Design of orthogonal genetic switches based on a crosstalk map of σs,anti‐σs,and promoters

Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) σ s as well as the specificity of their interaction with anti‐ σ s. DNA synthesis was used to build 86 ECF σ s (two from every subgroup), their promoters, and 62 anti‐ σ s identified from the genomes of diverse bacteria. A subset of 20 σ s and promoters were found to be highly orthogonal to each other. This set can be increased by combining the ?35 and ?10 binding domains from different subgroups to build chimeras that target sequences unrepresented in any subgroup. The orthogonal σ s, anti‐ σ s, and promoters were used to build synthetic genetic switches in Escherichia coli. This represents a genome‐scale resource of the properties of ECF σ s and a resource for synthetic biology, where this set of well‐characterized regulatory parts will enable the construction of sophisticated gene expression programs.     

The CarD/CarG regulatory complex is required for the action of several members of the large set of Myxococcus xanthus extracytoplasmic function σ factors

Extracytoplasmic function (ECF) σ factors are critical players in signal transduction networks involved in bacterial response to environmental changes. The Myxococcus xanthus genome reveals ～45 putative ECF‐σ factors, but for the overwhelming majority, the specific signals or mechanisms for selective activation and regulation remain unknown. One well‐studied ECF‐σ, CarQ, binds to its anti‐σ, CarR, and is inactive in the dark but drives its own expression from promoter P_QRS on illumination. This requires the CarD/CarG complex, the integration host factor (IHF) and a specific CarD‐binding site upstream of P_QRS. Here, we show that DdvS, a previously uncharacterized ECF‐σ, activates its own expression in a CarD/CarG‐dependent manner but is inhibited when specifically bound to the N‐terminal zinc‐binding anti‐σ domain of its cognate anti‐σ, DdvA. Interestingly, we find that the autoregulatory action of 11 other ECF‐σ factors studied here depends totally or partially on CarD/CarG but not IHF. In silico analysis revealed possible CarD‐binding sites that may be involved in direct regulation by CarD/CarG of target promoter activity. CarD/CarG‐linked ECF‐σ regulation likely recurs in other myxobacteria with CarD/CarG orthologous pairs and could underlie, at least in part, the global regulatory effect of the complex on M. xanthus gene expression.     

A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

Self‐association of TPR domains: Lessons learned from a designed,consensus‐based TPR oligomer

The tetratricopeptide repeat (TPR) motif is a protein–protein interaction module that acts as an organizing centre for complexes regulating a multitude of biological processes. Despite accumulating evidence for the formation of TPR oligomers as an additional level of regulation there is a lack of structural and solution data explaining TPR self‐association. In the present work we characterize the trimeric TPR‐containing protein YbgF, which is linked to the Tol system in Gram‐negative bacteria. By subtracting previously identified TPR consensus residues required for stability of the fold from residues conserved across YbgF homologs, we identified residues involved in oligomerization of the C‐terminal YbgF TPR domain. Crafting these residues, which are located in loop regions between TPR motifs, onto the monomeric consensus TPR protein CTPR3 induced the formation of oligomers. The crystal structure of this engineered oligomer shows an asymmetric trimer where stacking interactions between the introduced tyrosines and displacement of the C‐terminal hydrophilic capping helix, present in most TPR domains, are key to oligomerization. Asymmetric trimerization of the YbgF TPR domain and CTPR3Y3 leads to the formation of higher order oligomers both in the crystal and in solution. However, such open‐ended self‐association does not occur in full‐length YbgF suggesting that the protein's N‐terminal coiled‐coil domain restricts further oligomerization. This interpretation is borne out in experiments where the coiled‐coil domain of YbgF was engineered onto the N‐terminus of CTPR3Y3 and shown to block self‐association beyond trimerization. Our study lays the foundations for understanding the structural basis for TPR domain self‐association and how such self‐association can be regulated in TPR domain‐containing proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Discovery of the extracytoplasmic function σ factors

This special issue of Molecular Microbiology marks the 25^th anniversary of the discovery of the extracytoplasmic function (ECF) σ factors, proteins that subsequently emerged as the largest group of alternative σ factors and one of the three major pillars of signal transduction in bacteria, alongside one‐ and two‐component systems. A single bacterial genome can encode > 100 ECF σ factors, and combined with their cognate anti‐σ factors, they represent a modular design that primarily functions in transmembrane signal transduction. Here, we first describe the immediate events that led to the 1994 publication in the Proceeding of the National Academy of Sciences USA, and then set them in the broader context of key events in the history of σ biology research.     

