Despite this overall similarity, biotin-dependent carboxylases from the three domains of life carrying their reaction on different substrates adopt very diverse protein domain arrangements. This has made difficult the resolution of their evolutionary history up to now.Biotin-dependent carboxylases are a diverse family of carboxylating enzymes widespread in the three domains of life, and thus thought to be very ancient. This family includes enzymes that carboxylate acetyl-CoA, propionyl-CoA, methylcrotonyl-CoA, geranyl-CoA, acyl-CoA, pyruvate and urea. They share a common catalytic mechanism involving a biotin carboxylase domain, which fixes a COTaking advantage of the availability of a large amount of genomic data, we have carried out phylogenomic analyses to get new insights on the ancient evolution of the biotin-dependent carboxylases. This allowed us to infer the set of enzymes present in the last common ancestor of each domain of life and in the last common ancestor of all living organisms (the cenancestor). Our results suggest that the last common archaeal ancestor had two biotin-dependent carboxylases, whereas the last common bacterial ancestor had three. One of these biotin-dependent carboxylases ancestral to Bacteria most likely belonged to a large family, the CoA-bearing-substrate carboxylases, that we define here according to protein domain composition and phylogenetic analysis. Eukaryotes most likely acquired their biotin-dependent carboxylases through the mitochondrial and plastid endosymbioses as well as from other unknown bacterial donors. Finally, phylogenetic analyses support previous suggestions about the existence of an ancient bifunctional biotin-protein ligase bound to a regulatory transcription factor.2.The most parsimonious scenario for the early evolution of the biotin-dependent carboxylases, supported by the study of protein domain composition and phylogenomic analyses, entails that the cenancestor possessed two different carboxylases able to carry out the specific carboxylation of pyruvate and the non-specific carboxylation of several CoA-bearing substrates, respectively. These enzymes may have been able to participate in very diverse metabolic pathways in the cenancestor, such as in ancestral versions of fatty acid biosynthesis, anaplerosis, gluconeogenesis and the autotrophic fixation of CO They participate in many essential metabolic functions as diverse as the autotrophic fixation of CO2, the biosynthesis and degradation of fatty acids, the gluconeogenesis, the anaplerotic production of oxaloacetate or the degradation of some amino acids [Biotin-dependent carboxylases are a group of enzymes present in the three domains of life  able to catalyze the fixation of COno acids -10. Thesno acids . Members2 to the BCCP-bound biotin, and thus the intermediate formation of carboxybiotin. Finally, CT binds the carboxyl group from the carboxybiotin to the specific substrate of each carboxylase , a biotin carboxylase (BC) domain and a carboxyl transferase (CT) domain . BCCP ise Figure .Saccharomyces cerevisiae, a longer N-terminal domain interacting with biotin appears to be involved in the regulation of certain genes  is in charge of the post-transcriptionally attachment of the biotin to a specific conserved lysine residue of the BCCP ,13. BPL r et al. ).As previously mentioned, the BCCP domain is characteristic of all the biotin enzyme family, including the decarboxylases and transcarboxylases, whereas the BC domain is limited to the carboxylases. The CT domain gives its substrate specificity to each biotin-dependent carboxylase. Although all biotin-dependent carboxylases bear these three types of protein domains, their arrangement is unequal among different carboxylases and from one domain of life to another. This arrangement will be briefly summarized below Figure .Escherichia coli, which bears a model bacterial ACC, the three protein domains are encoded by four independent peptides: one BC subunit, one BCCP peptide and two distantly related CT subunits interacting together to ensure the carboxyl transfer function [2 fixation, in the synthesis of polyketides and in the degradation of fatty acids and branched-chain amino acids. In bacteria and eukaryotes, PCCs are made up of two polypeptides: a BC-BCCP α-subunit, and a CT-bearing β-subunit. Recent work has shown an additional domain  involved in subunit interactions and located between the BC and the BCCP motifs in the α-subunit [2 fixation of the archaeal order Sulfolobales [Acetyl-CoA carboxylases  catalyze the carboxylation of acetyl-CoA to malonyl-CoA in different metabolic processes, such as the first step of fatty acid synthesis or the autotrophic 3-hydroxyproprionate pathway. In function . It is n-subunit . Archaeaolobales ,29. This2 molecule to their respective substrates. MCC is involved in the breakdown of leucine and acyclic monoterpenes of the citronellol family, whereas GCC appears to participate only in the latter catabolism [The 3-methylcrotonyl-CoA carboxylase  and the geranyl-CoA carboxylase  synthesize 3-methylglutaconyl-CoA and γ-carboxygeranyl-CoA by adding one COtabolism . While Mtabolism . Both MCThe acyl-CoA carboxylases are a heterogeneous group of enzymes involved in the same pathways as ACC and PCC and also carrying out important functions in the synthesis of secondary metabolites in bacteria. Acyl-CoA carboxylases are basically made up of two types of subunits, the first bearing the BC and the BCCP domains and the second carrying the CT function . As prevRhizobium etli [Pyruvate carboxylases  catalyze the carboxylation of pyruvate to oxaloacetate, an important function for anaplerosis, gluconeogenesis and fatty acid synthesis. A significant feature concerning the protein domain composition of PYC is the homology of the BC and BCCP domains with those of the rest of biotin-dependent carboxylases, in contrast with the independent origin of the CT element. Most bacteria and eukaryotes bear a polypeptidic PYC carrying the BC domain in its N-terminal end, the CT in the central part and the BCCP domain in the C-terminus . Recent ium etli -38. The ium etli -42.2. While in Saccharomyces cerevisiae the allophanate hydrolase and the urea carboxylase are fused within the same polypeptide [Oleomonas sagaranensis and green algae these two functions are carried out by two independent enzymes homologous to the unique S. cerevisiae peptide [O. sagaranensis urea carboxylase is a polypeptide by itself containing a BC domain in its N-terminal end, a BCCP domain in its C-terminus and a predicted central CT domain non-homologous to the previously cited CTs.Urea carboxylases  fix a carboxyl group in urea to form allophanate, an intermediate product of a two-steps process of urea degradation. Allophanate is subsequently hydrolyzed by the allophanate hydrolase to ammonia and COypeptide , in the  peptide . The O. These examples illustrate the very complex modular architecture of the biotin-dependent carboxylases, which has been studied for long . This faAs mentioned above, the polypeptide composition can largely vary from one biotin-dependent carboxylase to another. Therefore, we have studied each functional protein domain separately. Unlike the biotin carboxylase (BC) domain, which is shared among all the biotin-dependent carboxylases, three non-homologous carboxyl transferase (CT) domains exist in these enzymes: one is common to the ACC, all the PCC, the MCC, the GCC and the acyl-CoA carboxylases and will be called here the CoA-substrate related carboxyl transferase (CCT); another is characteristic of the PYC and will be called the pyruvate carboxylase carboxyl transferase (PCT); finally, UCAs seem to use their own CT (UCT).The biotin carboxylase domain (BC) is the main distinctive feature shared among all the biotin-dependent carboxylases with respect to the biotin-dependent decarboxylases and transcarboxylases. A critical step in our study was the assignment of each one of the BC domain sequences to their specific function. As the BC domain is a very well conserved motif, it is difficult to distinguish the actual substrate specificity of one precise BC-encoding sequence only on the basis of primary sequence characteristics. Consequently, we constructed preliminary BC domain phylogenetic trees , Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Chlorobi, Deinococcus-Thermus group and one spirochete. In addition to these bacterial sequences, the eukaryotic BC sequences formed well defined functional groups closely related to their bacterial homologues. The only exception to this observation in our preliminary results were the very divergent eukaryotic polypeptidic ACCs, which formed an independent clade probably induced by a long branch attraction artifact due to the acceleration of evolutionary rates subsequent to the polypeptidic fusion of ACC. ACC, PYC, MCC and PCC are widely distributed throughout eukaryotes, while UCA and acyl-CoA carboxylases are limited to fungi and some green algae.In order to improve the resolution of the BC domain phylogeny, we selected representative sequences from the complete dataset to carry out maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses. The resulting trees were similar to each other, so only the ML tree was shown here , so archaeal sequences may be monophyletic, as it will be discussed later (see the PCT domain phylogeny section). Interestingly, the BC domain sequences of archaeal ACC-PCC sequences did not cluster among the acyl-CoA carboxylases as could have been expected, but branched among the archaeal PYC sequences. Several bacterial sequences from Aquificales and Epsilon-proteobacteria branched very close to the archaeal sequences, suggesting that they may have acquired these genes by horizontal gene transfer (HGT) from archaeal donors. One representative of these bacterial sequences (Aquifex aeolicus) has indeed been deeply studied and shown to adopt an archaeal PYC structure  and to actually carry out the PYC function [In all trees shown above, the archaeal sequences did not form a monophyletic group but were split in several clades within the paraphyletic group of acyl-CoA carboxylases. Nevertheless, these archaeal clades were close to each other, suggesting that they could actually be monophyletic but artificially divided into different clades because of a lack of phylogenetic signal or a phylogenetic reconstruction artifact. As there was little doubt about the position of the eukaryotic sequences with respect to the bacterial ones, we proceeded to the removal of all eukaryotic sequences in order to try to improve the resolution for the prokaryotic sequences, in particular the archaeal ones Figure . As in pfunction ,42.Since CCT is shared by enzymatic complexes catalyzing carboxylation of different substrates, the first stage of our analysis was to predict the function of each sequence. As for the BC domain analysis described above, a phylogenetic analysis  could be found in all the proteobacterial groups except the Epsilon-division, in Acidobacteria, Actinobacteria , Firmicutes, Bacteroidetes, Chloroflexi, some Chlorobi, the Deinococcus-Thermus group, Thermotogales and Fusobacteria. Despite a few differences between the BC-domain and the CCT-domain detection , with similar results between both techniques Figure . CCT seqP = 0.12), opening the possibility that the archaeal sequences may be monophyletic and that the topology observed could be affected by a phylogenetic reconstruction artifact.Finally, although CCT homologues could be detected in a wide diversity of archaeal genomes, we did not retrieve the monophyly of these archaeal sequences in the CCT phylogenies. The statistical support for the separation into several archaeal subgroups was weak, so we tested the possibility that they actually form a monophyletic assemblage by using an unbiased AU test to compare the tree shown in Figure + transport in some bacteria) is also known to use an α-subunit homologous to the PCT domain [Although the PCT domain is characteristic of PYC and is not shared by any other biotin-dependent carboxylase, the oxaloacetate decarboxylase  genomes. One of these genes was similar to the N-terminal region of the predicted UCT whereas the other was similar to the C-terminal region. These two genes were frequently found adjacent to each other in the concerned genomes, but none of them has been studied so far, and thus, functional data are not available.Fused Rhizobium etli, Staphylococcus aureus and Homo sapiens [Ruegeria pomeroyi [As mentioned above, extra protein domains involved in subunit interactions have been recently reported in PYC sequences of  sapiens -38 and ipomeroyi . In the pomeroyi , supportet