Search

Transcriptional regulation usually requires the action of several proteins that either repress or activate a promotor of an open reading frame. These proteins can counteract each other, thus allowing tight regulation of the transcription of the corresponding genes, where tight repression is often linked to DNA looping or cross-linking. Here, the tetramerization domain of the bacterial gene repressor Rco from Bacillus subtilis plasmid pLS20 (RcopLS20) has been identified and its structure is shown to share high similarity to the tetramerization domain of the well known p53 family of human tumor suppressors, despite lacking clear sequence homology. In RcopLS20, this tetramerization domain is responsible for inducing DNA looping, a process that involves multiple tetramers. In accordance, it is shown that RcopLS20 can form octamers. This domain was named TetDloop and its occurrence was identified in other Bacillus species. The TetDloop fold was also found in the structure of a transcriptional repressor from Salmonella phage SPC32H. It is proposed that the TetDloop fold has evolved through divergent evolution and that the TetDloop originates from a common ancestor predating the occurrence of multicellular life.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix–turn–helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness [BIO2016-77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU), to D.R.B., BFU2017-86378-P to F.dlC.] and by the Spanish Ministryof Science (MCI/AEI/FEDER,UE) [PGC2018-093885-BI00 and PID2021-122164NB-I00 to G.M., PID2020- 117028GB-I00 to D.R.B. and PID2019-110216GB-I00 to R. F-L.].

12 pags., 4 figs., 2 tabs. -- Addenda and errata: https://journals.iucr.org/s/issues/2022/03/00/rv5160/rv5160.pdf, The increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 μm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 μm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures., The following funding is acknowledged: Ayuda de Atracciony Retencion de Talento Investigador" from the Community of Madrid (scholarship No. 2019-T1/BMD-15552); STC Programof the National Science Foundation through BioXFEL (awardNo. 1231306); the Centre for Applied Structural Discovery(CASD) at the Biodesign Institute at Arizona State University; the Spanish Ministry of Science and Innovation, grants EQC2021-007532-P, PID2020-117028GB-I00, BIO2016-77883-C2-2-P

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF–ARF and ARF–DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

15 p.-6 fig.1 graph. abst., DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin., Ministry of Science and Innovation of Spain [BFU2017-83720-P, PID2020-120141GB-100 to M.C., 2022AEP028 (RT12018-097114-B-I00), BFU2010-19597 to G.dS.]; RYC fellowship [RYC-2011-09071 to C.M.]; CSIC [2020AEP116 to M.C.]. Funding for open access charge: CSIC.

The signaling molecule auxin is pivotal in coordinating many growth and development processes in plants mainly through the modulation of gene expression. The transcriptional response to auxin is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif (TGTC[TC]/[GG]) called the auxin-response element (AuxRE). ARFs can homodimerize through their DNA binding domains (DBD) thereby enabling cooperative binding for a bipartite inverted AuxRE (IR7). In addition to the DBD, most ARFs contain a C-terminal Phox and Bem1p (PB1) domain both capable of homotypic interactions, and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how each of these domains contributes to conferring DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of severalArabidopsis thalianaARFs with an IR7 AuxRE. We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining affinity and kinetics seen for the DBDs of different AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs towards composite DNA response elements can be tuned by small changes of their dimerization equilibrium suggesting that this effect has major implications for ARF-mediated transcriptional activity.

15 p.-4 fig.-3 tab., Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. RappLS20 is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by RcopLS20 in the absence of the mature pLS20 signaling peptide Phr*pLS20. We report here an in-depth structural study of apo and Phr*pLS20-bound states of RappLS20 at various levels of atomic detail. We show that apo-RappLS20 is dimeric and that Phr*pLS20-bound Rap forms NTD-mediated tetramers. In addition, we show that RappLS20 binds RcopLS20 directly in the absence of Phr*pLS20 and that addition of Phr*pLS20 releases RcopLS20 from RappLS20. This allows RcopLS20 to bind the promotor region of crucial conjugation genes blocking their expression., Ministry of Economy and Competitiveness of the Spanish Government [BFU2016-75471-C2-1-P (AEI/FEDER,EU) to C.A., BIO2013-41489-P (AEI/FEDER, EU) to W.M. which also funded AM-A, BIO2016-77883-C2-2-P (AEI/FEDER, EU) to R.B., BIO2016-77883-C2-2-P(AEI/FEDER, EU) also supported N.B.]; ‘Fundación Ramón Areces’ and ‘Banco de Santander’ to the Centro de Biología Molecular ‘Severo Ochoa’.Funding for open access charge: Ministry of Economy and Competitiveness of the Spanish Government [BIO2016-77883-C2-1-P and BIO2016-77883-C2-2-P].