Targeted Disruption of an EH‐domain Protein Endocytic Complex,Pan1‐End3

Pan1 is a multi‐domain scaffold that enables dynamic interactions with both structural and regulatory components of the endocytic pathway. Pan1 is composed of Eps15 Homology (EH) domains which interact with adaptor proteins, a central region that is responsible for its oligomerization and C‐terminal binding sites for Arp2/3, F‐actin, and type‐I myosin motors. In this study, we have characterized the binding sites between Pan1 and its constitutive binding partner End3, another EH domain containing endocytic protein. The C‐terminal End3 Repeats of End3 associate with the N‐terminal part of Pan1's central coiled‐coil region. These repeats appear to act independently of one another as tandem, redundant binding sites for Pan1. The end3‐1 allele was sequenced, and corresponds to a C‐terminal truncation lacking the End3 Repeats. Mutations of the End3 Repeats highlight that those residues which are identical between these repeats serve as contact sites for the interaction with Pan1.      

Fe2+ binding on amyloid β‐peptide promotes aggregation

The metal ions Zn²⁺, Cu²⁺, and Fe²⁺ play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06‐2X/6‐311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ_1‐42‐Zn²⁺, Aβ_1‐42‐Cu²⁺, and Aβ_1‐42‐Fe²⁺ systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N‐terminal. The present investigation, the Aβ_1‐42‐Zn²⁺ system possess three turn conformations separated by coil structure. Zn²⁺ binding caused the loss of the helical structure of N‐terminal residues which transformed into the S‐shaped conformation. Zn²⁺ has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu²⁺ binds with peptide, β sheet formation is observed at the N‐terminal residues of the peptide. Fe²⁺ binding is to promote the formation of Glu22‐Lys28 salt‐bridge which stabilized the turn conformation in the Phe19‐Gly25 residues, subsequently β sheets were observed at His13‐Lys18 and Gly29‐Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe²⁺ binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe²⁺ promotes the oligomerization by enhancing the peptide‐peptide interaction. Proteins 2016; 84:1257–1274. © 2016 Wiley Periodicals, Inc.     

Crystallographic structure of the tetratricopeptide repeat domain of Plasmodium falciparum FKBP35 and its molecular interaction with Hsp90 C‐terminal pentapeptide

Plasmodium falciparum FK506‐binding protein 35 (PfFKBP35) that binds to FK506 contains a conserved tetratricopeptide repeat (TPR) domain. Several known TPR domains such as Hop, PPP5, CHIP, and FKBP52 are structurally conserved and are able to interact with molecular chaperones such as Hsp70/Hsp90. Here, we present the crystal structure of PfFKBP35‐TPR and demonstrate its interaction with Hsp90 C‐terminal pentapeptide (MEEVD) by surface plasmon resonance and nuclear magnetic resonance spectroscopy‐based binding studies. Our sequence and structural analyses reveal that PfFKBP35 is similar to Hop and PPP5 in possessing all the conserved residues which are important for carboxylate clamping with Hsp90. Mutational studies were carried out on positively charged clamp residues that are crucial for binding to carboxylate groups of aspartate, showing that all the mutated residues are important for Hsp90 binding. Molecular docking and electrostatic calculations demonstrated that the MEEVD peptide of Hsp90 can form aspartate clamp unlike FKBP52. Our results provide insightful information and structural basis about the molecular interaction between PfFKBP35‐TPR and Hsp90.     

