Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1.

Casposase, a homolog of Cas1 integrase, is encoded by a superfamily of mobile genetic elements known as casposons. While family 2 casposase has been well documented in both function and structure, little is known about the other three casposase families. Here, we studied the family 1 casposase lacking the helix-turn-helix (HTH) domain from Candidatus Nitrosopumilus koreensis AR1 (Ca. N. koreensis). The determinants for integration by Ca. N. koreensis casposase were extensively investigated, and it was found that a 13-bp target site duplication (TSD) sequence, a minimal 3-bp leader and three different nucleotides of the TSD sequences are indispensable for target specific integration. Significantly, the casposase can site-specifically integrate a broad range of terminal inverted repeat (TIR)-derived oligonucleotides ranging from 7-nt to ∼4000-bp, and various oligonucleotides lacking the 5'-TTCTA-3' motif at the 3' end of TIR sequence can be integrated efficiently. Furthermore, similar to some Cas1 homologs, the casposase utilizes a 5'-ATAA-3' motif in the TSD as a molecular ruler to dictate nucleophilic attack at 9-bp downstream of the end of the ruler during the spacer-side integration. By characterizing the family 1 Ca. N. koreensis casposase, we have extended our understanding on mechanistic similarities and evolutionary connections between casposons and the adaptation elements of CRISPR-Cas immunity.

PoxCbh, a novel CENPB-type HTH domain protein, regulates cellulase and xylanase gene expression in Penicillium oxalicum.

The essential transcription factor PoxCxrA is required for cellulase and xylanase gene expression in the filamentous fungus Penicillium oxalicum that is potentially applied in biotechnological industry as a result of the existence of the integrated cellulolytic and xylolytic system. However, the regulatory mechanism of cellulase and xylanase gene expression specifically associated with PoxCxrA regulation in fungi is poorly understood. In this study, the novel regulator PoxCbh (POX06865), containing a centromere protein B-type helix-turn-helix domain, was identified through screening for the PoxCxrA regulon under Avicel induction and genetic analysis. The mutant ∆PoxCbh showed significant reduction in cellulase and xylanase production, ranging from 28.4% to 59.8%. Furthermore, PoxCbh was found to directly regulate the expression of important cellulase and xylanase genes, as well as the known regulatory genes PoxNsdD and POX02484, and its expression was directly controlled by PoxCxrA. The PoxCbh-binding DNA sequence in the promoter region of the cellobiohydrolase 1 gene cbh1 was identified. These results expand our understanding of the diverse roles of centromere protein B-like protein, the regulatory network of cellulase and xylanase gene expression, and regulatory mechanisms in fungi.

Conserved L464 in p97 D1-D2 linker is critical for p97 cofactor regulated ATPase activity.

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Šaway.

LOTUS domain is a novel class of G-rich and G-quadruplex RNA binding domain.

LOTUS domains are helix-turn-helix protein folds identified in essential germline proteins and are conserved in prokaryotes and eukaryotes. Despite originally predicted as an RNA binding domain, its molecular binding activity towards RNA and protein is controversial. In particular, the most conserved binding property for the LOTUS domain family remains unknown. Here, we uncovered an unexpected specific interaction of LOTUS domains with G-rich RNA sequences. Intriguingly, LOTUS domains exhibit high affinity to RNA G-quadruplex tertiary structures implicated in diverse cellular processes including piRNA biogenesis. This novel LOTUS domain-RNA interaction is conserved in bacteria, plants and animals, comprising the most ancient binding feature of the LOTUS domain family. By contrast, LOTUS domains do not preferentially interact with DNA G-quadruplexes. We further show that a subset of LOTUS domains display both RNA and protein binding activities. These findings identify the LOTUS domain as a specialized RNA binding domain across phyla and underscore the molecular mechanism underlying the function of LOTUS domain-containing proteins in RNA metabolism and regulation.

Structure and DNA damage-dependent derepression mechanism for the XRE family member DG-DdrO.

DdrO is an XRE family transcription repressor that, in coordination with the metalloprotease PprI, is critical in the DNA damage response of Deinococcus species. Here, we report the crystal structure of Deinococcus geothermalis DdrO. Biochemical and structural studies revealed the conserved recognizing α-helix and extended dimeric interaction of the DdrO protein, which are essential for promoter DNA binding. Two conserved oppositely charged residues in the HTH motif of XRE family proteins form salt bridge interactions that are essential for promoter DNA binding. Notably, the C-terminal domain is stabilized by hydrophobic interactions of leucine/isoleucine-rich helices, which is critical for DdrO dimerization. Our findings suggest that DdrO is a novel XRE family transcriptional regulator that forms a distinctive dimer. The structure also provides insight into the mechanism of DdrO-PprI-mediated DNA damage response in Deinococcus.