al. [Consequently, we attempted to identify BT/PT-like domains within other biotin-dependent carboxylases. High sequence similarity to BT and PT could only be identified among PCC and PYC sequences, respectively, confirming previous results ,38. In aet al. , for whiet al. . InteresThe biotin carboxyl carrier protein (BCCP) domain is a short biotinylated peptide (~ 90 amino acids) often fused to the other constitutive domains of the biotin-dependent carboxylases. Its short size and participation in very different polypeptide architectures make difficult to reconstruct accurate phylogenies using the BCCP sequences. Consequently, instead of analyzing the short BCCP domain itself, we decided to study the phylogeny of the biotin protein ligase (BPL) responsible of its biotinylation.et al. [Clostridium acetobutylicum, Lactococcus lactis, Halobacterium sp., Pyrococcus abyssii, Pyrococcus furiosus), our BPL-BirA searches confirmed the presence of only one BPL homologue in each prokaryotic genome. The BPL protein was widespread in Archaea, Bacteria and Eucarya and its phylogeny was congruent with the accepted main taxonomic groups within each domain  opens the possibility that they may have evolved after the last common ancestor of all Bacteria. Therefore, the analysis of the content of biotin-dependent carboxylases in the other domains of life is necessary to infer whether these enzymes are ancestral or not.In most of our biotin-dependent carboxylase phylogenies, eukaryotic sequences branch within their bacterial homologues, what strongly supports HGTs events from bacteria to an ancestor of the eukaryotes as the most likely way for them to have acquired these enzymes. Some of these HGTs probably took place through endosymbiosis, as suggested by the cellular localization of eukaryotic PCCs and MCCs in mitochondria ,57 and tet al. proposed that the most likely way to explain the unique domain composition of eukaryotic ACC is a fusion event between the α- and β-subunits of XCC on both sides of a central linker [Targeting sequences seem to be absent from eukaryotic PYC and ACC. Both BC- and PCT-domain phylogenies strongly support the branching of eukaryotic PYC sequences among their bacterial homologues, what indicates a bacterial origin even though the vector of this HGT cannot be specified. Surprisingly, concerning the eukaryotic ACCs, the phylogenetic reconstructions are at odds with protein domain comparison. On the one hand, eukaryotic ACCs branch among bacterial ACC sequences in preliminary CCT and accurate BC phylogenies , as well as for distantly related taxa . HGTs may have also been at the origin of completely new functions. For instance, bacterial ODC has been recently proposed to use one decarboxylating subunit common to all decarboxylases and a CT domain from any other origin . ODC-CT Contrarily to the rest of the protein domains involved in biotin-dependent carboxylation, we did not analyzed the evolution of the BCCP domain because of the limited phylogenetic signal due to its small size (~ 90 amino acids) and low sequence conservation, which make the BCCP phylogenies very sensitive to reconstruction artifacts. In addition, this domain can be found in many different polypeptide architectures, including transcarboxylases and decarboxylases that are not part of our work. A phylogenetic analysis of BCCPs published some years ago  supporteWe analyzed the biotin protein ligase (BPL) protein domain instead of the BCCP domain itself in order to reconstruct a more reliable phylogeny that could provide indirect evidence of the BCCP content in the cenancestor. BPL is related to the lipoyl protein ligases  and is rOur results confirmed previous observations of single BPL or BirA genes in each species. Except for some likely HGTs events, our phylogenetic reconstructions of the BPL protein domain showed a phylogeny congruent with the main accepted taxonomic groups in the three domains of life, suggesting a dominant vertical inheritance from the common ancestor to all living organisms Figure . The mosTo summarize, our data support that ACC, XCC and PYC are ancestral to Bacteria. MCC, PCC, GCC and UCA emerged subsequently from XCC in the bacterial lineage. Eukaryotes most likely inherited their biotin-dependent carboxylases through different HGTs from bacteria: MCC and PCC are related to alphaproteobacterial homologues and therefore seem to have been acquired through the mitochondrial endosymbiosis, whereas plant bacterial-like ACC is related to cyanobacterial homologues and thus of probable plastid origin. The remaining eukaryotic enzymes  have likely been also transferred from bacteria but their donors are unknown. The last common archaeal ancestor likely used a promiscuous BC subunit with PCT and CCT subunits to ensure PYC and ACC/PCC functions, respectively. Finally, the cenancestor likely bore a BirA gene able to biotinylate BCCPs. We used these results to infer the set of biotin-dependent carboxylases present in the cenancestor. It is important to note here that the precise nature of the cenancestor remains debated, in particular whether it was a single organism or a community of more or less related different organisms experiencing a high frequency of HGT ,62. WhenSince eukaryotes obtained their biotin-dependent carboxylases from bacteria, we ignore them for the discussion concerning the cenancestor complement and we focus specifically on the respective ancestors of Archaea and Bacteria as intermediate steps between present-day species and the cenancestor. The components of biotin-dependent carboxylases have been duplicated, recombined and fused many times across evolution and, thus, many different evolutionary scenarios can be proposed. As it would be too long to discuss all of them, we will focus only on the one that we consider to be the most parsimonious . Thaquestion -70.http://www.genome.jp/kegg) to be used as seeds for further similarity searches. Since some biotin-dependent carboxylases were absent from this database, we completed with archaeal ACC/PCC [http://www.ncbi.nlm.nih.gov/Genbank). Similarity searches with BLASTp [For each domain of life for which sequence data was available, we retrieved one representative of each of the different biotin-dependent carboxylases and BPL/BirA enzymes from the KEGG database  and GOR4 [Sequences for each protein domain found by these searches in the three domains of life were aligned with Muscle 3.6  or MAFFTand GOR4 .Preliminary trees based on the complete sequence dataset for each enzyme were constructed by the approximately maximum likelihood approach with FastTree 2.1.3  in orderJL and DM designed research; JL carried out phylogenetic analyses, and JL and DM wrote the manuscript. All authors read and approved the final version.Schematic phylogenetic trees of biotin carboxylase (BC) sequences and CoA-substrate related carboxyl transferase (CCT) sequences. These preliminary FastTree reconstructions allowed the functional assignment of each sequence according to a phylogenetic framework and functional data from the available literature.Click here for fileBiotin-dependent carboxylase content in bacterial complete genome sequences. This is an Excel file showing the presence or absence of homologues of the different biotin-dependent carboxylases in a collection of complete genome sequences.Click here for fileAlternative evolutionary scenarios for the early diversification of the biotin-dependent carboxylases. Colored blocks represent functional protein domains. The first scenario postulates the duplication of an ancient biotin-dependent carboxylase and the insertion of a pyruvate carboxylase carboxyl transferase (PCT) within the BT/PT domain predated the cenancestor. This organism had a polypeptidic PYC and a two-subunit XCC that evolved through several duplications and splits in Archaea and Bacteria. The second scenario explores the possibility that the cenancestor bore a CCT divided in two subunits as in contemporary bacterial ACC.Click here for fileList of all complete genome sequences used in this work. This is a text file containing the complete list of species upon which we carried out sequence similarity searches to detect biotin-dependent carboxylase homologues.Click here for fileMasked multiple sequence alignment of biotin carboxylase (BC) domain sequences (NEXUS format). This alignment was used to reconstruct the tree shown in Figure Click here for fileMasked multiple sequence alignment of archaeal and bacterial biotin carboxylase (BC) domain sequences (NEXUS format). This alignment was used to reconstruct the tree shown in Figure Click here for fileMasked multiple sequence alignment of CoA-substrate related carboxyl tranferase (CCT) domain sequences (NEXUS format). This alignment was used to reconstruct the tree shown in Figure Click here for fileMasked multiple sequence alignment of pyruvate carboxylase carboxyl tranferase (PCT) domain sequences (NEXUS format). This alignment was used to reconstruct the tree shown in Figure Click here for fileMasked multiple sequence alignment of biotin carboxyl ligase (BPL) sequences (NEXUS format). This alignment was used to reconstruct the tree shown in Figure Click here for file


Bacterial cells continuously sense and respond to their environment using their inherent signalling and gene regulatory networks. Cells are equipped with parallel signalling pathways, which can specifically cope with individual input signals, while interconnectivities between pathways lead to an enhanced complexity of regulatory responses that enable sophisticated adaptation. In principle, any cell signalling pathway may be rewired to respond to non-cognate signals by exchanging and recombining their underlying cognate signalling components. In the present article, we review the engineering strategies and use of chimaeric regulatory proteins in cell signalling pathways, especially the TCS  system in bacteria, to achieve novel customized signalling or regulatory functions. We envisage that engineered chimaeric regulatory proteins can play an important role to aid both forward and reverse engineering of biological systems for many desired applications. Most bacteria live in a continuously changing environment. In order to survive, they have evolved many signalling pathways to sense various extra- and intra-cellular signals and then respond accordingly by controlling their cognate downstream gene regulatory networks to adapt to the changes. Proteins involved in cell signalling pathways are typically modular, comprising highly conserved domains and residues. Therefore researchers have engineered variations of these proteins to endow them with customized characteristics. For example, both directed evolution and structure-based rational design approaches have been applied to modify scaffold  and reguTCS  transduction is the most prevalent signalling paradigm that bacterial cells use to sense and respond to the outside world . TCS typIn more complex signalling, additional transmitter and receiver modules are found in phosphorelays, which may serve to integrate additional inputs into the signalling pathway . ImportaIn order to propagate signals, modules of a signalling pathway have to physically interact. Hence binding specificity between modules is a key requirement for signalling fidelity. The specificity between modules is determined by the physicochemical complementarity of their interacting surfaces. Indeed, synthetic rewiring of signalling pathways is challenging because of our limited understanding of how to engineer novel complementary protein or domain surfaces with the desired functionalities. Despite a wealth of available structural data of complementary interfaces between proteins and domains, our knowledge remains largely descriptive and only a few generalizable patterns of protein and domain interfaces exist . ComputaTrans-acting modules are predicted to require high physicochemical specificity between them in order to avoid unwanted cross-talk between different signalling pathways comprising conserved domains. In cis-acting modules, where the components are fixed with one another and in stoichiometric ratios, high specificity may not be required due to the very high local concentrations of the linked modules. However, the covalent link between signalling modules may allow for direct and thus stringent output control. Although this hypothesis has yet to be experimentally proved, it is worth noting that cis-acting modules are much more prevalent at the end (receiver-output) of signalling pathways [Sinorhizobium meliloti [Another important consideration when designing synthetic signalling pathways is the identification of which is the relevant step of