Processing of cell‐surface signalling anti‐sigma factors prior to signal recognition is a conserved autoproteolytic mechanism that produces two functional domains

Cell‐surface signalling (CSS) enables Gram‐negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti‐sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP‐mediated proteolysis of the anti‐sigma factors is key to σ^ECF activation. Using the Pseudomonas aeruginosa FoxR anti‐sigma factor, we show here that RseP is responsible for the generation of an N‐terminal tail that likely contains pro‐sigma activity. Furthermore, it has been reported previously that this anti‐sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti‐sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti‐sigma factors.     

Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/Wrch‐1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N‐ and C‐terminal domains. The N‐terminal extension promotes binding to SH3 (Src homology 3)‐domain‐containing adaptors, whereas the C‐terminal extension mediates membrane targeting through palmitoylation of its non‐conventional CAAX box. RhoU/Wrch‐1 possesses transforming activity, which is negatively regulated by its N‐terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c‐Src‐expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N‐terminal extension and the palmitoylation site were dispensable, whereas the C‐terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked‐down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C‐terminal domains are critical for these functions.     

Exploring ligand recognition,selectivity and dynamics of TPR domains of chloroplast Toc64 and mitochondria Om64 from Arabidopsis thaliana

The study aims to gain insight into the mode of ligand recognition by tetratricopeptide repeat (TPR) domains of chloroplast translocon at the outer envelope of chloroplast (Toc64) and mitochondrial Om64, two paralogous proteins that mediate import of proteins into chloroplast and mitochondria, respectively. Chaperone proteins associate with precursor proteins in the cytosol to maintain them in a translocation competent conformation and are recognized by Toc64 and Om64 that are located on the outer membrane of the target organelle. Heat shock proteins (Hsp70) and Hsp90 are two chaperones, which are known to play import roles in protein import. The C‐termini of these chaperones are known to interact with the TPR domain of chloroplast Toc64 and mitochondrial Om64 in Arabidopsis thaliana (At). Using a molecular dynamics approach and binding energy calculations, we identify important residues involved in the interactions. Our findings suggest that the TPR domain from AtToc64 has higher affinity towards C‐terminal residues of Hsp70. The interaction occurs as the terminal helices move towards each other enclosing the cradle on interaction of AtHsp70 with the TPR domain. In contrast, the TPR domain from AtOm64 does not discriminate between the C‐termini of Hsp70 and Hsp90. These binding affinities are discussed with respect to our knowledge of protein targeting and specificity of protein import into endosymbiotic organelles in plant cells. Copyright © 2014 John Wiley & Sons, Ltd.     

NMR evidence for differential phosphorylation‐dependent interactions in WT and ΔF508 CFTR

The most common cystic fibrosis (CF)‐causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (ΔF508) in the first of two nucleotide‐binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild‐type and ΔF508 murine CFTR NBD1 with the C‐terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild‐type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N‐terminal intracellular domain (ICD). Phosphorylation of ΔF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full‐length CFTR and expand our understanding of the molecular basis of the ΔF508 defect.     

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

Human age‐onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens γD‐crystallin protein and its isolated domains. A partially unfolded folding intermediate of γD‐crystallin is detected in simulations with its C‐terminal domain (C‐td) folded and N‐terminal domain (N‐td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N‐td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C‐td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non‐native surface salt‐bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non‐native Glu135‐Arg142 salt‐bridge causes significant destabilization to the folding core of the isolated C‐td, which, in turn, induces unfolding of the N‐td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD‐crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD‐crystallin surface might lead to unfolding and subsequent aggregation.     

The ASPP interaction network: electrostatic differentiation between pro‐ and anti‐apoptotic proteins

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C‐terminal ankyrin repeats and SH3 domain (ANK‐SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl‐2, and NFκB. The structure of the complex between ASPP2_ANK‐SH3 and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2_ANK‐SH3 with Bcl‐2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2_ANK‐SH3 with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl‐2, and NFκB are different, yet lie on the same face of ASPP2_ANK‐SH3. The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein‐binding domains. A subset of surface‐exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl‐2, and NFκB. We also found a gain of positive charge at the non‐protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.