Heterogeneity in winged helix-turn-helix and substrate DNA interactions: Insights from theory and experiments.

Specific interactions between transcription factors (TFs) and substrate DNA constitute the fundamental basis of gene expression. Unlike in TFs like basic helix-loop-helix or basic leucine zippers, prediction of substrate DNA is extremely challenging for helix-turn-helix (HTH). Experimental techniques like chromatin immunoprecipitation combined with massively parallel DNA sequencing remains a viable option. We characterize the molecular basis of heterogeneity in HTH-DNA interaction using in silico tools and thence validate them experimentally. Given the profound functional diversity in HTH, we focus primarily on winged-HTH (wHTH). We consider 180 wHTH TFs, whose experimental three-dimensional structures are available in DNA bound/unbound conformations. Starting with PDB-wide scanning and curation of data, we construct a phylogenetic tree, which distributes 180 wHTH sequences under multiple sub-groups. Structure-sequence alignment followed by detailed intra/intergroup analysis, covariation studies and extensive network theory analysis help us to gain deep insight into heterogeneous wHTH-substrate DNA interactions. A central aim of this study is to find a consensus to predict the substrate DNA sequence for wHTH, amidst heterogeneity. The strength of our exhaustive theoretical investigations including molecular docking are successfully tested through experimental characterization of wHTH TF from Sulfurimonas denitrificans.

Assessing combinatorial diversity of aureochrome bZIPs through genome-wide screening.

Aureochromes are unique blue light-responsive LOV (Light Oxygen Voltage) photoreceptors cum basic leucine zipper (bZIP) transcription factors (TFs), present exclusively in photosynthetic marine stramenopiles. Considering the availability of the complete genome sequence, this study focuses on aureochromes from Ectocarpus siliculosus. Aureochromes mediate light-regulated developmental responses in this brown photosynthetic algae. Both the LOV sensor and the bZIP effector shows overall sequence-structure conservation. The structurally similar LOV+bZIP modules of aureochrome homologs/paralogs prefer a dimeric state. Besides a heterogeneous linker connecting the sensor-effector and a flexible N-terminal region, the sequence composition of both domains is vital. Aureochromes execute diverse cellular responses in different photosynthetic stramenopiles - though their activities can vary even within a given algal species. Therefore, it is important to understand whether aureochromes select dimerization partners from the same family or interact with other bZIPs as well. To regulate multifarious biological activities, it is possible that aureochromes activate the global TF interaction network. Following homo/heterodimer modeling, we address the compatibility of dimerization partners by screening through heptad repeats. We evaluate the dimer interface area in terms of gain in solvation energy and the number of hydrogen bonds/salt bridge interactions. We further explore the relative stability of these structures from a graph-theoretic perspective through well-studied measures such as the energy of the graph, average participation coefficient, and betweenness centrality. Furthermore, we also conduct an information-theoretic analysis using hitherto understudied measures such as network information centrality and Kullback-Leibler divergence. We find that all our investigations into the relative stability of the dimers using diverse methods from bioinformatics, network science, and, information theory are in harmonious agreement. Coupling preferences of monomers in aureochromes can be further translated to design novel optogenetic tools useful for understanding human development and disease.

Designing synthetic transcription factors: A structural perspective.

In this chapter, we discuss different design strategies of synthetic proteins, especially synthetic transcription factors. Design and engineering of synthetic transcription factors is particularly relevant for the need-based manipulation of gene expression. With recent advances in structural biology techniques and with the emergence of other precision biochemical/physical tools, accurate knowledge on structure-function relations is increasingly becoming available. Besides discussing the underlying principles of design, we go through individual cases, especially those involving four major groups of transcription factors-basic leucine zippers, zinc fingers, helix-turn-helix and homeodomains. We further discuss how synthetic biology can come together with structural biology to alter the genetic blueprint of life.

Identification and functional characterization of two bamboo FD gene homologs having contrasting effects on shoot growth and flowering.