Structural basis for feed-forward transcriptional regulation of membrane lipid homeostasis in Staphylococcus aureus.

The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and large (>30 Å) inter-domain movements; however, both conformational states can be populated by the ligand-free repressor species, as confirmed by the structure of SaFapR in two distinct crystal forms. Disruption of the ability of SaFapR to monitor malonyl-CoA compromises cell growth, revealing the essentiality of membrane lipid homeostasis for S. aureus survival and uncovering novel opportunities for the development of antibiotics against this major human pathogen.

Malleability of folding intermediates in the homeodomain superfamily.

Members of the homeodomain superfamily are three-helix bundle proteins whose second and third helices form a helix-turn-helix motif (HTH). Their folding mechanism slides from the ultrafast, three-state framework mechanism for the engrailed homeodomain (EnHD), in which the HTH motif is independently stable, to an apparent two-state nucleation-condensation model for family members with an unstable HTH motif. The folding intermediate of EnHD has nearly native HTH structure, but it is not docked with helix1. The determinant of whether two- or three-state folding was hypothesized to be the stability of the HTH substructure. Here, we describe a detailed Φ-value analysis of the folding of the Pit1 homeodomain, which has similar ultrafast kinetics to that of EnHD. Formation of helix1 was strongly coupled with formation of HTH, which was initially surprising because they are uncoupled in the EnHD folding intermediate. However, we found a key difference between Pit1 and EnHD: The isolated peptide corresponding to the HTH motif in Pit1 was not folded in the absence of H1. Independent molecular dynamics simulations of Pit1 unfolding found an intermediate with H1 misfolded onto the HTH motif. The Pit1 folding pathway is the connection between that of EnHD and the slower folding homeodomains and provides a link in the transition of mechanisms from two- to three-state folding in this superfamily. The malleability of folding intermediates can lead to unstable substructures being stabilized by a variety of nonnative interactions, adding to the continuum of folding mechanisms.

Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein.

Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface.

The linker sequence, joining the DNA-binding domain of the homologous transcription factors, Mlc and NagC, to the rest of the protein, determines the specificity of their DNA target recognition in Escherichia coli.

Protein-DNA recognition is fundamental to transcriptional regulation. Transcription factors must be capable of locating their specific sites situated throughout the genome and distinguishing them from related sites. Mlc and NagC control uptake and use of the sugars, glucose and N-acetylglucosamine. Both their helix-turn-helix motifs and their consensus binding sites on DNA are very similar. One distinguishing feature is that most NagC sites have a C/G bp at positions -11 and +11 from the centre of symmetry of the operator, while all Mlc sites have A/T. By constructing Mlc and NagC chimeras, we show that the helix-turn-helix motif per se is not responsible for specific recognition of Mlc or NagC sites, but that a linker, joining the DNA-binding domain to the rest of the protein, is the major determinant. We show that a change of just two amino acids in the NagC linker is sufficient to allow NagC to recognize an A/T bp at positions +/-11 and repress Mlc targets. Modelling of the NagC linker suggests that it forms an extended structure containing two arginines and we suggest that these arginines interact differently with the minor groove at positions +/-11 depending upon the presence of a C/G or A/T bp.

Mechanism for regulation of the putrescine utilization pathway by the transcription factor PuuR in Escherichia coli K-12.

In Escherichia coli, putrescine is metabolized to succinate for use as a carbon and nitrogen source by the putrescine utilization pathway (Puu pathway). One gene in the puu gene cluster encodes a transcription factor, PuuR, which has a helix-turn-helix DNA-binding motif. DNA microarray analysis of an E. coli puuR mutant, in which three amino acid residues in the helix-turn-helix DNA binding motif of PuuR were mutated to alanine to eliminate DNA binding of PuuR, suggested that PuuR is a negative regulator of puu genes. Results of gel shift and DNase I footprint analyses suggested that PuuR binds to the promoter regions of puuA and puuD. The binding of wild-type PuuR to a DNA probe containing PuuR recognition sites was diminished with increasing putrescine concentrations in vitro. These results suggest that PuuR regulates the intracellular putrescine concentration by the transcriptional regulation of genes in the Puu pathway, including puuR itself. The puu gene cluster is found in E. coli and closely related enterobacteria, but this gene cluster is uncommon in other bacterial groups. E. coli and related enterobacteria may have gained the Puu pathway as an adaptation for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations.