the pathway to be rewired. pathways , where cmeliloti . Interesmeliloti .Although building protein signalling circuits as a means to rewiring cell signalling is far from routine, several encouraging examples have demonstrated the feasibility and promise of synthetic protein-based signalling circuits to program cellular behaviours. To date, various signalling and regulatory proteins have been modified by domain combination, swapping or evolution to render them responsive to novel input signals or able to act on non-cognate target substrates. In the following sections, we discuss the current advances and challenges in this area where different chimaeric regulatory proteins are engineered alongside the various stages of the cell signalling pathways.Since the HKs in TCS systems have a modular structure comprising two conserved domains (the sensor and transmitter domains), we can hypothesize that the HK sensor domain can be swapped between different signalling proteins so that the downstream network of a signalling pathway can respond to a non-native input signal.Escherichia coli by fusing the photoreceptor domain of the phytochrome Cph1 protein from cyanobacterium Synechocystis to the intracellular signal transduction domain of the E. coli EnvZ kinase [lacZ reporter under the RR OmpR controlled promoter can express β-galactosidase to turn the cells black when exposed to red light. Using this chimaeric light sensor kinase, the authors then followed up this work by subsequently creating a bacteria-based dark/light edge detection programme [In a pioneering proof-of-principle bacterial photographing system, a synthetic light-sensitive chimaeric sensor kinase (Cph1–EnvZ) was made in Z kinase , yieldinrogramme .E. coli EnvZ kinase can be rationally altered to phosphorylate the non-cognate RRs CpxR and RstA instead of the cognate OmpR in vitro. In another study, Xu et al. [Myxococcus xanthus to chemotactically respond to nitrate, which the wild-type strain cannot respond to, independently of cellular metabolism and physiology. Both of these examples indicate the possibility of rewiring the cell native TCS by rationally designing the signalling kinase to act on a non-cognate RR, thus initiating a reprogrammed signalling response.Alternatively, swapping or modifying the transmitter domains, the downstream RRs can be activated by an alternative input signal. As a proof-of-concept of this idea, Skerker et al.  showed tu et al.  created The output domain of a RR is at the end of TCS pathways and is typically a DNA-binding module needed to trigger transcription of downstream genes A. TherefIn principle, any two or more domains can be fused by introducing tethering linkers between them. This is readily done and of great value for instance in co-localization studies, by fusing GFP to a protein of interest. However, in signal transduction pathways, specificity between modules is essential for signalling fidelity and control stringencies. In the aforementioned Cph8 chimaera, signalling fidelity downstream of the EnvZ module was provided by the native EnvZ/OmpR intracellular signalling pathway. The functional rewiring relied on exchanging the extracellular sensor moiety in a way that it could still communicate signal perception to the HK domain, where the transmembrane domain must play a structural coupling role across the membrane. The authors constructed a small chimaeric library after defining domain boundaries based on sequence alignments and tested a number of constructs in which the Chp1 moiety length differed while the EnvZ transmembrane moiety was fixed. This was necessary to obtain a functional chimaera, indicating that precise design of the domain architecture underlies rewired signalling.Box 1•Study protein functioning and evolutionThe major challenge in rewiring signalling pathways is to design novel interactions capable of faithfully communicating between modules. The nature of the functional engineered constructs can reveal design principles, i.e. how module specificity is achieved and how it may have evolved. Rewiring the phosphotransfer reaction between transmitter and receiver modules in TCS systems  has esta•Inform protein engineering approachesAs the number of synthetically rewired signalling pathways increases, so will our understanding of the principles governing signal transduction pathways, facilitating future approaches. Although every signalling connection is different, the component parts are conserved and the underlying biochemistry is similar, enabling rational design that can be based on first principles.•Design proteins with bespoke functionsIn the present article, we have only discussed the strategies that are currently employed to generate interoperability between signalling modules, in particular those culminating in gene transcription. Such systems may be more accessible since the engineering approach still comprises an element of trial and error and transcriptional output can be readily selected for. However, the potential to recombine other domain functionalities in novel combinations may apply more generally, for example combining metabolic pathways that are hitherto not connected.•Perturbation tools that act rapidly, reversibly and are tunableRewiring signalling pathways offers new ways to perturb and study cells. By making new network connections that are not present in the native cell, we can investigate how the cell copes. Such perturbations are conceptually different from conventional gene deletions of transcription factors, where the information flow downstream of the deleted gene is disrupted. Rewired signalling pathways can allow for temporal and tunable  transcri•Allow regulon-wide transcription controlOur approach aims to rewire signalling that would allow the control of transcription of the extensive nitrogen regulon. Most synthetic biology approaches have focused on changing gene regulatory DNA sequences (mainly promoters) for transcription control. To do so for entire regulons would involve the introduction of many such regulatory elements and also require knowledge of all operons of the regulon. Rewired synthetic transcription factors may therefore represent a more accessible and comprehensive mean for regulon-wide transcription regulation.•Applicable for metabolic engineeringOne of the challenges in metabolic engineering is the complexity of feedback loops that operate in homoeostasis, impairing efforts to optimize the production of desired natural products. Rewired signalling can uncouple such feedback loops while retaining transcription control of genes involved in the pathways leading to the desired product, providing considerable scope for their application in biotechnology.bEBPs are modular, typically comprising a C-terminal DNA HTH (helix–turn–helix) binding domain, a central catalytic AAA+ (ATPase associated with various cellular activities) domain and an N-terminal regulatory domain  Figure A. Upon pin vivo  functional modules is an attractive possibility, in accordance with the conceptual approach in synthetic biology to rationally engineer systems through the assembly of predefined components. Such approaches are also attractive because the majority of proteins in Nature have evolved through the recombination of genetic elements that code for functional protein modules . However


A general paradigm to understand protein function is to look at properties of isolated well conserved domains, such as SH3 or PDZ domains. While common features of domain families are well understood, the role of subtle differences among members of these families is less clear. Here, molecular dynamics simulations indicate that the binding mechanism in PSD95-PDZ3 is critically regulated via interactions outside the canonical binding site, involving both the poorly conserved  Protein interactions play crucial roles in all biological processes. A common way of studying them is to focus on sub-parts of proteins, called domains, that mediate specific types of interactions. For instance, it is known that most PDZ domains mediate protein interactions by binding to the C-terminus of other proteins. Humans have more than 200 slightly different copies of these domains. At the level of the binding site, PDZ domains look quite similar. This is in apparent contradiction with their heterogeneous binding specificity. Using detailed molecular dynamics simulations in conjunction with statistical analysis, we predict that contacts outside of the canonical binding site play important roles in regulating protein interactions. Some of these contacts influence the overall dynamics of PDZ domains, providing an explanation for their allosteric effect. These interactions involve regions of the PDZ domains that are much less conserved, suggesting that they can help in differentiating selectivity in this large domain family. Molecular dynamics (MD) simulations indicate that residues upstream of the 4th C-terminal amino acid are crucial for binding. Specifically, lysines residues at position −4 and −7 in the CRIPT peptide are observed to dynamically interact with the Seminal X-ray crystallography experiments on the third PDZ domain of PSD95 in complex with the CRIPT C-terminal peptide indicated that peptide binding is realized through the last four residues (peptide positions 0 to −3), while the rest of the peptide is mostly disordered PDZ3 see  [1], [17These observations suggested that interactions beyond the canonical class-I motif are needed to achieve stable binding in native conditions , possibly with a major role of the Structural cluster analysis provides a quantitative classification of the non-canonical interactions  in the WT showed that the flexibility of the bound form is not affected by helix truncation . HoweverThese observations have important consequences for the interpretation of the entropic penalty upon binding to The important role of backbone dynamics is in contrast with recent NMR relaxation experiments which found a negligible contribution of the backbone compared to side chains flexibility Stabilization of the extra-domain helix is further mediated by a hydrophobic patch, formed by he  loop , it is fTo further investigate this hypothesis, we used PSIPRED The PSD95-PDZ3 To provide a dynamical picture of the process, we performed additional simulations of the DLG1-PDZ2:E6 complex see . Our calIn PDZ binding, the relatively limited information about peptide amino acids more distant from the C-terminus prevented a clear structural understanding of the effect and importance of these upstream side chains. Our work aims to fill this gap by providing calculations with both a canonical 5-mer CRIPT peptide as well as a longer 9-mer peptide in complex with PSD95-PDZ3. Three main results emerge from our work.First, we observe in our simulations that peptide binding is mediated by ionic interactions with the loop following the binding site, referred to here as the Second, we propose a mechanistic explanation for the microscopic origin of the binding entropic penalty in absence of the extra-domain helix of PSD95-PDZ3. In the apo form, the helix plays a crucial role in stabilizing both the carboxylate binding loop and the Third, analysis of 258 human PDZ domains as well as MD simulations of single-mutants allowed for the identification of an amino acid at the beginning of the Molecular dynamics simulations were performed using the GROMACS implementation is 2I0L  . The firfour-digits code. The first three digits describe the peptide-loop interactions, e.g. “110”. The last digit represents an id, encoding the peptide structural conformation . The latter was obtained by running a leader-based cluster-analysis on the ligand backbone  Distance between the 5-mer peptide side chain nitrogen of (TIF)Click here for additional data file.Figure S2CRIPT peptide affinity scheme for PSD95-PDZ3. (Top) Schematic representation of the domain-peptide complex. Experimental binding affinities from Ref. (TIF)Click here for additional data file.Figure S3Amino acid frequencies at position 328 in human PDZ domains. Contribution of paralog domains have been weighted as described in (TIF)Click here for additional data file.Figure S4Conservation of the (TIF)Click here for additional data file.Table S1Details of the simulations performed.(PDF)Click here for additional data file.Table S2Examples of PDZ structures in complex with ligands with unresolved residues between position −4 and −7.(XLSX)Click here for additional data file.Table S3Manually curated multiple sequence alignment of the 258 human PDZ domains used in this work.(XLSX)Click here for additional data file.Table S4Clusters of human PDZ paralogs.(XLSX)Click here for additional data file.