Bamboos, member of the family Poaceae, represent many interesting features with respect to their fast and extended vegetative growth, unusual, yet divergent flowering time across species, and impact of sudden, large scale flowering on forest ecology. However, not many studies have been conducted at the molecular level to characterize important genes that regulate vegetative and flowering habit in bamboo. In this study, two bamboo FD genes, BtFD1 and BtFD2, which are members of the florigen activation complex (FAC) have been identified by sequence and phylogenetic analyses. Sequence comparisons identified one important amino acid, which was located in the DNA-binding basic region and was altered between BtFD1 and BtFD2 (Ala146 of BtFD1 vs. Leu100 of BtFD2). Electrophoretic mobility shift assay revealed that this alteration had resulted into ten times higher binding efficiency of BtFD1 than BtFD2 to its target ACGT motif present at the promoter of the APETALA1 gene. Expression analyses in different tissues and seasons indicated the involvement of BtFD1 in flower and vegetative development, while BtFD2 was very lowly expressed throughout all the tissues and conditions studied. Finally, a tenfold increase of the AtAP1 transcript level by p35S::BtFD1 Arabidopsis plants compared to wild type confirms a positively regulatory role of BtFD1 towards flowering. However, constitutive expression of BtFD1 had led to dwarfisms and apparent reduction in the length of flowering stalk and numbers of flowers/plant, whereas no visible phenotype was observed for BtFD2 overexpression. This signifies that timely expression of BtFD1 may be critical to perform its programmed developmental role in planta.

Residue interaction dynamics in Vaucheria aureochrome1 light-oxygen-voltage: Bridging theory and experiments.

Allosteric communication is the basis of signaling and information transfer. Collective interactions between amino acid residues, which are spatially distributed in the three dimensional structure of a protein molecule, form the basis of allosteric network. While the construction of residue interaction graphs (RIG) is based on static crystal structures of proteins, it is important to extract information on protein dynamics to understand allostery. Therefore, quantitative analysis of RIG based on the framework of differential network (DN), is immensely helpful in identifying key amino acid residue interactions within such communication pathways. While the simultaneous availability of protein structures from two different states is essential for DN, there are additional challenges. Crystallographic artifacts like nonbiological dimeric arrangements within the crystal lattice automatically influence the construction and eventually the interpretation of RIG. Therefore, experimental validation of predictions from the analyses of RIG is naturally scarce in the literature. Herein, we study the photo sensor domain of the signaling photoreceptor transcription factor, aureochrome1, to understand light-driven signaling. We perform direct experiments to verify the predictions from RIG using the machinery of DN. However, the agreement leaves scope for improvement. We then discuss the notion of quaternary structure alignment to obtain a biologically meaningful dimer. Thence, we reconstruct the RIG and reanalyze the modified structure. Results of these reanalyses render far superior agreement with experiments. Therefore, this notion of addressing crystallographic biases provides a fresh yet general approach for reconciliation of theory and experiments. It is applicable beyond the present case to all signaling proteins in general.

Structural Basis of Design and Engineering for Advanced Plant Optogenetics.

In optogenetics, light-sensitive proteins are specifically expressed in target cells and light is used to precisely control the activity of these proteins at high spatiotemporal resolution. Optogenetics initially used naturally occurring photoreceptors to control neural circuits, but has expanded to include carefully designed and engineered photoreceptors. Several optogenetic constructs are based on plant photoreceptors, but their application to plant systems has been limited. Here, we present perspectives on the development of plant optogenetics, considering different levels of design complexity. We discuss how general principles of light-driven signal transduction can be coupled with approaches for engineering protein folding to develop novel optogenetic tools. Finally, we explore how the use of computation, networks, circular permutation, and directed evolution could enrich optogenetics.

Identification, characterization and gene expression analyses of important flowering genes related to photoperiodic pathway in bamboo.