The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins.

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

Systematic mutagenesis of all predicted gntR genes in Xanthomonas campestris pv. campestris reveals a GntR family transcriptional regulator controlling hypersensitive response and virulence.

The GntR family is one of the most abundant and widely distributed groups of helix-turn-helix transcriptional regulators in bacteria. Six open reading frames in the genome of the plant pathogen Xanthomonas campestris pv. campestris were predicted to encode GntR regulators. All six of the predicted GntR-encoding genes were individually mutagenized and mutants from five of them were successfully obtained. Plant disease response assays revealed that one, whose product belongs to the YtrA subfamily and has been named HpaR1, is involved in the hypersensitive response (HR) and virulence. Electrophoretic mobility shift assays and in vitro transcription assays revealed that HpaR1 could repress its own transcription level through binding to its promoter sequence, indicating an autoregulatory feedback inhibition mechanism for HpaR1 expression. Promoter-gusA reporter and reverse-transcription polymerase chain reaction analyses revealed that HpaR1 positively and negatively affects the expression of HR and pathogenicity (hrp) genes in host plant and standard media, respectively. Constitutive expression of the key hrp regulator, hrpG, in the hpaR1 mutant could bypass the requirement of HpaR1 for the induction of wild-type HR, suggesting that HpaR1 regulates the expression of hrp genes that encode the type III secretion system via hrpG.

Diversity and distribution of transcription factors: their partner domains play an important role in regulatory plasticity in bacteria.

The ability of bacteria to deal with diverse environmental changes depends on their repertoire of genes and their ability to regulate their expression. In this process, DNA-binding transcription factors (TFs) have a fundamental role because they affect gene expression positively and/or negatively depending on operator context and ligand-binding status. Here, we show an exhaustive analysis of winged helix-turn-helix domains (wHTHs), a class of DNA-binding TFs. These proteins were identified in high proportions and widely distributed in bacteria, representing around half of the total TFs identified so far. In addition, we evaluated the repertoire of wHTHs in terms of their partner domains (PaDos), identifying a similar trend, as with TFs, i.e. they are abundant and widely distributed in bacteria. Based on the PaDos, we defined three main groups of families: (i) monolithic, those families with little PaDo diversity, such as LysR; (ii) promiscuous, those families with a high PaDo diversity; and (iii) monodomain, with families of small sizes, such as MarR. These findings suggest that PaDos have a very important role in the diversification of regulatory responses in bacteria, probably contributing to their regulatory complexity. Thus, the TFs discriminate over longer regions on the DNA through their diverse DNA-binding domains. On the other hand, the PaDos would allow a great flexibility for transcriptional regulation due to their ability to sense diverse stimuli through a variety of ligand-binding compounds.

The ChiS-Family DNA-Binding Domain Contains a Cryptic Helix-Turn-Helix Variant.

Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a ß-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

Hybrid Ptr2-like activators of archaeal transcription.

Methanocaldococcus jannaschii Ptr2, a member of the Lrp/AsnC family of bacterial DNA-binding proteins, is an activator of its eukaryal-type core transcription apparatus. In Lrp-family proteins, an N-terminal helix-turn-helix DNA-binding and dimerizing domain is joined to a C-terminal effector and multimerizing domain. A cysteine-scanning surface mutagenesis shows that the C-terminal domain of Ptr2 is responsible for transcriptional activation; two types of DNA binding-positive but activation-defective mutants are found: those unable to recruit the TBP and TFB initiation factors to the promoter, and those failing at a post-recruitment step. Transcriptional activation through the C-terminal Ptr2 effector domain is exploited in a screen of other Lrp effector domains for activation capability by constructing hybrid proteins with the N-terminal DNA-binding domain of Ptr2. Two hybrid proteins are effective activators: Ptr-H10, fusing the effector domain of Pyrococcus furiosus LrpA, and Ptr-H16, fusing the P. furiosus ORF1231 effector domain. Both new activators exhibit distinguishing characteristics: unlike octameric Ptr2, Ptr-H10 is a dimer; unlike Ptr2, the octameric Ptr-H16 poorly recruits TBP to the promoter, but more effectively co-recruits TFB with TBP. In contrast, the effector domain of Ptr1, the M. jannaschii Ptr2 paralogue, yields only very weak activation.

