Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR^Sl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR^Sl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR^Sl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoR^Sl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control.     

Conformational analysis of bacterial cell wall peptides indicates how particular conformations have influenced the evolution of penicillin-binding proteins,beta-lactam antibiotics and antibiotic resistance mechanisms

Our aim was to use a conformational analysis technique developed for peptides to identify structural relationships between bacterial cell wall peptides and beta-lactam antibiotics that might help to explain their different actions as substrates and inhibitors of penicillin binding proteins (PBPs). The conformational forms of the model cell wall peptide Ac-L-Lys(Ac)-D-Ala-D-Ala are described by just a few backbone torsion combinations: three C-terminal carboxylate regions, with Tor8 (psi(i+1)) ranges of D3 region (50 degrees to 70 degrees ), D6 region (140 degrees to 170 degrees ) and D9 region (-50 degrees to -70 degrees ) are combined with either of two Tor6 (phi(i))-Tor4 (psi(i)) combinations, C4 region (-50 degrees to -80 degrees ) with B8 region (-40 degrees to -70 degrees ) or C11 region (30 degrees to 50 degrees ) with B2 region (30 degrees to 70 degrees ). From these results, and comparisons with conformational analyses of various beta-lactams and Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that molecular recognition of cell wall peptide substrates by PBPs requires conformers with backbone torsion angles of D3C4B8. beta-Lactam antibiotics are constrained compounds with fewer conformational forms; these match well the backbone torsions of cell wall peptides at D3C4, allowing their recognition and acylation by PBPs, whereas their unique Tor4 produces differently orientated CO and N atoms that appear to prevent subsequent deacylation, leading to their action as suicide substrates. The results are also related to the selective pressures involved in evolution of beta-lactamases from PBPs. From analysis of conformers of Ac-L-Lys(Ac)-D-Ala-D-Ala and the vancomycin-resistant analogue Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that vancomycin may recognise D6C11B2 conformers, giving it complementary substrate specificity to PBPs. This approach could have applications in the rational design of antibiotics targeted against PBPs and their substrates.     

Conformational changes of the BS2 operator DNA upon complex formation with the Antennapedia homeodomain studied by NMR with 13C/15N-labeled DNA.

The NMR structures have been determined for a 13C/15N doubly labeled 14 base-pair DNA duplex comprising the BS2 operator sequence both free in solution and in the complex with the Antennapedia homeodomain. The impact of the DNA labeling is assessed from comparison with a previous structure of the same complex that was determined using isotope labeling only for the protein. Differences between the two structure determinations are nearly completely limited to the DNA, which retains the global B -conformation of the free DNA also in the complex. Local protein-induced conformational changes are a narrowing of the minor groove due to the interaction with the N-terminal arm of the homeodomain, and changes of the sugar puckers of the deoxyriboses G5 and C6, which are apparently induced by van der Waals interactions with Tyr25, and with Gln50 and Arg53, respectively. The high conservation of these amino acid residues in homeodomains suggests that protein-induced shifts in some sugar puckers contribute to the affinity of homeodomains to their cognate DNA. The data obtained here with the Antennapedia homeodomain-DNA complex clearly show that nucleic acid isotope-labeling can support detailed conformational characterization of DNA in complexes with proteins, which will be indispensable for structure determinations of complexes containing globally distorted DNA conformations.     

Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor.

Fur (ferric uptake regulation protein) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. It has been suggested that metal binding induces a conformational change in the protein, which is subsequently able to recognize DNA. This mechanism of activation has been investigated here using selective chemical modification monitored by mass spectrometry. The reactivity of each lysine residue of the Fur protein was studied, first in the apo form of the protein, then after metal activation and finally after DNA binding. Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of target DNA. Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding. On the basis of these results, together with a secondary structure prediction, the presence in Fur of a non-classical helix-turn-helix motif is proposed. Experimental results show that activation upon metal binding induces conformational modification of this specific motif. The recognition helix, interacting directly with the major groove of the DNA, would include the domain [Y55-F61]. This helix would be followed by a small "wing" formed between two beta strands, containing Lys76, which might interact directly with DNA. These results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentration regulators, and the structure of their DNA-binding domain.     

Comparison of the structures of operator DNA free and in complex with lambda repressor

The structures of operator DNA unbound and in complex with lambda repressor protein are compared. The conformation of the left 10 base pairs of a lambda right regulatory operator DNA sequence has been previously determined in solution using nuclear magnetic resonance techniques and the structure of a homologous left regulatory operator DNA bound to lambda repressor N-terminal domain had been previously solved using X-ray crystallography. The DNA adopts an overall linear B-form DNA both in the absence and presence of lambda repressor. Superimpositioning of the DNA structures reveals small differences between them that are due to the binding of protein and not to the different techniques used for their determination.     