BACKGROUND: Bamboo is an important member of the family Poaceae and has many inflorescence and flowering features rarely observed in other plant groups. It retains an unusual form of perennialism by having a long vegetative phase that can extend up to 120 years, followed by flowering and death of the plants. In contrast to a large number of studies conducted on the annual, reference plants Arabidopsis thaliana and rice, molecular studies to characterize flowering pathways in perennial bamboo are lacking. Since photoperiod plays a crucial role in flower induction in most plants, important genes involved in this pathway have been studied in the field grown Bambusa tulda, which flowers after 40-50 years. RESULTS: We identified several genes from B. tulda, including four related to the circadian clock [LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), ZEITLUPE (ZTL) and GIGANTEA (GI)], two circadian clock response integrators [CONSTANS A (COA), CONSTANS B (COB)] and four floral pathway integrators [FLOWERING LOCUS T1, 2, 3, 4 (FT1, 2, 3, 4)]. These genes were amplified from either gDNA and/or cDNA using degenerate as well as gene specific primers based on homologous sequences obtained from related monocot species. The sequence identity and phylogenetic comparisons revealed their close relationships to homologs identified in the temperate bamboo Phyllostachys edulis. While the four BtFT homologs were highly similar to each other, BtCOA possessed a full-length B-box domain that was truncated in BtCOB. Analysis of the spatial expression of these genes in selected flowering and non-flowering tissue stages indicated their possible involvement in flowering. The diurnal expression patterns of the clock genes were comparable to their homologs in rice, except for BtZTL. Among multiple BtCO and BtFT homologs, the diurnal pattern of only BtCOA and BtFT3, 4 were synchronized in the flower inductive tissue, but not in the non-flowering tissues. CONCLUSION: This study elucidates the photoperiodic regulation of bamboo homologs of important flowering genes. The finding also identifies copy number expansion and gene expression divergence of CO and FT in bamboo. Further studies are required to understand their functional role in bamboo flowering.

Mapping networks of light-dark transition in LOV photoreceptors.

MOTIVATION: In optogenetics, designing modules of long or short signaling state lifetime is necessary for control over precise cellular events. A critical parameter for designing artificial or synthetic photoreceptors is the signaling state lifetime of photosensor modules. Design and engineering of biologically relevant artificial photoreceptors is based on signaling mechanisms characteristic of naturally occurring photoreceptors. Therefore identifying residues important for light-dark transition is a definite first step towards rational design of synthetic photoreceptors. A thorough grasp of detailed mechanisms of photo induced signaling process would be immensely helpful in understanding the behaviour of organisms. RESULTS: Herein, we introduce the technique of differential networks. We identify key biological interactions, using light-oxygen-voltage domains of all organisms whose dark and light state crystal structures are simultaneously available. Even though structural differences between dark and light states are subtle (other than the covalent bond formation between flavin chromophore and active site Cysteine), our results successfully capture functionally relevant residues and are in complete agreement with experimental findings from literature. Additionally, using sequence-structure alignments, we predict functional significance of interactions found to be important from network perspective yet awaiting experimental validation. Our approach would not only help in minimizing extensive photo-cycle kinetics procedure but is also helpful in providing first-hand information on the fundamentals of photo-adaptation and rational design of synthetic photoreceptors in optogenetics. CONTACT: devrani.dbs@presiuniv.ac.in or soumen@jcbose.ac.in SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Characterization of [4Fe-4S] cluster vibrations and structure in nitrogenase Fe protein at three oxidation levels via combined NRVS, EXAFS, and DFT analyses.

Azotobacter vinelandii nitrogenase Fe protein (Av2) provides a rare opportunity to investigate a [4Fe-4S] cluster at three oxidation levels in the same protein environment. Here, we report the structural and vibrational changes of this cluster upon reduction using a combination of NRVS and EXAFS spectroscopies and DFT calculations. Key to this work is the synergy between these three techniques as each generates highly complementary information and their analytical methodologies are interdependent. Importantly, the spectroscopic samples contained no glassing agents. NRVS and DFT reveal a systematic 10-30 cm(-1) decrease in Fe-S stretching frequencies with each added electron. The "oxidized" [4Fe-4S](2+) state spectrum is consistent with and extends previous resonance Raman spectra. For the "reduced" [4Fe-4S](1+) state in Fe protein, and for any "all-ferrous" [4Fe-4S](0) cluster, these NRVS spectra are the first available vibrational data. NRVS simulations also allow estimation of the vibrational disorder for Fe-S and Fe-Fe distances, constraining the EXAFS analysis and allowing structural disorder to be estimated. For oxidized Av2, EXAFS and DFT indicate nearly equal Fe-Fe distances, while addition of one electron decreases the cluster symmetry. However, addition of the second electron to form the all-ferrous state induces significant structural change. EXAFS data recorded to k = 21 Å(-1) indicates a 1:1 ratio of Fe-Fe interactions at 2.56 Å and 2.75 Å, a result consistent with DFT. Broken symmetry (BS) DFT rationalizes the interplay between redox state and the Fe-S and Fe-Fe distances as predominantly spin-dependent behavior inherent to the [4Fe-4S] cluster and perturbed by the Av2 protein environment.

