Side-chain-to-tail thiolactone peptide inhibitors of the staphylococcal quorum-sensing system

The expression of many staphylococcal virulence factors are regulated by the agr locus via a two-component signal transduction system (TCSTS), which is activated in response to a secreted autoinducer peptide (AIP). By exploiting the unique chemical architecture of the naturally occurring AIP-1, several potent inhibitors of staphylococcal TCSTS were designed and synthesized using either a linear or branched solid-phase approach. These inhibitors are competitive binders and contain the crucial 16-membered side-chain-to-tail thiolactone peptide pharmacophore.     

RNA recognition by a Staufen double-stranded RNA-binding domain

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix α1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.     

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain.

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.     

Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase

Methylation of CpG dinucleotides is the major epigenetic modification of mammalian genomes, critical for regulating chromatin structure and gene activity. The mixed-lineage leukaemia (MLL) CXXC domain selectively binds nonmethyl-CpG DNA, and is required for transformation by MLL fusion proteins that commonly arise from recurrent chromosomal translocations in infant and secondary treatment-related acute leukaemias. To elucidate the molecular basis of nonmethyl-CpG DNA recognition, we determined the structure of the human MLL CXXC domain by multidimensional NMR spectroscopy. The CXXC domain has a novel fold in which two zinc ions are each coordinated tetrahedrally by four conserved cysteine ligands provided by two CGXCXXC motifs and two distal cysteine residues. We have identified the CXXC domain DNA binding interface by means of chemical shift perturbation analysis, cross-saturation transfer and site-directed mutagenesis. In particular, we have shown that residues in an extended surface loop are in close contact with the DNA. These data provide a template for the design of specifically targeted therapeutics for poor prognosis MLL-associated leukaemias.     

In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone

In Pseudomonas aeruginosa , synthesis of the quorum-sensing signal molecules N -butanoyl- L -homoserine lactone (BHL) and N -hexanoyl- L -homoserine lactone (HHL) requires the LuxI homologue RhlI(VsmI). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified RhlI can catalyse the biosynthesis of BHL and HHL using either S -adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli , we conclude that SAM is the natural substrate for RhlI-directed N -acylhomoserine lactone (AHL) biosynthesis. The N -acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n -butanoyl-CoA or n -hexanoyl-CoA). The specificity of RhlI for charged CoA derivatives is demonstrated as RhlI was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n -octanoyl-CoA, n -decanoyl-CoA, DL-β-hydroxybutanoyl-CoA or crotonoyl-CoA. RhlI was also unable to use N -acetyl- S -3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the RhlI-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.     

Structural basis of p63α SAM domain mutants involved in AEC syndrome

p63 is a member of the p53 tumour suppressor family that includes p73. The p63 gene encodes a protein comprising an N-terminal transactivation domain, a DNA binding domain and an oligomerization domain, but varies in the organization of the C-terminus as a result of complex alternative splicing. p63α contains a C-terminal sterile α motif (SAM) domain that is thought to function as a protein-protein interaction domain. Several missense and heterozygous frame shift mutations, encoded within exon 13 and 14 of the p63 gene, have been identified in the p63α SAM domain in patients suffering from ankyloblepharon-ectodermal dysplasia-clefting syndrome. Here we report the solution and high resolution crystal structures of the p63α SAM domain and investigate the effect of several mutations (L553F/V, C562G/W, G569V, Q575L and I576T) on the stability of the domain. The possible effects of other mutations are also discussed.     

Crystal structure of ribosomal protein L30e from the extreme thermophile Thermococcus celer: thermal stability and RNA binding

We report here the high-resolution crystal structure of the ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer determined at cryo-temperature. When it is compared with its mesophilic homologue, L30e from yeast, a number of structural features that can enhance thermostability are revealed. Disordered residues corresponding to a large RNA-binding loop in yeast L30e are well structured in the T. celer protein. The overall charge of T. celer L30e is near neutral, whereas that of the yeast homologue is highly positive. This is the result of an increase in the number of acidic residues at the expense of polar residues, Asn, Ser, and Thr. Extensive ion pair networks are found on the molecular surface. Exposed nonpolar surface areas are reduced in the T. celer protein. Its side chain atoms preferably form hydrogen bonds with main chain atoms. Taken together, these factors contribute to high protein stability. The roles of well-conserved L30e residues are studied and found to be important in defining a very compact overall structure and in maintaining the structure of the RNA binding site. By comparing it with the yeast homologue, we also identified the residues that are responsible for RNA binding and built a model to illustrate how L30e binds to an RNA kink turn motif.     

The structure of a tunicate C-type lectin from Polyandrocarpa misakiensis complexed with D -galactose.

C-type lectins are calcium-dependent carbohydrate-recognising proteins. Isothermal titration calorimetry of the C-type Polyandrocarpa lectin (TC14) from the tunicate Polyandrocarpa misakiensis revealed the presence of a single calcium atom per monomer with a dissociation constant of 2.6 microM, and confirmed the specificity of TC14 for D -galactose and related monosaccharides. We have determined the 2.2 A X-ray crystal structure of Polyandrocarpa lectin complexed with D -galactose. Analytical ultracentrifugation revealed that TC14 behaves as a dimer in solution. This is reflected by the presence of two molecules in the asymmetric unit with the dimeric interface formed by antiparallel pairing of the two N-terminal beta-strands and hydrophobic interactions. TC14 adopts a typical C-type lectin fold with differences in structure from other C-type lectins mainly in the diverse loop regions and in the second alpha-helix, which is involved in the formation of the dimeric interface. The D -galactose is bound through coordination of the 3 and 4-hydroxyl oxygen atoms with a bound calcium atom. Additional hydrogen bonds are formed directly between serine, aspartate and glutamate side-chains of the protein and the sugar 3 and 4-hydroxyl groups. Comparison of the galactose binding by TC14 with the mannose binding by rat mannose-binding protein reveals how monosaccharide specificity is achieved in this lectin. A tryptophan side-chain close to the binding site and the distribution of hydrogen-bond acceptors and donors around the 3 and 4-hydroxyl groups of the sugar are essential determinants of specificity. These elements are, however, arranged in a very different way than in an engineered galactose-specific mutant of MBPA. Possible biological functions can more easily be understood from the fact that TC14 is a dimer under physiological conditions.