Conformational changes in the bacterial SRP receptor FtsY upon binding of guanine nucleotides and SRP

In cotranslational preprotein targeting in Escherichia coli, the signal recognition particle (SRP) binds to the signal peptide emerging from the ribosome and, subsequently, interacts with the signal recognition particle receptor, FtsY, at the plasma membrane. Both FtsY and the protein moiety of the signal recognition particle, Ffh, are GTPases, and GTP is required for the formation of the SRP-FtsY complex. We have studied the binding of GTP/GDP to FtsY as well as the SRP-FtsY complex formation by monitoring the fluorescence of tryptophan 343 in the I box of mutant FtsY. Thermodynamic and kinetic parameters of the FtsY complexes with GDP, GTP, and signal recognition particle are reported. Upon SRP-FtsY complex formation in the presence of GTP, the fluorescence of tryptophan 343 increased by 50 % and was blue-shifted by 10 nm. We conclude that GTP-dependent SRP-FtsY complex formation leads to an extensive conformational change in the I box insertion in the effector region of FtsY.     

The absence of non-local conformational changes in OR3 operator DNA on complexing with the cro repressor

The Interaction of the cro protein of lambda phage with a synthetic OR3 operator having 17 base pairs in length and with its 9 bp fragment has been studied using the circular dichroism (CD) method. In both cases, a considerable change in the CD of the samples was found in the region 260-300 nm upon the addition of the cro protein. The stoichiometry obtained by the CD titration was identical for OR3 and its 9 bp fragment: one duplex per dimeric cro. NaCl addition makes the complexes dissociate so that the 9 bp fragment becomes free at [NaCl] greater than 0.2 M while the whole OR3 becomes free at [NaCl] greater than 0.5 M. The CD spectra of both the free duplexes show a typical B-form conservative pattern with a positive CD band (270 nm) and a negative one (250 nm). The specific complexing of both the duplexes results in a substantial CD depression in the positive band. The most pronounced effect occurs at 280 nm. This spectral change is quite distinct from those in the B to A transition and in the non-cooperative winding of the DNA within the B-family of forms. The interaction of the cro protein with the non-operator DNAs, calf thymus DNA and a synthetic 10 bp duplex, reveals no visible CD changes at all. An inference is drawn that the CD change in the specific complexes is mainly due to the induced CD in tyr-26 upon its interaction with a specific base pair in the operator or its fragment, the operator DNA conformation being conserved in a B-like form as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the complex between lac repressor headpiece and operator DNA from measurements of the orientation relaxation and the electric dichroism.

The complex between lac repressor headpiece and short rodlike DNA fragments containing the lac operator sequence is characterised by measurements of the rotation diffusion. Using the method of electric dichroism we measure the rotation relaxation and determine changes in the length of the DNA upon ligand binding with high accuracy. According to these measurements any change in the length of the operator DNA upon binding of the first two headpiece molecules remains below 1A; the electric dichroism also remains virtually unchanged. At high degrees of (unspecific) binding we observe an increase in the rotation relaxation time, which is attributed to an increase of the apparent mean radius of the complex. As a control of our procedure for the determination of length changes we use the intercalation of ethidium bromide and arrive at an increase of the DNA length per bound ethidium of 3.2A (at 3.4A rise per base pair). The results obtained for the headpiece operator complex are not consistent with models assuming large changes of the DNA structure or intercalation of tyrosine residues.     

Structural organization and mechanisms of mobility of the gene cassette coding for resistance to antibiotics and bacterial virulence factors

The review is focussed on two types of gene cassettes which are significant in bacterial variability. The first type are cassettes with antibiotic resistance genes; these are the smallest mobile genetic elements including a gene (most commonly an antibiotic resistance gene) and a short sequence acting as a recombination site. Sometimes these cassettes contain genes not responsible for antibiotic resistance but their functions are not yet known. The second type contains large clusters of genes coding for bacterial virulence factors. They were termed "pathogenicity islands" due to their difference in the percentage of G-C pairs in comparison with bacterial chromosomes, in which they are contained. The structural organization and mechanisms of mobility of various types of gene cassettes are discussed.     

Human discrimination and modeling of high-frequency complex tones shed light on the neural codes for pitch

Accurate pitch perception of harmonic complex tones is widely believed to rely on temporal fine structure information conveyed by the precise phase-locked responses of auditory-nerve fibers. However, accurate pitch perception remains possible even when spectrally resolved harmonics are presented at frequencies beyond the putative limits of neural phase locking, and it is unclear whether residual temporal information, or a coarser rate-place code, underlies this ability. We addressed this question by measuring human pitch discrimination at low and high frequencies for harmonic complex tones, presented either in isolation or in the presence of concurrent complex-tone maskers. We found that concurrent complex-tone maskers impaired performance at both low and high frequencies, although the impairment introduced by adding maskers at high frequencies relative to low frequencies differed between the tested masker types. We then combined simulated auditory-nerve responses to our stimuli with ideal-observer analysis to quantify the extent to which performance was limited by peripheral factors. We found that the worsening of both frequency discrimination and F0 discrimination at high frequencies could be well accounted for (in relative terms) by optimal decoding of all available information at the level of the auditory nerve. A Python package is provided to reproduce these results, and to simulate responses to acoustic stimuli from the three previously published models of the human auditory nerve used in our analyses.     