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1–3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems. The minimal regulatory logic and the evolutionary history of the auxin signalling pathway are explored by studying the much simpler auxin network in Marchantia with a combination of genetic, domain shuffling and biochemical strategies.

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named RelpLS20, and two auxiliary DNA-binding factors, named Aux1pLS20 and Aux2pLS20. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1pLS20 and the atomic X-ray structure of the C-terminal domain of Aux2pLS20. We also study the interactions between the auxiliary proteins and the full-length RelpLS20, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1pLS20 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2pLS20 shows that it forms a tetramer and suggests that it is an analog of TraMpF of plasmid F. This is the first evidence of the existence of a TraMpF analog in gram positive conjugative systems, although, unlike other TraMpF analogs, Aux2pLS20 does not interact with the relaxase. Aux1pLS20 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase RelpLS20. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.

Significance The plant hormone auxin controls many aspects of growth and development. It does so by changing the activity of AUXIN RESPONSE FACTOR (ARF) proteins that recognize specific DNA elements in plant genes and switch gene expression on or off. A major question in plant biology is how these ARF proteins bind unique DNA sequences and thus select which genes are regulated by auxin. Here, the authors systematically study the DNA architecture required for high-affinity binding of ARF proteins, and for auxin-dependent gene regulation in the plant Arabidopsis thaliana. This work shows that repeats of ARF recognition sites are critical for function, and reveals the molecular basis for the distinct activities of different repeat structures., The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF–DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

The plant hormones auxin affects most aspects of plant growth and development. The auxin transport and signaling are regulated by different factors that modulate plant morphogenesis and respond to external environments. The modulation of gene expression by Auxin Response Factors (ARFs) and inhibiting Auxin/Indole-3-Acetic Acid (Aux/IAA) proteins are involved in auxin signaling pathways. These components are encoded by gene families with numerous members in most flowering plants.However, there is no genome-wide analysis of the expression profile and the structural and functional properties of theHere, we discovered 63This study will improve our understanding of the auxin signaling system and its regulatory role in plant growth and development.

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum- signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed., Ministry of Science and Innovation of the Spanish Government (PID2019 108778GB C21 (AEI/FEDER, EU)) to WM, (BIO2016-77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU)), to DB, (PID2019-104544GBI00 (AEI/FEDER/EU)) to CA, (PID2019-109320GB-100/AEI/10.13039/501100011033) to SA, (PGC2018-094143-B-I00, (AEI/FEDER, EU)) to FR, and (FIS2016-78313-P) to SA. The Ministry of Science and Innovation of the Spanish Government also funded the Centro Nacional de Biotecnología (CNB) by means of the “Severo Ochoa” Center of Excellence grant SEV 2017-0712. A Wellcome Investigator grant (209500) to Jeff Errington supported LW. Institutional grants from the ‘Fundación Ramón Areces’ and ‘Banco de Santander’ supported

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition., Economy and Competitiveness of the Spanish Government [BFU2016-75471-C2-1-P (AEI/FEDER, EU) to C.A., BIO2013-41489-P (AEI/FEDER, EU) and BIO2016-77883-C2-1-P (AEI/FEDER, EU) to W.M., BIO2016-77883-C2-2-P (AEI/FEDER, EU) to R.B., BIO-2015-66203-P (AEI/FEDER, EU) to F.R., FIS2016-78313-P (AEI/FEDER, EU) to S.A.]; BIO2013-41489-P (AEI/FEDER, EU) and BIO2016-77883-C2-1-P (AEI/FEDER, EU) also supported J.V., A.M., and C.G.; Wellcome Investigator Award [209500 to Jeff Errington] supported L.W; ‘Ramón y Cajal’ Contract Supported S.A.; ‘Agencia Estatal de Investigaci ´on’ (AEI); ‘Fondo Europeo de Desarrollo Regional (FEDER); European Union (EU)