Bacillus anthracis, and she received no first-class letters from facilities known to have processed intentionally contaminated letters. Environmental sampling in the regional Connecticut postal facility yielded B. anthracis spores from 4 (31%) of 13 sorting machines. One extensively contaminated machine primarily processes bulk mail. A second machine that does final sorting of bulk mail for her zip code yielded B. anthracis on the column of bins for her carrier route. The evidence suggests she was exposed through a cross-contaminated bulk mail letter. Such cross-contamination of letters and postal facilities has implications for managing the response to future B. anthracis–contaminated mailings.On November 20, 2001, inhalational anthrax was confirmed in an elderly woman from rural Connecticut. To determine her exposure source, we conducted an extensive epidemiologic, environmental, and laboratory investigation. Molecular subtyping showed that her isolate was indistinguishable from isolates associated with intentionally contaminated letters. No samples from her home or community yielded  Bacilus anthracis infection reported to the CTDPH since 1968 and the eleventh inhalational anthrax case in the United States since October 4, 2001   on employee absenteeism . For first-class letters, canceling machines apply both an identification code (orange bar code on the back of the envelope) and a postnet code (black bar code on the front of the envelope) to envelopes. Bulk letters are not processed on canceling machines because they are presorted with the postnet code preapplied. When the identification code is used, canceling machines record the day of the month, the time of day, and the sequence in which each first-class letter was processed during a given half-hour interval. Sorting machines then use the postnet code to group both first-class and bulk letters that go to a particular address. Sorting machines record the total count of all letters processed on each sorting machine, but only first-class letters with identification codes include time of day and identity of the particular sorting machine. Consequently, identifying information on bulk letters is not recorded in the mail tracing system. All letters are processed on sorting machines approximately two to five times, progressing from an initial sort, to the five-digit zip code, through a specific final sort in which letters are sorted to collecting bins in the delivery sequence for the given postal carrier route . We chose October 9 as a starting date because the only recovered intentionally contaminated letters that resulted in cases of inhalational anthrax were postmarked on this date and because 36 days had already elapsed between this date and the onset of the patient’s symptoms. Information on the date, time, and machines involved in processing these first-class letters was retrieved.We searched the patient’s home for letters she received since September 1, 2001, and recovered letters from her office, personal files, and trash bins. The recovered letters were categorized as first-class or bulk and submitted to the CTDPH laboratory for culture. First-class letters were checked against the USPS database for date and location of cancelation. To determine whether the patient received first-class letters from any PDCs that processed an intentionally contaminated letter , we examined USPS data of outgoing mail from these facilities for first-class letters with Connecticut destination addresses. Data were reviewed from October 9 until either the date the particular PDC closed because of To identify all first-class letters the patient received, regardless of point of origin, we examined USPS data for first-class letters sorted in the Southern Connecticut PDC that served her local post office from October 9 to November 13. We retrieved available information on the date, time, and identity of a letter’s originating PDC; however, information on the machines that processed a particular letter in the originating PDC is not available. To check for bulk letters sent to the patient’s zip code from October 9 to October 16, 2001, we contacted mailing companies that used the Trenton PDC. To check for bulk letters sent to the patient’s zip code during October 9 to October 16, 2001, we contacted mailing companies that used the Trenton PDC.B. anthracis, we collected additional moist surface wipe samples from each column of collecting bins on the three letter-sorting machines with samples yielding B. anthracis and from the sorting machine that completed the final sort of letters that included the patient’s mail carrier route. We also collected nasal swabs for culture from employees of the Southern Connecticut PDC during November 21 to 24.Environmental samples were collected at the Southern Connecticut PDC on five occasions. On November 11, 2001, 2 days before the patient’s symptoms began, an independent contractor took surface samples from various locations with dry synthetic swabs as part of a nationwide USPS effort to identify contamination of selected PDCs. On November 21, a second independent contractor hired by USPS collected additional dry swab samples of surfaces, including 29 letter-canceling and -sorting machines, 4 flat- (magazine) sorting, and 4 parcel-sorting machines; air-handling units; and vacuum cleaner filters from different facilities. On November 25, we inspected and repeated sampling of similar locations using moist synthetic swabs. On November 28, guided by findings from epidemiologic investigations at the Brentwood PDC, we collected vacuum and moist synthetic 2x2-inch surface wipe samples from all letter-canceling and -sorting machines , goat hair, or leather. A family member assisted her in paying bills, reading unfamiliar letters, and handling bank transactions. In the 60 days preceding symptom onset, her only visitors besides family and close friends were her church pastor, approximately 25 Halloween trick-or-treaters, and two persons from her cleaning service. No unusual visitors or solicitors were noted by friends or neighbors.She did not drive; family and friends provided transportation. She was always accompanied when outside the home and limited her time away from home to <2 hours to prevent fatigue. Her activities outside her home consisted of weekly hair salon appointments, routine physician visits, lunch outings with friends, weekly church attendance, visiting another church for a Christmas fair, and voting at the town hall. No unusual persons or occurrences were noted by those who accompanied her during these outings.B. anthracis). They had no brand of medication, physicians, hobbies, social networks, or geographic area in common.When she was compared with the 10th case-patient with inhalational anthrax in New York City, limited similarities were found: both were women >60 years of age, lived alone in clean homes, wore hats, and had a bottle of the same brand of perfume  also cultured negative for B. anthracis. Cultures of the 17 samples from the selected outdoor locations and 16 nasal swabs from visitors to her home also were negative for B. anthracis.Cultures of all 258 samples and 84 personal effects from the patient’s home were negative for B. anthracis.We recovered 29 letters from her home postmarked after September 1, 2001: 7 canceled first-class letters and 23 presorted bulk letters. These 29 letters likely did not represent the entire number of letters she had received since September 1. Of the six first-class letters, only one was postmarked after October 9 (October 26). All first-class letters were sliced open along the top border of the envelope, whereas bulk letters, mainly solicitations or credit-card offers recovered from her garbage, had been torn in half. Samples from all 29 recovered letters were negative for B. anthracis contamination). Five first-class letters from the Trenton PDC and three first-class letters from the Brentwood PDC were sent to her postal carrier route during this time. Two of the letters from the Trenton PDC were processed approximately 3 hours after the Daschle and Leahy letters and on the same canceling machine. None of these letters were delivered to an address on her street, none coincided with a first-class letter to the patient, and none were recovered.The Morgan and West Palm Beach PDCs did not save records of outgoing mail for the period of interest. The Trenton and Brentwood PDCs did not send first-class letters directly to her address during October 9 to October 21  that was sorted in the Trenton PDC 283 letters (approximately 15 s) after the intentionally contaminated letter to Senator Leahy. Letter A was processed in the Southern Connecticut PDC and the patient’s local post office and delivered to an address approximately 4 miles away on a different mail carrier route. Three separate moist swab samples taken from the outside of letter A’s envelope yielded 1, 3, and 7 CFU of Overall, the Trenton PDC processed approximately 5 million letters between October 9–October 16, and the Brentwood PDC processed 13 million letters between October 9 and October 21, when they were closed. Of these, approximately 1.1 million letters were processed at each facility within 24 hours after the letters to Senators Daschle and Leahy passed through, half of which were bulk letters .Of 33 bulk mailing companies that sent letters through the Trenton PDC, 19 sent letters to Connecticut; 17 of these companies had no records of bulk letters sent to the address of the patient or her close contacts.The Southern Connecticut PDC is approximately 350,000 square feet. It has 11 machines to cancel and code letters originating there and 18 machines to sort letters originating in and arriving at the Southern Connecticut PDC. Each letter-sorting machine has 48–52 columns of permanent collecting bins arranged in columns four bins high . Flats aB. anthracis. The strain was indistinguishable from that of the bioterrorism-related isolates (B. anthracis CFU in 0.53 g of paper dust collected. Wipe samples of 30 (58%) of 52 columns of bins from this machine yielded B. anthracis. Of 65 swab, wipe, or vacuum samples collected from the sorting machine (no. 6) that completed the final sort of letters that included the patient’s mail carrier route, only the wipe sample taken from the column of bins that held sorted letters for the patient’s postal carrier route were positive for B. anthracis (B. anthracis.Forty-one (7%) of 590 samples from the Southern Connecticut PDC yielded nthracis . CultureThis report describes the epidemiologic and environmental investigation of a case of inhalational anthrax in a 94-year-old woman from rural Connecticut. Molecular subtyping and antibiotic susceptibility testing of the isolate from blood culture demonstrated that it was indistinguishable from the other bioterrorism-related isolates, establishing a link to the cases that were caused by the mailing of intentionally contaminated letters  was actively shedding or transferring spores at any point in Connecticut. Thus, cross-contaminated letters can have markedly different levels of cross-contamination and potential to shed spores. An individual assessment is necessary to determine the magnitude of risk.The possibility of cross-contaminated letters as a cause of anthrax illness has been postulated (If we assume that the case in Connecticut, and possibly the unexplained case of inhalational anthrax in New York City (B. anthracis spores is recognized, persons could be advised to open letters in a well-ventilated area, avoid tearing envelopes, discard envelopes after opening, and wash hands after handling envelopes (If low-dose exposure occurred through the mail, numerous persons might have had such exposures. Why illness developed in the Connecticut patient and not in others is unknown, but the reasons might include her habit of tearing mail in half before disposal, her advanced age, her history of obstructive lung disease, and her use of inhaled bronchodilators (The finding of the approximate equivalent of 3 million spores from a vacuum sample on one sorting machine underscores the importance of maintaining the revised postal facility cleaning procedures geared to minimize aerosolization of dust (B. anthracis spores in these locations. Nonetheless, locations that processed or received intentionally contaminated letters had widespread contamination (A number of key limitations exist to this investigation, however. First, we do not know the threshold for detecting spores in the environment. The absence of positive cultures in the home and other places sampled in her town does not exclude the presence of