The complex regulation of ferredoxin/thioredoxin-related genes by light and the circadian clock

The biochemical properties of the ferredoxin/thioredoxin transduction pathway regulating the activity of key carbon-fixation enzymes through post-translational modifications are well characterized but little is known about the regulation of the different genes. In the present study, we investigated in Chlamydomonas reinhardtii the regulation of the expression of ferredoxin, thioredoxin m, ferredoxin-NADP reductase, phosphoribulokinase, as well as that of cytosolic thioredoxin h, the function of which is still largely unknown. The effects of light, the circadian clock and active cell division were investigated by northern blotting. The five genes were found to be regulated by light and the circadian clock but with different kinetics and amplitudes. This leads for the first time to the proposal that an extra-chloroplastic thioredoxin is possibly implicated in light and/or circadian-related processes. An interplay between several light-transduction pathways in controlling the expression of the genes is suggested by the expression studies and the theoretical analysis of the promoters. Received: 2 December 1998 / Accepted: 19 March 1999     

Understanding the changes in the circular dichroism of light harvesting complex II upon varying its pigment composition and organization

In this work we modeled the circular dichroism (CD) spectrum of LHCII, the main light harvesting antenna of photosystem II of higher plants. Excitonic calculations are performed for a monomeric subunit, taken from the crystal structure of trimeric LHCII from spinach [Liu, Z. F., Yan, H. C., Wang, K. B., Kuang, T. Y., Zhang, J. P., Gui, L. L., An, X. M., and Chang, W. R. (2004) Nature 428, 287-292]. All of the major features of the CD spectrum above 450 nm are satisfactorily reproduced, and possible orientations of the Chl and carotenoid transition dipole moments are identified. The obtained modeling parameters are used to simulate the CD spectra of two complexes with altered pigment composition: a mutant lacking Chls a 611-612 and a complex lacking the carotenoid neoxanthin. By removing the relevant pigment(s) from the structure, we are able to reproduce their spectra, which implies that the alteration does not disturb the overall structure. The CD spectrum of trimeric LHCII shows a reversed relative intensity of the two negative bands around 470 and 490 nm as compared to monomeric LHCII. The simulations reproduce this reversal, indicating that it is mainly due to interactions between chromophores in different monomeric subunits, and the trimerization does not induce observable changes in the monomeric structure. Our simulated spectrum resembles one of two different trimeric CD spectra reported in literature. We argue that the differences in the experimental trimeric CD spectra are caused by changes in the strength of the monomer-monomer interactions due to the differences in detergents used for the purification of the complexes.     

Model membranes to shed light on the biochemical and physical properties of ezrin/radixin/moesin

Ezrin, radixin and moesin (ERM) proteins are more and more recognized to play a key role in a large number of important physiological processes such as morphogenesis, cancer metastasis and virus infection. Recent reviews extensively discuss their biological functions 1, 2, 3 and 4. In this review, we will first remind the main features of this family of proteins, which are known as linkers and regulators of plasma membrane/cytoskeleton linkage. We will then briefly review their implication in pathological processes such as cancer and viral infection. In a second part, we will focus on biochemical and biophysical approaches to study ERM interaction with lipid membranes and conformational change in well-defined environments. In vitro studies using biomimetic lipid membranes, especially large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) and recombinant proteins help to understand the molecular mechanism of conformational activation of ERM proteins. These tools are aimed to decorticate the different steps of the interaction, to simplify the experiments performed in vivo in much more complex biological environments.     

Broad-complex functions in postembryonic development of the cockroach Blattella germanica shed new light on the evolution of insect metamorphosis

Background

Insect metamorphosis proceeds in two modes: hemimetaboly, gradual change along the life cycle; and holometaboly, abrupt change from larvae to adult mediated by a pupal stage. Both are regulated by 20-hydroxyecdysone (20E), which promotes molts, and juvenile hormone (JH), which represses adult morphogenesis. Expression of Broad-complex (BR-C) is induced by 20E and modulated by JH. In holometabolous species, like Drosophila melanogaster, BR-C expression is inhibited by JH in young larvae and enhanced in mature larvae, when JH declines and BR-C expression specifies the pupal stage.

Methods

Using Blattella germanica as a basal hemimetabolous model, we determined the patterns of expression of BR-C mRNAs using quantitative RT-PCR, and we studied the functions of BR-C factors using RNA interference approaches.

Results

We found that BR-C expression is enhanced by JH and correlates with JH hemolymph concentration. BR-C factors appear to be involved in cell division and wing pad growth, as well as wing vein patterning.

Conclusions

In B. germanica, expression of BR-C is enhanced by JH, and BR-C factors appear to promote wing growth to reach the right size, form and patterning, which contrast with the endocrine regulation and complex functions observed in holometabolous species.

General significance

Our results shed new light to the evolution from hemimetaboly to holometaboly regarding BR-C, whose regulation and functions were affected by two innovations: 1) a shift in JH action on BR-C expression during young stages, from stimulatory to inhibitory, and 2) an expansion of functions, from regulating wing development, to determining pupal morphogenesis.