23 p.-5 fig.-2 tab., Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site-and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named rel(LS20), is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the Rel(LS20) shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research., Work in the Meijer lab was funded by the Spanish government through grant Bio2013- 41489-P of the Ministry of Economy and Competitiveness, and through grant Bio2016- 77883-C2-1-P of the Ministry of Economy, Industry and Competitiveness; the former grant also funded AMA and CGC. The Spanish government also supported DRB, JRLO, and CA.DRB was funded by grant Bio2016-77883-C2-2-P of the Ministry of Economy, Industry and Competitiveness, and JRLO and CA were supported by grant BFU2014-52070-C2-2-P of the Ministry of Economy and Competitiveness to CA.LJW's work was supported by Wellcome Trust grant WT098374AIA to Jeff Errington.

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOBV family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOBV relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOBV histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria., The Institute for Research in Biomedicine Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Spanish Ministry of Economy and Competitiveness (MINECO). The Molecular Biology Institute, Barcelona Spanish Research Council’s Structural Biology Unit is the recipient of a Maria de Maeztu Award of Excellence from MINECO. This work was supported by grants from MINECO (BFU2008-02372/ BMC, CSD-2006-23, BFU2011-22588, CSD-2008/00013, BIO2013-49148-C2-2-R, BFU2014-53550-P, BIO2015-69085-REDC, and BIO2015-64802), the Generalitat de Catalunya (2014-SGR134 and 2014-SGR1530), and the European Commission [project no. 260644, ERC_SimDNA, and the H2020 program (MuG and BioExcel Projects)]. R.P. and H.G. received a La Caixa/Institute for Research in Biomedicine Barcelona International PhD and Juan de La Cierva fellowship, respectively. M.O. is a Catalan Institution for Research and Advanced Studies Academia researcher.

The MobM relaxase (494 amino acids) encoded by the promiscuous streptococcal plasmid pMV158 recognizes the plasmid origin of transfer, oriTpMV158, and converts supercoiled pMV158 DNA into relaxed molecules by cleavage of the phosphodiester bond of a specific dinucleotide within the sequence 5'-GTGTG/TT-3' (>/> being the nick site). After cleavage, the protein remains stably bound to the 5'-end of the nick site. Band-shift assays with single-stranded oligonucleotides and size-exclusion chromatography allowed us to show that MobM was able to generate specific complexes with one of the inverted repeats of the oriTpMV158, presumably extruded as stem-loop structure. A number of tests have been performed to attain a better characterization of the nicking activity of MobM and its linkage with its target DNA. The optimal pH for DNA relaxation was found to be 6.5. Upon nicking, gel retardation assays showed that MobM formed stable complexes with its target DNA. Moreover, MobM bound to relaxed pMV158 molecules were visualized by electron microscopy. The staphylococcal plasmids pUB110 and pE194, and the streptococcal plasmid pDL287 harbour putative oriTs and may encode Mob proteins homologous to MobM. The oriTpUB110, oriTpDL287, and oriTpE194 sequences share 100%, 70%, and 67% (in a 43-nucleotide stretch and allowing a 3-bp gap) identity to oriTpMV158, respectively. Nicking assays using supercoiled DNAs from pUB110, pDL287, and pE194 showed that MobM was able to relax, to differing degrees, all plasmid DNAs. Our results suggest that cross-recognition of heterologous oriTs by Mob proteins could play an important role in the plasmid spreading between bacteria. © 2013 Elsevier Inc., Research financed by the Spanish Ministry of Economy and Competitiveness (grants CSD-2008-00013-INTERMODS and BFU2010-19597 to M.E., BFU2009-11868 to A.B., and BFU2008-02372/BMC, CONSOLIDER CSD 2006-23, and BFU2011-22588 to M.C), the Generalitat de Catalunya (SGR2009-1309 to M.C.), and the European Commission (FP7 Cooperation Project SILVER - GA No. 260644, to M.C.).