Bordetella pertussis is controlled by the two-component system BvgAS. Atypically, the sensor-kinase BvgS is active without specific stimuli at 37°C in laboratory conditions and is inactivated by the addition of negative chemical modulators. The structure of BvgS is complex, with two tandem periplasmic Venus flytrap domains and a cytoplasmic PAS domain that precedes the kinase domain, which is followed by additional phosphotransfer domains. PAS domains are small, ubiquitous sensing or regulatory domains. The function of the PAS domain in BvgS remains unknown.In bacteria, signal-transduction two-component systems are major players for adaptation to environmental stimuli. The perception of a chemical or physical signal by a sensor-kinase triggers its autophosphorylation. The phosphoryl group is then transferred to the cognate response regulator, which mediates the appropriate adaptive response. Virulence of the whooping cough agent We showed that recombinant BvgS PAS proteins form dimers that are stabilized by α helical regions flanking the PAS core. A structural model of the PAS domain dimer was built and probed by site-directed mutagenesis and by biochemical and functional analyses. Although we found no ligands for the PAS domain cavity, its integrity is required for signaling. We also showed that the structural stability of the PAS core and its proper coupling to its flanking N- and C-terminal α helices are crucial for BvgS activity.We propose that a major function of the BvgS PAS domain is to maintain conformational signals arising from mechanical strain generated by the periplasmic domain. The tight structure of the PAS core and its connections with the upstream and downstream helices ensure signaling to the kinase domain, which determines BvgS activity. Many mild substitutions that map to the PAS domain keep BvgS active but make it unresponsive to negative modulators, supporting that modulation increases conformational strain in the protein. Two-component systems (TCS) are one of the predominant signal transduction systems in bacteria, which are often essential to enable microorganisms to adapt to changes of their environment . They reBordetella pertussis colonizes the upper respiratory tract of humans. Its virulence regulon is controlled by the TCS BvgAS. At 37°C and in laboratory growth conditions, the BvgAS system is activated, leading to the transcription of a number of genes coding for virulence factors, necessary for infection [- phase can be triggered by the addition of chemical modulators, such as nicotinate or sulfate ions. However, the authentic negative modulators that might act at specific stage(s) of the bacterium’s life cycle remain to be identified. BvgS is a hybrid sensor-kinase harboring several cytoplasmic domains that mediate a complex phospho-transfer cascade [The whooping cough agent  cascade . It also cascade . We have cascade . BvgS isBvg). PAS domains are structurally conserved, 100- to 120-residue-long signaling modules with sensory and regulatory functions, present in kinases, chemoreceptors and other types of proteins in all branches of the phylogenetic tree [in vitro and in vivo[2[In this work, we characterized the PAS domain of BvgS  and sequenced. The resulting plasmids encode recombinant PASBvg proteins with an N-terminal His tag in all cases.The sequence coding for the PAS core domain was amplified by PCR using the PAScore UP and PAScore LO oligonucleotides as primers  and the avirulent B. pertussis BPSMΔbvgS were described in [ΔbvgA harbour a chromosomal deletion of bvgA. It was constructed by allelic replacement using homologous recombination as follows. DNA fragments flanking the bvgA gene were amplified from the BPSM chromosome using the pairs of oligonucleotides BvgA-UP1 and BvgA-LO1, and BvgA-UP2 and BvgA-LO2, respectively. The amplicons were used as templates for an overlapping PCR, and the resulting amplicon was introduced as an XbaI-HindIII restriction fragment into pSS1129 restricted with the same enzymes [The virulent BPSMribed in . BPSMΔbv enzymes . The res enzymes .To introduce the substitutions of interest into the recombinant protein, the N2C3 UP and N2C3 LO primers were used to amplify the relevant gene portion from the mutagenized plasmids described above. The amplicons were then introduced into pASK-IBA35+ in the same manner as for the wt gene fragment.BvgS core from the pQE and pGEV derivatives were performed in Escherichia coli SG13009(pREP4) (Qiagen) and BL21(DE3), respectively. pREP4 harbors a lacIQ gene for repression of the lac promoter prior to induction with IPTG.Productions of the PASE. coli BL21 (DE3). A number of inductions conditions were also tested, and the following one was identified as the most suitable. A 50-ml overnight culture in LB medium supplemented with 150 μg/ml ampicillin (LB-Amp100) was used to inoculate 1 liter of LB-Amp150 to an OD600 of 0.05. The culture was incubated at 37°C in a rotary shaker at 220 rpm. Expression of recombinant PASBvg was induced at an OD600 of 0.4 by the addition of 200 μg/L anhydrotetracycline (IBA). After 5 h of incubation under the same conditions, the cells were harvested by centrifugation at 8,000 × g for 20 min at 4°C. Hemin (Sigma) or 5-aminolevulinic acid (Sigma) were added at a concentration of 10 mM one hour before induction in the relevant cultures. For N2C3 production at 16°C, the cultures were grown at 37°C until they reached an OD600 of 0.4, then switched to 16°C 30 min before addition of the inducer. Induction was performed for 16 hours.A number of conditions were tested to optimize protein production, by varying the temperature of the cultures, the absorbance at 600 nm of the culture at the time of induction, the concentration of inducer and the duration of the induction. Production of the 9 recombinant proteins from the pIBA derivatives was performed in g. The supernatant was loaded onto a Ni2+-Sepharose affinity column (GE Life Sciences) pre-equilibrated with the binding buffer. Two washing steps were performed by using successively 10 mM and 50 mM of imidazole in the binding buffer, followed by an elution step with 200 mM imidazole. The protein was further purified by gel filtration in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl through a HiLoad 16/60 Superdex 75 column . All purification steps were carried out at 4°C or 12°C.In all cases, the cell pellets were resuspended in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM imidazole (binding buffer) with 5 μg/ml of DNase I (Sigma) and EDTA-free protease inhibitor cocktail (Roche). Cells were disrupted by three passages in a French pressure cell, and the bacterial debris was removed by centrifugation for 20 min at 10,000 × Mass spectrometry analyses were performed on an ESI-Q-TOF spectrometer  in positive ion mode by GIGA Proteomics at the University of Liège, Belgium. Purified N2C3 was used at a concentration of 10 μM in 27 mM ammonium acetate for native conditions, or in 31.25 mM ammonium acetate, 30% acetonitrile and 0.5% formic acid for denaturing conditions.The delipidation treatment of the purified protein was performed as described in . The proBvg proteins  were concentrated using a centrifugal filter unit . Crystallization screening was carried out using the sitting-drop, vapor-diffusion technique in 96-well microplates. Trays were set using a Phenix crystallization robot (Art Robbins instrument) and commercial crystallization kits . The drops were set up by mixing equal volumes (0.1 μl) of the protein and the precipitant solutions equilibrated against 75 μl of the precipitant solution. The protein concentrations ranged from 10 to 80 mg/ml for PASBvg N2C3 and N2C2 and from 10 to 30 mg/ml for PASBvg N3C2 and N3C3.For the crystallogenesis attempts, purified PASB. pertussis strains, the bacteria were grown in modified Stainer-Scholte medium (SS) [600 of 5 and broken using a Hybaid Ribolyser apparatus (30 s at speed 6 in tubes containing 0.1 mm silica spheres as the lysing matrix). The lysates were clarified by centrifugation , and the membrane fractions were pelleted from 1 ml of supernatants by ultracentrifugation . The pellets were resuspended in 100 μl PBS and used for denaturing electrophoresis in 4-8% gradient polyacrylamide gels . The proteins were then transferred electrophoretically to nitrocellulose membranes for immunoblotting. Polyclonal antibodies against BvgS were raised in rats  and used at a 1:500 dilution in PBS + 0.1% Tween 20. The secondary antibody was an anti-rat immunoglobulin- alkaline phosphatase conjugate (Promega) at a 1:7,500 dilution in the same buffer. Revelation of the blots was performed using the BCIP/NBT Color Development Substrate (Promega).To prepare the membrane fractions of the various ium (SS)  containiA similarity search using PSI-BLAST  was perfB. pertussis strains harboring specific mutations in bvgS and a ptx-lacZ fusion were grown in modified SS medium containing 100 μg/ml streptomycin and 10 μg/ml gentamycin. After 24 h at 37°C under rotating agitation as above, the bacterial suspension was used to initiate cultures in 10 ml of medium either not supplemented or containing the desired concentration of modulators. The inoculation volume was adapted to compensate for slower growth in the presence of high concentrations of nicotinate. The bacteria were grown until the cultures reached an OD600 of 1.5, harvested by centrifugation, resuspended in Z buffer  to an OD600 of 5 and broken as described above. ß-galactosidase activities were determined as described [ptx operon codes for pertussis toxin, a virulence factor whose expression is positively regulated by BvgAS. The ptx-lacZ transcriptional fusion interrupts the first gene of the operon and places lacZ under the control of the Bvg-regulated ptx promoter. Thus, the levels of β galactosidase activity measured after growth in virulent, Bvg+ conditions reflect the activity of BvgS, while those under modulating conditions reflect the ability of BvgS to respond to the negative modulators.The various escribed . The expAmong the hundreds of predicted VFT sensor-kinases many, including BvgS, harbor in their cytoplasmic moiety PAS, GAF, receiver or Hpt domains in addition to the His-kinase module . When prBvg as a recombinant protein in E. coli. The PAS core domain . Denaturation of the recombinant proteins using a thermal shift assay (TSA) [In order to study its function in BvgS and perform its biochemical characterization, we produced PASay (TSA)  indicateBvg produced in E. coli is dimeric, and the flanking helices predicted to form coils that precede and follow the PAS core appear to stabilize it. Most kinases of two–component systems work as dimers, and therefore the finding that the domain immediately preceding the kinase in BvgS also dimerises is not unexpected. In addition, PAS domains of other proteins frequently form dimers. It is thus