DNA binding site, made by lysine 349. This contact was confirmed by titration calorimetry and mutagenesis, with a contribution of 1.0 kcal mol(-)(1) to binding energy. HPV16 E2 has the highest DNA binding affinity and exerts a strict transcriptional control, translated into the repression of the E6 and E7 oncogenes. These novel features provide the structural and thermodynamic basis for this tight transcriptional control, the loss of which correlates with carcinogenesis.

The mode of atrazine [6‐chloro‐N‐ethyl‐N′‐(1‐methylethyl)‐1,3,5‐triazine‐2,4‐diamine] adsorption to humic substances is still uncertain, mainly because of the large heterogeneity of humus and its poor molecular characterization. In this study, four humic fractions were isolated from a peat and a volcanic soil by three different extraction mechanisms: charge repulsion (NaOH extraction), ligand exchange (pyrophosphate extraction, Na4P2O7), and hydrogen‐bonding disruption (extractions by aqueous mixture of acetone and dymethyl sulphoxide, DMSO). These four humic fractions showed consistent differences in molecular structure between the two soils. Humic fractions extracted by acetone had the largest content of aliphatic C, whereas those obtained by the pyrophosphate solution were richest in aromatic C. Sorption isotherms of 14C‐labeled atrazine conducted on the four humic fractions showed that, for both soils, the order of adsorption was acetone > NaOH > DMSO > Na4P2O7, whereas the order for desorption was the reverse. The relation between results of atrazine interaction and the molecular structure of humic matter indicates that the aliphatic C content of soil organic matter may be one of the parameters controlling atrazine adsorption to soils. Hydrophobic interactions and conformational flexibility in the aliphatic portions of humic matter controlled the adsorption of atrazine in the interior of humic self‐associated aggregates and the degree of desorption found both in water and methanol. Conversely, the conformational rigidity conferred to humic fractions by a large content of aromatic moieties appeared conducive only to surface adsorption and thus to easier herbicide desorption.