The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2120-128) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2120-128 region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2., This work was supported by the Spanish Ministry of Economy, and Competitivity (MINECO) grant BIO2013-45993R and Almirall S.A to MB, PP and BC, and by MINECO grant BIO2013– 45793R, cofinanced with structural funds (FEDER) from the European Union, to JT and MP. MP, JT, MM and GK were also supported by the European Commission’s Framework Program 7 (BioNMR, contract 261863). RB and MC were supported by MINECO grants BFU2011-22588, BFU2014-53550-P, MDM-2014-0435.

Alba is a 3rd-generation 3 GeV synchrotron facility with an emittance of 4.6nm·rad which has been operational since 2011 and has recently started top-up operation. Photons in a broad energy range of 0.08-80 keV are served to seven beamlines dedicated to a large variety of scientific fields. The portfolio includes two beamlines, XALOC and MSPD, fully dedicated to X-ray crystallography. BL13-XALOC is currently the only macromolecular crystallography beamline. The end-station includes a high-accuracy single-axis diffractometer with a removable minikappa stage, a sample-mounting robot and a large-area, photon-counting detector. The beamline optics, fed by an in-vacuum undulator, deliver a tunable photon beam between 5.5 and 22 keV. The beam size at the sample position can be adjusted by defocusing the mirrors in a range of 50-300μm in the horizontal direction and 5.5-300μm in the vertical direction. Beamline BL04-MSPD, which is fed by a superconducting wiggler, has two in-line end-stations. The first station is devoted to high-pressure/microdiffraction. It offers a $ 15\times 15$ μm beam in the range 20-50 keV, particularly suited for powder diffraction studies requiring a very small beam, e.g. mapping of cultural heritage samples and high-pressures studies. The second station is dedicated to high-resolution/high-throughput powder diffraction. It covers the 8-50 keV range and includes a heavy-duty 3-circle diffractometer equipped with a 13-channel multianalyzer detector with high-angular resolution ( $ \Delta 2\theta=0.004^{\circ}$ FWHM) and a high-throughput, position-sensitive detector spanning $ 40^{\circ}$ in 2 $ \theta$ range allowing millisecond data acquisitions.

TrwC is a DNA strand transferase that catalyzes the initial and final stages of conjugative DNA transfer. We have solved the crystal structure of the N-terminal relaxase domain of TrwC in complex with a 27 base-long DNA oligonucleotide that contains both the recognition hairpin and the scissile phosphate. In addition, a series of ternary structures of protein-DNA complexes with different divalent cations at the active site have been solved. Systematic anomalous difference analysis allowed us to determine unambiguously the nature of the metal bound. Zn2+, Ni2+ and Cu2+ were found to bind the histidine-triad metal binding site. Comparison of the structures of the different complexes suggests two pathways for the DNA to exit the active pocket. They are probably used at different steps of the conjugative DNA-processing reaction. The structural information allows us to propose (i) an enzyme mechanism where the scissile phosphate is polarized by the metal ion facilitating the nucleophilic attack of the catalytic tyrosine, and (ii) a probable sequence of events during conjugative DNA processing that explains the biological function of the relaxase. © 2006 Elsevier Ltd. All rights reserved., This study was supported by the Ministerio de Educación y Ciencia of Spain (grants BIO2002-03964, GEN2003-20642 and BFU2005-06758/BMC to M.C.; grant BMC2002-00379 to F.C.) and by the Generalitat de Catalunya (grant 2001SGR-346 to M.C.)

The crystal structure of the Δ,Δ enantiomer of the binuclear >light-switch> ruthenium complex [μ-(11,11′-bidppz)(1,10- phenanthroline)4 Ru2]4+ bound to the oligonucleotide d(CGTACG) shows that one dppz moiety of the dumbbell-like compound inserts into the DNA stack through the extrusion of an AT base pair. The second dppz moiety recruits a neighboring DNA molecule, and the complex thus cross-links two adjacent duplexes by bridging their major grooves. Pinned together by molecular rivets: The crystal structure of the complex between double-stranded DNA and the binuclear complex [μ-(11,11′-bidppz)(1,10- phenanthroline)4 Ru2]4+ (1, shown in blue; bidppz=bis(dipyridophenazine)), showed that one half of the bidppz ligand inserts into a DNA duplex, and the other half stacks onto the blunt end of a nearby DNA duplex. Two molecules of 1 with the same binding mode effectively cross-link two DNA duplexes. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., This research was supported by the Spanish Ministry of Science and Innovation (grants BFU2008-02372/BMC, CONSOLIDER CSD 2006-23, and BFU2011-22588), the Generalitat de Catalunya (grant SGR2009-1309), and the European Commission (FP7 Cooperation Project SILVER, GA No. 260644)