likely that PASBvg dimerises in the context of the full-length protein as well.Thus, recombinant PASBvg. However, none of the four soluble recombinant proteins yielded diffracting crystals in spite of repeated attempts. We therefore searched for a homolog of known structure in the protein structure database, on the basis of which a 3-dimensional model of PASBvg could be built. The closest PAS domain of known structure, PASHm (pdb code: 3BWL), found in an Htr-like protein of Haloarcula marismortui has been crystallized in a structural genomic program. PASHm is dimeric and harbors a long α helix N terminal to the PAS core, like that predicted to link the transmembrane segment and PAS core domain in BvgS. Interestingly, the physico-chemical properties of these N-terminal flanking α helices are very similar between PASHm and PASBvg, with a number of charged residues in both cases. In the full-length protein of H. marismortui, the PASHm domain is followed by a predicted α helix and a histidine-kinase domain, like in BvgS. However, PASHm was crystallized without this C-terminal α helix. The features of PASHm – dimerisation and the presence of flanking helical extensions at both extremities are in agreement with the predictions and available data for PASBvg, indicating that the former represents a reasonable structural template for the latter.We next attempted to obtain the X-ray structure of recombinant PASBvg was thus built in silico  is located in the long α helix F, with its side chain pointing to the cavity in an appropriate position to interact with a putative heme co-factor . Furthermore, when production of PASBvg was performed with the addition of hemin or the heme precursor levulinate to the growth medium, no absorbance peak indicative of a heme protein was observed for the purified protein.PAS meliloti-31. In FBvg harbors a heme co-factor or a related molecule when present in the full-length BvgS protein in B. pertussis by replacing His643 with Ala. In bona fide heme-PAS domains, replacement of the His residue abolishes heme binding [B. pertussis is virulent in aerobic growth conditions, we reasoned that O2 would most likely be a positive signal for BvgS if the PAS domain harbored an O2-sensing heme, and therefore that a substitution abolishing heme binding should inactivate BvgS. The mutation was introduced into the chromosome of the B. pertussis Tohama I derivative BPSME705 by allelic exchange, and the activity of BvgAS was assessed by using a lacZ reporter under the control of the ptx promoter, which is positively controlled by BvgAS. The mutated strain expressed ß-galactosidase activity at a level similar to that of the strain containing wt BvgS . Using the thermal shift assay its Tm was determined to be 7°C lower than its wt counterpart .To probe for the presence of another potential co-factor or ligand in the PASBvg cavity between the inner surface of the β sheet and the helices of the PAS core. These residues were chosen on the basis of the structural model and of sequence alignments. PASBvg harbours a unique Cys residue (Cys607) in a short loop bordering the cavity. Cys residues have been implicated in co-factor binding in other types of PAS (e.g. LOV domains) [607 by an Ala residue in full-length BvgS did not modify its basal activity in B. pertussis . The activity of BvgSCys607Ala was modulated only at much higher modulator concentrations than those required for the wild type control, indicating that this variant has an intermediate rather than a non-responsive modulation phenotype.We then targeted other residues of the PASdomains) . In addidomains) , a functdomains) . The subs Figure . InteresBvg cavity whose replacement decreases both the denaturation temperature of the recombinant protein and the ability of BvgS to respond to nicotinic acid and related molecules that are perceived by the periplasmic domain.The corresponding recombinant protein was produced, purified and analyzed by TSA. Its Tm was 8°C lower than that of wt N2C3 Table . AltogetMycobacterium tuberculosis Rv1364c protein (pdb code 3K3C) shows an Arg residue in the cavity that is essential for the binding of a C16-fatty acid ligand [Bvg at a corresponding position (Arg670), and its side chain appears to be oriented in the same manner in the PASBvg model as that in PASRv1364c . Similarly, the Tm of the N2C3 protein harboring the Arg670Ala substitution was only moderately decreased compared to that of its wt counterpart . We thus verified that BvgSTyr596Ala+Asn631Ala was produced in a stable form in the recombinant B. pertussis strain by preparing membrane extracts and subjecting them to immuno-blotting using polyclonal anti-BvgS antibodies , and in the PAS596Ala + Asn631Ala substitutions on the thermal stability of the recombinant protein. Surprisingly, although N2C3Tyr596Ala+Asn631Ala was purified in a soluble and dimeric form in good amounts, no cooperative denaturation profile was obtained by TSA, and thus no Tm could be calculated. This suggested a significantly looser structure of the PAS core even at lower temperatures. The observations that the joint replacements of Tyr596 and Asn631 in the PASBvg cavity both abolished BvgS activity and considerably destabilized PASBvg argue that the structural stability of the PAS core domain is important for BvgS function. Of note, mutations in the PAS core have been shown to affect the stability and function of other PAS domains as well [We next determined the effect of the Tyr as well ,36.Hm structure, a highly conserved Asn residue corresponding to Asn608 in BvgS forms hydrogen bonds within the PAS core and docks the core to the flanking N-terminal α helix . The three variants were nevertheless detected in membrane extracts of the recombinant strains  also decreases BvgS sensitivity to nicotinate. These effects might be explained either by a moderate loosening of the PAS core because the small Ala side chain replaces a larger one, which disrupts the transmission of negative signals, or by a defect in binding a potential intracellular ligand required for transmission of negative signals. The double Tyr596Ala + Asn631Ala substitutions in the PAS cavity that abolish BvgS activity and strongly decrease the PAS thermal stability might also disable ligand binding in vivo. Binding of a cytoplamic ligand by the PAS domain would be consistent with the established link between the nutritional state of B. pertussis and its virulence [Bvg domain might sense intracellular molecule(s) whose abundance reflect(s) the metabolic state of the bacterium, and changes to the concentration of these components might affect signaling. Such a scenario would be compatible with the ‘rheostat’ behavior attributed to BvgS [Our results do not support the hypothesis that PASirulence  and withirulence ,50. ThusAlthough substantial information has been gathered about how the cytoplasmic domains of BvgS work, the function of its PAS domain has remained unknown. In this work, we performed its characterization, which represents new information that contributes to our understanding of VFT-containing sensor-kinases. We showed that the recombinant PAS domain of the sensor-kinase BvgS dimerises, and that the N- and C-terminal α-helical regions that flank the PAS core are critical for dimer stabilization. We identified specific amino acid residues in the PAS domain that are essential for BvgS activity, located in the PAS core and at the junctions between it and its flanking α helices. We thus propose a mechanical role for the PAS domain in BvgS, which is to maintain the conformational tension imposed by the periplasmic moiety of BvgS. The degree of tension in the protein determines the activity of the kinase, and modulation corresponds to an increased tension. Our model thus explains for the first time the phenotypes of a number of BvgS variants that harbor mild substitutions in the PAS domain and are unable to respond to negative modulation.TCS: Two-component system; PAS: Per/ARNT/Sim; VFT: Venus flytrap domain; TSA: Thermal shift assay; Tm: Melting (denaturation) temperatureThe authors declare no competing interests.ED, JH, FJ-D and RA designed the study; ED, JH, AW performed the experiments; ED, JH, AW, CL, FJ-D and RA wrote the paper. All authors have read and approved the final version of manuscript.Oligonucleotides used in this study.Click here for file


Multidomain proteins incorporating interaction domains are central to regulation of cellular processes. The elucidation of structural organization and mechanistic insights into many of these proteins, however, remain challenging due to their inherent flexibility. Here, we describe the organization and function of four interaction domains in PLCγ1 using a combination of structural biology and biochemical approaches. Intramolecular interactions within the regulatory region center on the cSH2 domain, the only domain that also interacts with the PLC-core. In the context of fibroblast growth-factor receptor signaling, the coordinated involvement of nSH2 and cSH2 domains mediates efficient phosphorylation of PLCγ1 resulting in the interruption of an autoinhibitory interface by direct competition and, independently, dissociation of PLCγ1 from the receptor. Further structural insights into the autoinhibitory surfaces provide a framework to interpret gain-of-function mutations in PLCγ isoforms linked to immune disorders and illustrate a distinct mechanism for regulation of PLC activity by common interaction domains. ► Domains from PLCγ regulatory region show structural and functional integration ► Only cSH2 domain interacts with the PLC-core forming a high affinity surface ► Activation involves removal of autoinhibition and dissociation from the receptor ► Disease-linked mutations map to the autoinhibitory interface Structural studies of multi-domain proteins provide a considerable challenge due to the presence of flexible regions not amenable to crystallography. Bunney et al. describe a strategy that generated new insights into the structural organization and integration of individual domains comprising PLCγ. Selective intermolecular interactions are crucial to cellular regulatory processes and are usually mediated by modular protein domains. There are around 100 families of interaction domains in humans, each of which can be found in many copies; these therefore represent a prevalent feature of the human proteome . ModulesTaking SH2 domains as an example, there is a notable difference in the number of structures reported for the isolated domains, free or bound to phosphotyrosine (pY)-containing polypeptide ligands, compared to larger structures of proteins encompassing or interacting with this domain . The impMost multidomain proteins have at least some degree of segmental mobility facilitated by the flexibility of the interdomain linkers . This al2) incorporate a number of different protein modules. PLCγ enzymes (PLCγ1 and PLCγ2), for example, contain eight domains, four of which are unique to this PLC family  enzymes that generate second messengers from phosphatidylinositol 4,5-bisphosphate  in the unstructured receptor C-terminal tail has been shown to be necessary and sufficient for the recruitment of PLCγ isoforms , the structure of this entire regulatory region has not been reported and γSA does not appear to be amenable to crystallographic approaches; this could be due to relative mobility of its modular components. To address this, we have used a combination of methods including NMR, SAXS and crystallography of regions within the γSA to obtain insights into the overall structural and dynamic characteristics of this regulatory region.The key regulatory elements for activation  and prev15N,1H-HQSC NMR spectroscopy. In addition, we solved the