Observation of the Fe-CN and Fe-CO vibrations in the active site of [NiFe] hydrogenase by nuclear resonance vibrational spectroscopy.

Nuclear inelastic scattering of (57)Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes.

EXAFS and NRVS reveal a conformational distortion of the FeMo-cofactor in the MoFe nitrogenase propargyl alcohol complex.

We have used EXAFS and NRVS spectroscopies to examine the structural changes in the FeMo-cofactor active site of the α-70(Ala) variant of Azotobacter vinelandii nitrogenase on binding and reduction of propargyl alcohol (PA). The Mo K-edge near-edge and EXAFS spectra are very similar in the presence and absence of PA, suggesting PA does not bind at Mo. By contrast, Fe EXAFS spectra show a clear and reproducible change in the long Fe-Fe interaction at ~3.7 Å on PA binding with the apparent appearance of a new Fe-Fe interaction at 3.99 Å. An analogous change in the long Mo-Fe 5.1 Å interaction is not seen. The NRVS spectra exclude the possibility of large-scale structural change of the FeMo-cofactor involving breaking the µ(2) Fe-S-Fe bonds of the Fe(6)S(9)X core. The simplest chemically consistent structural change is that the bound form of PA is coordinated at Fe atoms (Fe6 or Fe7) adjacent to the Mo terminus, with a concomitant movement of the Fe away from the central atom X and along the Fe-X bond by about 0.35 Å. This study comprises the first experimental evidence of the conformational changes of the FeMo-cofactor active site on binding a substrate or product.

Crystal structures of Aureochrome1 LOV suggest new design strategies for optogenetics.

Aureochrome1, a signaling photoreceptor from a eukaryotic photosynthetic stramenopile, confers blue-light-regulated DNA binding on the organism. Its topology, in which a C-terminal LOV sensor domain is linked to an N-terminal DNA-binding bZIP effector domain, contrasts with the reverse sensor-effector topology in most other known LOV-photoreceptors. How, then, is signal transmitted in Aureochrome1? The dark- and light-state crystal structures of Aureochrome1 LOV domain (AuLOV) show that its helical N- and C-terminal flanking regions are packed against the external surface of the core ß sheet, opposite to the FMN chromophore on the internal surface. Light-induced conformational changes occur in the quaternary structure of the AuLOV dimer and in Phe298 of the Hß strand in the core. The properties of AuLOV extend the applicability of LOV domains as versatile design modules that permit fusion to effector domains via either the N- or C-termini to confer blue-light sensitivity.

Dynamics of the [4Fe-4S] cluster in Pyrococcus furiosus D14C ferredoxin via nuclear resonance vibrational and resonance Raman spectroscopies, force field simulations, and density functional theory calculations.

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ∼400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.

Photolysis of Hi-CO Nitrogenase - Observation of a Plethora of Distinct CO Species using Infrared Spectroscopy.

Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibited Azotobacter vinelandii nitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm-1 and 1679 cm-1 together with positive bands at 1711 cm-1, 2135 and 2123 cm-1. The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm-1 is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species 'Hi-1' and 'Lo-1' respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol-1. Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative 'Hi-2' bands at 1936 and 1858 cm-1 with a positive 'Lo-2' band at 1780 cm-1, while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative 'Hi-3' bands at 1938 and 1911 cm-1 and a positive feature 'Lo-3' band at 1921 cm-1. These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo-cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.

An oligopeptide transporter of Mycobacterium tuberculosis regulates cytokine release and apoptosis of infected macrophages.

BACKGROUND: The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA(MTB). METHODOLOGY/PRINCIPAL FINDINGS: OppA(MTB) was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA(MTB) confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1beta, IL-6 and TNF-alpha was also compromised after inactivation of oppD. CONCLUSIONS: Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.

Identification of protein-bound dinitrosyl iron complexes by nuclear resonance vibrational spectroscopy.