SuperStar is an empirical method for identifying interaction sites in proteins, based entirely on the experimental information about non-bonded interactions, present in the IsoStar database. The interaction information in IsoStar is contained in scatterplots, which show the distribution of a chosen probe around structure fragments. SuperStar breaks a template molecule (e.g. a protein binding site) into structural fragments which correspond to those in the scatterplots. The scatterplots are then superimposed on the corresponding parts of the template and converted into a composite propensity map. The original version of SuperStar was based entirely on scatterplots from the CSD. Here, scatterplots based on protein-ligand interactions are implemented in SuperStar, and validated on a test set of 122 X-ray structures of protein-ligand complexes. In this validation, propensity maps are compared with the experimentally observed positions of ligand atoms of comparable types. Although non-bonded interaction geometries in small molecule structures are similar to those found in protein-ligand complexes, their relative frequencies of occurrence are different. Polar interactions are more common in the first class of structures, while interactions between hydrophobic groups are more common in protein crystals. In general, PDB and CSD-based SuperStar maps appear equally successful in the prediction of protein-ligand interactions. PDB-based maps are more suitable to identify hydrophobic pockets, and inherently take into account the experimental uncertainties of protein atomic positions. If the protonation state of a histidine, aspartate or glutamate protein side-chain is known, specific CSD-based maps for that protonation state are preferred over PDB-based maps which represent an ensemble of protonation states.

Heats of formation of five series of diene sterol isomers were calculated and compared with synthetic yields of acid-catalysed isomerisation reactions starting from Δ5,7 isomers. Calculations were based on molecular mechanics, using the MM3 program package. For each of the five Δ5,7 starting compounds, three possible reaction paths were considered, in which heats of formation were calculated for theoretically possible intermediate double bond isomers. Similar results are found for all five series. The starting compounds are found to have the unfavourable heats of formation compared to all other isomers considered within one series. In general, isomerisation reactions of diene sterols ultimately yield spiro compounds when allowed to proceed for a sufficient amount of time. These compounds are found to have the lowest heats of formation in each series. However, they were not formed in the reactions considered in this paper, because the reactions were stopped after the desired isomer was formed in excess, before the spiro compounds could occur. Most compounds identified as products in the syntheses have a favourable heat of formation compared to the isomers preceding the (most stable) spiro compounds. However, when the reactions were carried out at low temperature, isomers with less favourable heats of formation could be trapped. We show that calculated heats of formation correspond well with synthetic yields and we suggest they can be a useful tool in planning syntheses.

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N-and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself. © 2013, American Society for Microbiology. [Journal], This work was supported by the Spanish Ministry of Science and Innovation (grants BFU2008-02372/BMC, CONSOLIDER CSD 2006-23, and BFU2011-22588 to M.C. and grants CSD2008-00013, INTERMODS, and BFU2010-19597 to M.E.), the Generalitat de Catalunya (grant SGR2009-1309 to M.C.), the “La Caixa”/IRB Barcelona International PhD Programme Fellowship (IRB Barcelona call 01/09/FLC to R.P.), and the European Commission (FP7 Cooperation Project SILVER-GA no. 260644 to M.C.)

The crystal structures of five follicular fluid meiosis-activating sterol-related Δ8,14-sterol compounds are presented. These are 4,4-di­methyl-23-phenyl-24-nor-5α-chola-8,14-dien-3β-ol, C31H44O, 4,4-di­methyl-22-phenyl-23,24-dinor-5α-chola-8,14-dien-3β-ol, C30H42O, (20R)-4,4-di­methyl-22-oxa-5α,20-chol­es­ta-8,14,24-trien-3β-ol, C28H44O2, 4,4-di­methyl-23-phenyl-22-oxa-24-nor-5α-chola-8,14-dien-3β-ol–water (4/1), 4C30H42O2·H2O, and 4,4-di­methyl-5α-cholesta-8,14-dien-3-one, C29H46O. Two of the derivatives are inactive and three are active as agonists. Preliminary structure–activity relationship studies showed that the positions of the double bonds in the skeleton and the structures of the side chains are important determinants for activity. The conformations of the skeletons were compared with double-bond isomers retrieved from the Cambridge Structural Database [Allen & Kennard (1993). Chem. Des. Autom. News, 8, 1, 31–37]; no significant differences were found. Thus, conformational changes induced by the double bonds are not discriminative with respect to the activity of the compounds. Comparisons of the side-chain conformations of active and inactive structures revealed that the crystal structures were not conclusive as far as correlation of conformation and activity of the side chains were concerned.