structure of the tandem nSH2-cSH2 pair by X-ray crystallography  obtained for 68 resolved cross peaks, is substantially larger than would be expected for the isolated domains. The corresponding derived isotropic rotational correlation time  is closer to that predicted from the X-ray crystal structure using hydrodynamic modeling  applied to residues 549–772 of the tandem than to the prediction for the isolated domains (7.4 and 8 ns)  C, right.The comparative analysis of the NMR spectra combined with backbone NMR assignments, also revealed surfaces of individual domains likely to be involved in weak interactions within the γSA. Comparison of the γSA with deletion variants lacking either the SH3 domain (γSAΔSH3) or both the SH3 and nSH2 domains (spPH-cSH2) revealed that chemical shifts of subsets of spPH and cSH2 cross peaks are perturbed relative to the isolated domains D. These In order to model γSA, SAXS data were recorded for the γSA polypeptide and a variety of related constructs . The radGiven the potential for dynamic disorder suggested by NMR , to asseTo further probe the domain organization of γSA we performed analysis of SAXS data obtained for γSA itself, γSAΔSH3 and constructs containing domain pairs nSH2-cSH2, cSH2-SH3 and spPH-cSH2. We applied MONSA , a progrGiven the limited flexibility of γSA suggested by the EOM analysis, we further generated a model of its domain structure in terms of the orientation and position of the different domains. We employed a rigid body docking protocol implemented in the program XPLOR-NIH that can incorporate a variety of experimental data types, including SAXS data and other structural information B. For thTaken together with the relatively narrow line widths in the NMR spectra for the 52 kDa γSA construct  and S2 tY771F,Y775F (545–790) following phosphorylation on Y783  construct in the same crystal form shows general similarity with some subtle differences in angle and distance between the domains (not shown).766, binding site for PLCγ1; D of 5 ± 1 nM, while the lower affinity (binding to cSH2) shows endothermic behavior and a KD of 81 ± 16 nM. Measurements performed with nSH2-cSH2Y771F,Y775F tandem phosphorylated to a high level at Y783 show a strikingly different binding isotherm. In this case, a dominant equimolar interaction attributed to interaction of FGFR1-1p with the nSH2 domain is maintained. However, the second binding phase is highly suppressed due to occupancy in cis of the canonical pY-binding site on the cSH2 by the pY783-linker region. Interestingly, in this latter instance the binding of FGFR1-1p to the nSH2 domain (KD 44 ± 11 nM) is nine-times weaker than in the case of the non-phosphorylated tandem construct.To test the functional implications of this structural integration of the nSH2 and cSH2 domains and changes that accompany binding of pY783 (and surrounding residues), we compared nonphospho and phospho forms of the tandem nSH2-cSH2 for their ability to interact with FGFR1. Isothermal titration calorimetry (ITC) was used to determine the relative affinity of tandem nSH2-cSH2 constructs for FGFR1-1p  completely reversed the broadening of cross peaks in the spectrum. Importantly, analysis of the spectrum at different titration points identified chemical shift changes for residues positioned on a region of the surface of the cSH2 domain that partly overlaps with the phosphopeptide-binding groove  or the mammalian cell line Freestyle 293F (Invitrogen) were used following manufacturer's instructions. Purification was performed using Ni2+ chelating chromatography with cleavable His-tags. An expanded set of procedures can be found in the Genes or their fragments to be expressed were cloned into pOPINS (Oxford Protein Production Facility), pTriEx4 or pTriEx6 (Novagen). Proteins were prepared as outlined in the All NMR experiments were carried out at 25°C on Varian INOVA or Bruker AVANCE spectrometers operating at 14.1 T or 16.5 T equipped with a cryoprobe. Details of data processing, semiquantitative analysis of cross-peak broadening and molecular modeling are given in the Synchrotron SAXS data were collected on the EMBL X33 camera with a Pilatus detector on the storage ring DORIS III  and ESRF ID14-3 camera with a Pilatus detector . Details of data analysis with DAMMIN and MONSA software packages are supplied in the 200 system . Experimental details are supplied in either figure legends or the ITC experiments were carried out with either a Microcal VP-ITC machine (as described in Pull-down assays with immobilized FGFR1 and various constructs of PLCγ1 were performed with Streptactin Macropore beads following manufacturer's instructions. Specific experimental conditions can be found in the Assays of PLC activity in COS7 and PAE cells were carried out as outlined in  with exp


Drosophila, the transport regulator Klar displays tissue-specific localization: In photoreceptors, it is abundant on the nuclear envelope; in early embryos, it is absent from nuclei, but instead present on lipid droplets. Differential targeting of Klar appears to be due to isoform variation. Droplet targeting, in particular, has been suggested to occur via a variant C-terminal region, the LD domain. Although the LD domain is necessary and sufficient for droplet targeting in cultured cells, lack of specific reagents had made it previously impossible to analyze its role in vivo.In klar with a lesion specifically in the LD domain; this lesion abolishes both droplet localization of Klar and the ability of Klar to regulate droplet motion. It does not disrupt Klar's function for nuclear migration in photoreceptors. Using a GFP-LD fusion, we show that the LD domain is not only necessary but also sufficient for droplet targeting in vivo; it mediates droplet targeting in embryos, in ovaries, and in a number of somatic tissues.Here we describe a new mutant allele of Drosophila.Our analysis demonstrates that droplet targeting of Klar occurs via a cis-acting sequence and generates a new tool for monitoring lipid droplets in living tissues of  Lipid droplets are the intracellular sites for storage of neutral lipids. Long dismissed as inert inclusions, they are now recognized as dynamic organelles with a myriad of functions well beyond fat storage ]. I]. I2]]. e.g., .Drosophila embryos, droplet motion is controlled by Klarsicht (Klar) [At least in part, specificity of droplet motion is achieved via distinct motor regulators that are present exclusively on lipid droplets. For example, in both mammals and flies, members of the Perilipin family modulate droplet motion ,13,20; tt (Klar) , a protet (Klar) . PresumaKlar's role is not limited to lipid droplets; in different cells, Klar controls distinct transport processes. In early embryos, it regulates the bidirectional motion of lipid droplets , which aklar locus encodes at least three protein isoforms that are due to the use of multiple promoters and Poly A sites [This tissue-specific targeting of Klar correlates with isoform variation . The kla A sites . The alp A sites ,24; the  A sites ; no func A sites .For Klar α, there is ample support for this model. Its C-terminal region contains a 60 aa KASH (Klarsicht/ANC-1/Syne Homology) domain. KASH domains typically localize to the outer nuclear envelope, and together with SUN domain proteins in the inner nuclear envelope establish a bridge linking cytoplasmic and nucleoplasmic proteins ,29. Indein vivo has not been clearly established. For one, the exact distribution of LD fusion proteins in cultured cells is distinct from the distribution of endogenous Klar in vivo: in embryos, Klar β is present in one or a few dots per lipid droplet, suggesting that it may be recruited to privileged sites [in vivo droplet localization requires C-terminal regions of Klar β [klar alleles either only disrupt Klar α or truncate both Klar α and Klar β within the shared N-terminal region. Therefore, they cannot be used to distinguish if droplet targeting is due to the Klar β-specific LD domain or due to regions shared between Klar α and Klar β.Available evidence is consistent with the notion that the unique C-terminal region of Klar β has an analogous role in targeting to lipid droplets. For example, in cultured cells, this so-called LD domain is sufficient to recruit RFP to lipid droplets . Howevered sites . In cultf Klar β . Howeverklar allele that deletes the last 64 aa of this domain. This allele abolishes droplet localization entirely. We also expressed a GFP-LD fusion protein and find that it targets to lipid droplets in early embryos, in the female germ line, and in somatic tissues. This analysis establishes that in vivo the LD domain is necessary and sufficient for targeting to lipid droplets.To examine the role of specifically the LD domain, we identified a new klar alleles with lesions in Klar β remove not only the LD domain (114 amino acids), but also at least 128 amino acids encoded by exons 14 and 15, exons shared with Klar α. Thus, these reagents do not allow a conclusive test if targeting to embryonic lipid droplets requires the LD domain (unique to Klar β) or is mediated by regions common to both Klar α and Klar β.The transcripts for Klar α and β diverge after exon 15, followed either by exons 16, 17 and 18 (Klar α) or exon 15ext (Klar β) . Exon 15klar alleles with lesions specifically in the LD domain. Since Klar β null alleles are viable [klar exon 15ext. We recovered two candidate lines  with predicted sequence changes in exon 15ext. We focused on the potential klar mutation in line Z3-3772 since it was predicted to result in deletion of roughly half of the LD domain.To address this issue, we searched for new e viable , we tooke viable . We emple viable -34 to fiMany lines from the mutant collection are mixtures of homozygotes and heterozygotes; the latter carry not only the mutagenized chromosome but also a balancer chromosome. In particular, for strain Z3-3772, the TILLING analysis had employed DNA from heterozygotes as in the extant stock homozygotes are rare to non-existent. When we extracted the non-balancer chromosome from this stock and sequenced exon 15ext, the predicted protein sequence of the corresponding LD domain was identical to the canonical sequence of Klar Figure .E12Df(3L)emc, a large deletion that removes - among many other genes - the entire klar locus. Genomic sequencing revealed the absence of a contiguous 25 bp stretch in the middle of exon 15ext.To reconcile this observation with the TILLING results, we hypothesized that the mutation in the parental line might exist on the balancer chromosome, TM6B. We generated flies heterozygous for the TM6B chromosome from line Z3-3772 and for MW and to the corresponding klar allele as MWklar. The TM6B chromosome without this deletion and the corresponding klar allele will be called TM6BZ and Zklar, respectively.This deletion is not a general feature of the TM6B balancer chromosome. Although many of the lines from the mutant collection were analyzed as heterozygotes, the deletion was recovered only once. In particular, our screen identified sequence variations for 19 other lines for which DNA from heterozygous flies had been characterized; but for none of these lines was the 25 bp deletion identified. We also extracted a TM6B chromosome from a different stock of the collection and found that the 25 bp deletion was not present. Thus, this deletion in exon 15ext did not pre-exist on the TM6B chromosome and apparently arose spontaneously since the mutagenesis scheme employed to generate the collection  did not What is the consequence of this change in the genomic sequence? The 25 bp deletion in exon 15ext shifts the open reading frame and should result in a truncated LD domain: While the initial 50 amino acids are unchanged, the