We have applied (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to identify protein-bound dinitrosyl iron complexes. Intense NRVS peaks due to vibrations of the N-Fe-N unit can be observed between 500 and 700 cm(-1) and are diagnostic indicators of the type of iron dinitrosyl species present. NRVS spectra for four iron dinitrosyl model compounds are presented and used as benchmarks for the identification of species formed in the reaction of Pyrococcus furiosus ferredoxin D14C with nitric oxide.

Cyclosporin A binding to Mycobacterium tuberculosis peptidyl-prolyl cis-trans isomerase A--investigation by CD, FTIR and fluorescence spectroscopy.

Circular dichroism and resolution-enhanced Fourier transform infrared reveal induction of secondary structural elements on peptidyl-prolyl cis-trans isomerase A (PpiA) from Mycobacterium tuberculosis upon binding cyclosporin A (CsA). Thermal denaturation shows aggregation of PpiA at higher temperatures (>70 degrees C) and CsA fails to impart stabilization in protein structure. However, CsA stabilizes PpiA structure in urea denaturation. In presence/absence of CsA, urea-induced reversible unfolding of secondary and tertiary structures follows two-state and three-state transition, respectively. The chemical unfolding results also demonstrate that loss in the tertiary structure precedes the loss in secondary structure both in presence and absence of CsA at the initial stages. Fluorescence quenching suggests presence of a positive barrier around tryptophan microenvironment of PpiA.

Crystal structure of low-molecular-weight protein tyrosine phosphatase from Mycobacterium tuberculosis at 1.9-A resolution.

The low-molecular-weight protein tyrosine phosphatase (LMWPTPase) belongs to a distinctive class of phosphotyrosine phosphatases widely distributed among prokaryotes and eukaryotes. We report here the crystal structure of LMWPTPase of microbial origin, the first of its kind from Mycobacterium tuberculosis. The structure was determined to be two crystal forms at 1.9- and 2.5-A resolutions. These structural forms are compared with those of the LMWPTPases of eukaryotes. Though the overall structure resembles that of the eukaryotic LMWPTPases, there are significant changes around the active site and the protein tyrosine phosphatase (PTP) loop. The variable loop forming the wall of the crevice leading to the active site is conformationally unchanged from that of mammalian LMWPTPase; however, differences are observed in the residues involved, suggesting that they have a role in influencing different substrate specificities. The single amino acid substitution (Leu12Thr [underlined below]) in the consensus sequence of the PTP loop, CTGNICRS, has a major role in the stabilization of the PTP loop, unlike what occurs in mammalian LMWPTPases. A chloride ion and a glycerol molecule were modeled in the active site where the chloride ion interacts in a manner similar to that of phosphate with the main chain nitrogens of the PTP loop. This structural study, in addition to identifying specific mycobacterial features, may also form the basis for exploring the mechanism of the substrate specificities of bacterial LMWPTPases.

Predicted molecular structure of the mammalian cell entry protein Mce1A of Mycobacterium tuberculosis.

The proposed role of the mammalian cell entry protein 1A (Mce1A) of Mycobacterium tuberculosis is to facilitate invasion of host cells. The structure of Mce1A was modelled on the basis of the crystal structure of Colicin N of Escherichia coli by fold prediction and threading. Mce1A, as the model predicts, is an alpha/beta protein consisting of two major (alpha and beta) domains, connected by a long alpha helix. The model further revealed that the protein contains 12 helices, 9 strands, and 1 turn. The final model of Mce1A was verified through the program VERIFY 3D and more than 90% of the residues were in the favourable region. A mouse monoclonal antibody, TB1-5 76C, is directed to an epitope within a 60-mer peptide that has been shown to promote uptake of bacteria in mammalian cells. We show here that the epitope could be narrowed down to a core of 4 amino acids, TPKD. Upstream flanking residues, KRR also contributed to binding. Mce2A does not promote uptake in mammalian cells and sequence comparison of Mce1A and Mce2A indicates that the epitope mediates uptake. The epitope was located at the surface of the Mce1A model at the distal beta strand-loop region in the beta domain. The localization of this epitope in the model confirms its potential role in promoting uptake of M. tuberculosis in host cells.