Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers.

The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.

Heat Shock Proteins Revisited: Using a Mutasynthetically Generated Reblastatin Library to Compare the Inhibition of Human and Leishmania Hsp90s.

Thirteen new reblastatin derivatives, with alkynyl, amino and fluoro substituents on the aromatic ring, were prepared by a chemo-biosynthetic approach using an AHBA(-) mutant strain of Streptomyces hygroscopicus, the geldanamycin producer. The inhibitory potencies of these mutaproducts and of an extended library of natural products and derivatives were probed with purified heat shock proteins (Hsps), obtained from Leishmania braziliensis (LbHsp90) as well as from human sources (HsHsp90). We determined the activities of potential inhibitors by means of a displacement assay in which fluorescence-labelled ATP competes for the ATP binding sites of Hsps in the presence of the inhibitor in question. The results were compared with those of cell-based assays and, in selected cases, of isothermal titration calorimetry (ITC) measurements. In essence, reblastatin derivatives are also able to bind effectively to the ATP-binding site of LbHsp90, and for selected derivatives, moderate differences in binding to LbHsp90 and HsHsp90 were encountered. This work demonstrates that parasitic heat shock proteins can be developed as potential pharmaceutical targets.

New, non-quinone fluorogeldanamycin derivatives strongly inhibit Hsp90.

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.

Insights into the biosynthesis and assembly of cryptophycean phycobiliproteins.

Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes, and cryptophytes for light-harvesting and consist of apoproteins covalently associated with open-chain tetrapyrrole chromophores. Although the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single type of phycobiliprotein that is localized in the thylakoid lumen. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. In this study, we identified and characterized enzymes involved in PEB biosynthesis. In addition, we present the first in-depth biochemical characterization of a eukaryotic phycobiliprotein lyase (GtCPES). Plastid-encoded HO (GtHo) was shown to convert heme into biliverdin IXα providing the substrate with a putative nucleus-encoded DHBV:ferredoxin oxidoreductase (GtPEBA). A PEB:ferredoxin oxidoreductase (GtPEBB) was found to convert DHBV to PEB, which is the substrate for the phycobiliprotein lyase GtCPES. The x-ray structure of GtCPES was solved at 2.0 Å revealing a 10-stranded ß-barrel with a modified lipocalin fold. GtCPES is an S-type lyase specific for binding of phycobilins with reduced C15=C16 double bonds (DHBV and PEB). Site-directed mutagenesis identified residues Glu-136 and Arg-146 involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apophycobiliprotein CpeB is proposed and discussed.

Cooperative binding of PhoB(DBD) to its cognate DNA sequence-a combined application of single-molecule and ensemble methods.

A combined approach based on isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) experiments, circular dichroism spectroscopy (CD), atomic force microscopy (AFM) dynamic force spectroscopy (DFS), and surface plasmon resonance (SPR) was applied to elucidate the mechanism of protein-DNA complex formation and the impact of protein dimerization of the DNA-binding domain of PhoB (PhoB(DBD)). These insights can be translated to related members of the family of winged helix-turn-helix proteins. One central question was the assembly of the trimeric complex formed by two molecules of PhoB(DBD) and two cognate binding sites of a single oligonucleotide. In addition to the native protein WT-PhoB(DBD), semisynthetic covalently linked dimers with different linker lengths were studied. The ITC, SPR, FRET, and CD results indicate a positive cooperative binding mechanism and a decisive contribution of dimerization on the complex stability. Furthermore, an alanine scan was performed and binding of the corresponding point mutants was analyzed by both techniques to discriminate between different binding types involved in the protein-DNA interaction and to compare the information content of the two methods DFS and SPR. In light of the published crystal structure, four types of contribution to the recognition process of the pho box by the protein PhoB(DBD) could be differentiated and quantified. Consequently, it could be shown that investigating the interactions between DNA and proteins with complementary techniques is necessary to fully understand the corresponding recognition process.

Evolution from the prokaryotic to the higher plant chloroplast signal recognition particle: the signal recognition particle RNA is conserved in plastids of a wide range of photosynthetic organisms.

The protein targeting signal recognition particle (SRP) pathway in chloroplasts of higher plants has undergone dramatic evolutionary changes. It disposed of its RNA, which is an essential SRP component in bacteria, and uses a unique chloroplast-specific protein cpSRP43. Nevertheless, homologs of the conserved SRP54 and the SRP receptor, FtsY, are present in higher plant chloroplasts. In this study, we analyzed the phylogenetic distribution of SRP components in photosynthetic organisms to elucidate the evolution of the SRP system. We identified conserved plastid SRP RNAs within all nonspermatophyte land plant lineages and in all chlorophyte branches. Furthermore, we show the simultaneous presence of cpSRP43 in these organisms. The function of this novel SRP system was biochemically and structurally characterized in the moss Physcomitrella patens. We show that P. patens chloroplast SRP (cpSRP) RNA binds cpSRP54 but has lost the ability to significantly stimulate the GTPase cycle of SRP54 and FtsY. Furthermore, the crystal structure at 1.8-Å resolution and the nucleotide specificity of P. patens cpFtsY was determined and compared with bacterial FtsY and higher plant chloroplast FtsY. Our data lead to the view that the P. patens cpSRP system occupies an intermediate position in the evolution from bacterial-type SRP to higher plant-type cpSRP system.

PEX14 is required for microtubule-based peroxisome motility in human cells.

We have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.

CpeS is a lyase specific for attachment of 3Z-PEB to Cys82 of {beta}-phycoerythrin from Prochlorococcus marinus MED4.

In contrast to the majority of cyanobacteria, the unicellular marine cyanobacterium Prochlorococcus marinus MED4 uses an intrinsic divinyl-chlorophyll-dependent light-harvesting system for photosynthesis. Despite the absence of phycobilisomes, this high-light adapted strain possesses ß-phycoerythrin (CpeB), an S-type lyase (CpeS), and enzymes for the biosynthesis of phycoerythrobilin (PEB) and phycocyanobilin. Of all linear tetrapyrroles synthesized by Prochlorococcus including their 3Z- and 3E-isomers, CpeS binds both isomers of PEB and its biosynthetic precursor 15,16-dihydrobiliverdin (DHBV). However, dimerization of CpeS is independent of bilins, which are tightly bound in a complex at a ratio of 1:1. Although bilin binding by CpeS is fast, transfer to CpeB is rather slow. CpeS is able to attach 3E-PEB and 3Z-PEB to dimeric CpeB but not DHBV. CpeS transfer of 3Z-PEB exclusively yields correctly bound ßCys(82)-PEB, whereas ßCys(82)-DHBV is a side product of 3E-PEB transfer. Spontaneous 3E- and 3Z-PEB addition to CpeB is faulty, and products are in both cases ßCys(82)-DHBV and likely a PEB bound at ßCys(82) in a non-native configuration. Our data indicate that CpeS is specific for 3Z-PEB transfer to ßCys(82) of phycoerythrin and essential for the correct configuration of the attachment product.