This research was supported by the EU Marie Curie Research Training Network (HPRN-CT-2002-00175) and by grants from the Spanish Ministerio de Ciencia y Innovación (BFU2008-02372, BFU2005-24122-E, BFU2005-24123-E, and CSD2006-00023 to M.C., BIO2006-14139 to I.U.), the Generalitat de Catalunya (grant 2009-SGR-1309 to M.C.), the Fundació La Marató de TV3 (grant 052810 to M.C.), and the EU projects SPINE2-Complexes and 3D-Repertoire. The research was conducted in the context of the EU COST Action D39. Synchrotron data collection was supported by the ESRF (ID29 and ID14-4) and the EU.

In this paper, we review recent DNA-binding agents that are expected to influence the field of DNA-targeting. We restrict ourselves to binders for which the three-dimensional structure in complex with DNA or RNA has been determined by X-ray crystallography or NMR. Furthermore, we primarily focus on unprecedented ways of targeting peculiar DNA structures, such as junctions, quadruplexes, and duplex DNAs different from the B-form. Classical binding modes of small molecular weight compounds to DNA, i.e. groove binding, intercalation and covalent addition are discussed in those cases where the structures represent a novelty. In addition, we review 3D structures of triple-stranded DNA, of the so-called Peptide Nucleic Acids (PNAs), which are oligonucleotide bases linked by a polypeptide backbone, and of aptamers, which are DNA or RNA receptors that are designed combinatorially. A discussion on perspectives in the field of DNA-targeting and on sequence recognition is also provided. © 2009 The Royal Society of Chemistry.

(Figure Presented) Three point turn: A metallosupramolecular helicate with trigonal-antiprismatic geometry fits perfectly into the central hydrophobic cavity of a three-way DNA junction allowing a new mode of DNA recognition. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA., This work was supported by an EU Marie Curie Research Training Network (HPRN-CT-2002-00175) and by grants (to M.C.) from the Ministerio de Educación y Ciencia and the Generalitat de Catalunya. M.J.H. is the Royal Society of Chemistry Sir Edward Frankland Fellow 2004-5. A.G.B. is the recipient of a fellowship from the Ministerio de Sanidad y Consumo

Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map. publishersversion published

The crystal structures of five follicular fluid meiosis-activating sterol-related Delta(8,14)-sterol compounds are presented. These are 4,4-dimethyl-23-phenyl-24-nor-5alpha-chola-8,14-dien-3beta-ol, C(31)H(44)O, 4,4-dimethyl-22-phenyl-23,24-dinor-5alpha-chola-8,14-dien-3beta-ol, C(30)H(42)O, (20R)-4,4-dimethyl-22-oxa-5alpha,20-cholesta-8,14,24-trien-3beta-ol, C(28)H(44)O(2), 4,4-dimethyl-23-phenyl-22-oxa-24-nor-5alpha-chola-8,14-dien-3beta-ol-water (4/1), 4C(30)H(42)O(2).H(2)O, and 4,4-dimethyl-5alpha-cholesta-8,14-dien-3-one, C(29)H(46)O. Two of the derivatives are inactive and three are active as agonists. Preliminary structure-activity relationship studies showed that the positions of the double bonds in the skeleton and the structures of the side chains are important determinants for activity. The conformations of the skeletons were compared with double-bond isomers retrieved from the Cambridge Structural Database [AllenKennard (1993). Chem. Des. Autom. News, 8, 1, 31-37]; no significant differences were found. Thus, conformational changes induced by the double bonds are not discriminative with respect to the activity of the compounds. Comparisons of the side-chain conformations of active and inactive structures revealed that the crystal structures were not conclusive as far as correlation of conformation and activity of the side chains were concerned.