The pathway-specific regulator AveR from Streptomyces avermitilis positively regulates avermectin production while it negatively affects oligomycin biosynthesis.

The function of the regulatory protein AveR in Streptomyces avermitilis was examined. An aveR deletion mutant abolished avermectin production and produced more oligomycin, and its phenotype was complemented by a single copy of the aveR gene. Removal of the C-terminal HTH domain of AveR abolished avermectin biosynthesis, indicating the importance of HTH domain for AveR function. Promoter titration and promoter probe assays suggested that the transcription of aveA1, encoding polypeptide AVES1 of avermectin PKS, was activated by AveR. Chromatin immunoprecipitation (ChIP) assay showed that the predicted promoter regions of both the ave cluster and the olm cluster were target sites of AveR, and the DNA-binding activity of AveR was dependent on its HTH domain. RT-PCR analysis revealed that the transcriptions of ave structural genes were dependent on AveR, but that of olm structural genes and putative pathway-specific regulatory genes increased in the aveR mutants. Consistent with these observations, overexpression of aveR successfully increased avermectin production. These results indicated that aveR encodes a pathway-specific activator essential for avermectin biosynthesis and it also negatively affects oligomycin biosynthesis.

Solution structure of the HsapBK K+ channel voltage-sensor paddle sequence.

Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.

Mechanism of ligand-induced folding of a natively unfolded helixless variant of rabbit I-BABP.

Substitution of the helix-turn-helix capping motif (residues 9-35) of rabbit I-BABP with a flexible Gly-Gly-Ser-Gly linker results in the loss of stabilizing hydrophobic contacts and renders the beta-clamshell structure of this steroidal bile acid transport protein unfolded. However, in the presence of a bile acid ligand, we observe strong coupling between binding and folding, resulting in an enthalpy-driven high-affinity interaction (K(A) approximately 4 x 10(5) M(-1)) that "rescues" the native state. We investigate the mechanism of induced folding using fluorescence stopped-flow kinetic measurements to distinguish between conformational selection and induced-fit models. We observe both ligand-dependent and -independent kinetic phases which, together with their relative amplitudes, we attribute to an induced-fit "fly casting" type of model in which transient encounter complexes between the ligand and the extended polypeptide chain may act as nucleation sites for folding. An initial fast ligand-dependent kinetic process appears to be consistent with formation of a hydrophobically collapsed intermediate state which slowly rearranges to a nativelike beta-clamshell structure. We show that the intermediate forms at a rate 1000 times slower than the rate of ligand association with wild-type I-BABP, reflecting the large configurational entropic barrier to the coupled binding and folding steps of Deltaalpha-I-BABP. We have provided mechanistic insights into how natively disordered states, now commonly identified in biology, may fold on binding a target substrate or ligand.

Structure of the restriction-modification controller protein C.Esp1396I.

The controller protein of the Esp1396I restriction-modification (R-M) system binds differentially to three distinct operator sequences upstream of the methyltransferase (M) and endonuclease (R) genes to regulate the timing of gene expression. The crystal structure of a complex of the protein with two adjacent operator DNA sequences has been reported; however, the structure of the free protein has not yet been determined. Here, the crystal structure of the free protein is reported, with seven dimers in the asymmetric unit. Two of the 14 monomers show an alternative conformation to the major conformer in which the side chains of residues 43-46 in the loop region flanking the DNA-recognition helix are displaced by up to 10 A. It is proposed that the adoption of these two conformational states may play a role in DNA-sequence promiscuity. The two alternative conformations are also found in the R35A mutant structure, which is otherwise identical to the native protein. Comparison of the free and bound protein structures shows a 1.4 A displacement of the recognition helices when the dimer is bound to its DNA target.

Chapter 1: Variation in form and function the helix-turn-helix regulators of the GntR superfamily.

One of the most abundant and widely distributed groups of Helix-turn-helix (HTH) transcription factors is the metabolite-responsive GntR family of regulators (>8500 members in the Pfam database; Jan 2009). These proteins contain a DNA-binding HTH domain at the N terminus of the protein and an effector-binding and/or oligomerisation domain at the C terminus, where upon on binding an effector molecule, a conformational change occurs in the protein which influences the DNA-binding properties of the regulator resulting in repression or activation of transcription. This review summarises what we know about the distribution, structure, function and classification of these regulators and suggests that they may have a future role in biotechnology.