C-terminal 64 residues are replaced by an unrelated five-amino-acid sequence Figure .MW klarcarried additional lesions, we sequenced all the coding exons of klar in this allele. The predicted Klar protein(s) display a number of differences to the canonical Klar sequence available on FlyBase emconly have a single copy of the klar locus, and they express Klar β at reduced levels compared to the wild type. Klar1 protein lacks the C-terminal 286 amino acids, and therefore migrates slightly faster than the wild-type protein [MW was expressed at similar levels to the single copy of wild-type KlarZ. Its apparent molecular weight was similar to that of KlarZ, but larger than that of Klar1; these observations are consistent with a lack of ~60 amino acids in KlarMW.We first asked if the mutant Klars Figure . The Kla klarYG3  served a protein . Finallyin-vivo centrifugation [Z klarKlar was highly enriched on the droplet layer, Klar signal was absent from the droplet layer in the LD truncation mutant MWklar. We conclude that truncation of the LD domain abolishes targeting of Klar to lipid droplets. Thus, the LD domain is indeed necessary for droplet localization in vivo.To test if the mutant Klar was present on lipid droplets, we employed the assay previously used  to demonfugation ,35. Whenfugation . We therDuring early embryogenesis, lipid droplets display stereotyped shifts in their overall distribution as the relative balance of plus- and minus-end motion changes in a temporally controlled manner . In wildMW klarand Z klarembryos. In both genotypes, the periphery turned clear in Phase II (not shown), just like for all other klar alleles previously characterized. In Phase III, Z klarembryos were opaque, like the wild type; a single copy of klar is indeed sufficient to support normal net droplet transport in Phase III [MW klarembryos were clear, very similar to embryos lacking Klar β altogether  with mutations in exon 15ext. We verified those changes by genomic sequencing; they are predicted to result in amino-acid changes at five positions Figure . In addiMW klarallele , nuclei were present in both apical and basal sections, indicating disrupted nuclear migration; nuclei were also found in the optical nerve. In MW klareye discs, nuclei were apical and not present basally nor in the optical nerve. Thus, Klar β is not essential for apical positioning of photoreceptor nuclei.To reveal the position of photoreceptor nuclei in third-instar eye discs, we employed the marker Elav Figure . We thenin vivo for the droplet localization of Klar as well as for Klar's role in regulating droplet transport. To address whether the LD domain is sufficient for these functions, we generated transgenes to allow Gal4-mediated expression of a GFP-LD fusion protein. Since the lipid droplets of the early embryo originate during oogenesis, we combined these transgenes with Gal4 drivers specifically expressed in the female germ line , so that the fusion protein could be produced when those lipid droplets are first generated. Western analysis confirmed that GFP-LD was expressed in ovaries and embryos  accumulates during oogenesis Figure , and is de-novo in these tissues, but localizes to pre-existing droplets.Using the ubiquitously active Act5C-Gal4 driver, we also expressed GFP-LD in somatic cells. In a number of adult and larval tissues, we found discrete GFP puncta, including in follicle cells in the adult ovary, larval salivary glands, and imaginal discs. These structures are lipid droplets, as they appear as rings at higher magnification and co-label with Nile Red Figure . Since iDrosophila cells [In summary, targeting of the LD domain to lipid droplets is not an embryo-specific phenomenon. GFP-LD also localizes to lipid droplets in the female germ line and in a number of somatic tissues. In addition, an analogous RFP-LD fusion protein localizes to lipid droplets in cultured la cells . Thus, Gklar locus encodes at least three proteins with different exon content, different lesions in klar have distinct effects on the isoforms and thus on distinct biological processes [MW klardisrupts Klar β but not Klar α; it alters droplet motion but not nuclear migration. It constitutes a new type of allele (class III) that selectively impairs the β isoform.Because the rocesses . LesionsMW Klarprovides a unique tool to separately examine the functions of Klar α and Klar β. Klar is widely expressed [xpressed  and has xpressed ,44-48. IOur analysis of GFP-LD fusions suggests that targeting of Klar β to lipid droplets is a multi-step process. In many cells, GFP-LD strongly accumulates on lipid droplets Figure , 7. In mNevertheless, GFP-LD clearly accumulates strongly on lipid droplets in many tissues, forming characteristic ring structures. Intriguingly, this distribution is distinct from the distribution of full-length Klar β, which is present in discrete dots on embryonic droplets , a distre.g. just like hormone-sensitive lipase is recruited to droplets by binding to PLIN1 [e.g., general hydrophobicity versus specific residues) are essential for proper droplet targeting. These studies can be conducted in the more accessible cultured-cell system, since the LD domain shows the same targeting properties in vivo . We therefore conclude that proper regulation of droplet motors requires the N-terminal region of Klar β and not just the LD domain. This model is further supported by the fact that Klar α shares those N-terminal regions and is also involved in motor regulation.e.g., the exact distances traveled in a single run or the frequency of pausing [Whether the LD domain simply targets Klar β to lipid droplets or has additional functions is not yet clear. For example, because in yeast the LD domain can interact with the droplet protein LSD-2, it has been suggested that it participates in transmitting developmental signals from LSD-2 via Klar to motors . In the  pausing .However, our data suggest that such effects, if they exist at all, do not alter the net outcome of transport. GFP-LD expressing embryos displayed the same transparency changes as the wild type: the embryo periphery became transparent in Phase II and cloudy in Phase III Figure  and outwDrosophila, droplet motion has so far been described only in early embryos [in vivo [Since LD has the ability to bind to lipid droplets in a wide range of cells, Klar β may control droplet motility in many tissues. In  embryos  and in o embryos , but no [in vivo ,56, inclklar allele that specifically disrupts the LD domain. Using these tools, we demonstrate that the LD domain is both necessary and sufficient for droplet targeting in vivo. We conclude that Klar β is targeted to lipid droplets via an isoform-specific protein motif, just like Klar α is targeted to the nuclear envelope via the KASH domain. Thus, it is controlled inclusion of cis-acting targeting sequences that mediates the differential intracellular localization of Klar in distinct tissues. Although Klar's LD domain is necessary for Klar β to act in the regulation of lipid-droplet transport, by itself it does not mediate Klar's transport functions. Likely it is the N-terminal regions shared between Klar α and Klar β that mediate motor regulation. In this model, variable C-terminal targeting sequences control Klar's intracellular distribution and thus dictate which subset of intracellular motors is controlled by Klar.To test the function of the LD domain of Klar β, we generated inducible GFP-LD fusion constructs and identified a klar alleles YG3 klarand 1 klarand the deficiency E12 Df(3L)emcwere described previously [D. melanogaster strain isolated in 1983 at Ivory Coast [Oregon R was used as the wild-type stock. The stocks carrying eviously ,22. The eviously . Line Z3eviously , and it ry Coast ; it dispry Coast .KpnI and NotI sites of pUASp [Drosophila embryos by Genetic Services, Inc. . Transgenic flies were selected by eye color. In total, twenty lines mapping to the X, second or third chromosome were established. To express the fusion proteins, transgenic animals were crossed with lines carrying matβ4-Gal4-VP16 or Act5C-Gal4 drivers.To generate the GFP-LD expressing flies, GFP was amplified from pEGP-C1 (Clontech) and cloned into the of pUASp . A tobacof pUASp  and clonTILLING analysis was performed by the Fly-TILL service as described . In ~600klar piecemeal using annealing temperatures and GC content to determine the most effective oligo sequences (primer sequences available upon request). Purified DNA from adult flies was used to individually PCR amplify these genomic fragments. PCR products were depleted of free nucleotides with ExoSAP-IT and sequenced at the Life Sciences Core Laboratories Center at Cornell University. Sequencing results were compared to the canonical sequences available on FlyBase.DNA primers were created to bookend individual exons of klar exon 15 in the available genome annotations for the Drosophila and mosquito species accessible via the FlyBase BLAST server. We also identified related sequences from the G. morsitans genome project (Sanger Institute) as well as in a cDNA from the medfly C. capitata (GenBank # FG077614.1). In Drosophila melanogaster, exon 15ext follows immediately downstream of exon 15. In all 16 additional fly species examined, the corresponding DNA sequences downstream of exon 15 have the potential to encode proteins with significant similarity to the D. melanogaster LD domain  was used at 1:10,000, and mouse anti-GFP (Roche) at 1:1000. LSD-2 was detected with a rabbit polyclonal anti-serum  at 1:20,2) containing 1 M sucrose plus protease inhibitor cocktail (Sigma-Aldrich), and then mechanically disrupted on ice. The lysate was transferred to a fresh 1.5 ml tube and overlaid sequentially with 200 μl TKM containing 0.5 M sucrose, 200 μl TKM containing 0.25 M sucrose, and 400 μl TKM. After centrifugation at 1000 g for 5 min, 5000 g for 5 min and 13400 g for 10 min, the buoyant lipid droplets were collected from the top of the gradient. The amount of protein in the isolated lipid droplets was measured by Bradford protein assay  before solubilizing the lipid droplets in SDS-containing buffer for subsequent SDS-PAGE analysis. Fractions such prepared are similar to those described by Cermelli et al. [To generate samples enriched in lipid droplets, we adapted the protocol described previously . For eaci et al. : the ove2PO4, 1 mM EDTA, pH 7.4), and mechanically disrupted. 40 μl of this lysate were mixed with 1 ml activated Triglyceride Reagent , and triglyceride levels were determined according to the manufacturer's instructions. For each experiment, three independent samples were analyzed per genotype, and the data shown in Figure Embryos were collected for 1.5 hr, dechorionated with 50% bleach, and resuspended in triton salt solution ,60. 200 MWklar, consulted on a range of technical issues, and helped to draft the manuscript. ZL performed the biochemical purification of lipid droplets and the triglyceride measurements. NPR performed Western analysis, immunostaining, and genomic sequencing. JE performed the bulk of genomic sequencing, including primer design. MAW conceived and designed the study, performed the imaging analysis, aligned sequences and drafted the manuscript. All authors read and approved the final manuscript.YVY generated and characterized the GFP-LD transgenes, contributed to the functional analysis of Motion of GFP-LD puncta in living embryos. Images were acquired by confocal microscopy at 2.5 images per second. Playback at 12 frames per second. Scale bar = 7.5 μm.Click here for fileMotion of GFP-LD puncta in living embryos. Images were acquired by confocal microscopy at 1.35 images per second. Playback at 6 frames per second. Scale bar = 7.5 μm.Click here for file