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Introduction In this chapter, we explore how changing political visions, socio-cultural imaginaries and hydro-territorial configurations interact with shifting practices of water justice. In Plato’s Republic, Socrates comments that justice is what those in power consider just. Over the centuries, this statement has haunted any discussions and efforts to create a fairer society. Recent social-justice debate has extended to include the physical world as an integral component in structuring just/unjust socio-ecological relations. This chapter examines how hydro-territorial politics finds expression in the diverse actors’ confluences and encounters with spatial and political-geographical projects that compete, superimpose and align their territorialization strategies to strengthen their governance positions, ideologies and water-control claims. This continuously transforms the territory’s hydraulic grid, cultural reference frames, economic base structures and political relationships. Territorial struggles go beyond battles over natural resources per se, as they also involve conflicts over meaning, norms, knowledge, decision-making authority, representations and discourses. Policy actors commonly tend to present socio-natural, geopolitical territories as mere biophysical “nature” or legal-administrative “governance units,” portraying water problems and solutions as politically neutral, technical and managerial issues to be objectively managed through “rational water use” and “good governance” - a conscious or unconscious veil to legitimize deeply political choices sustaining specific political orders (Harris, 2009; Hommes et al., 2016; Perreault, 2014). Challenging such powerful conventions, we examine the contradictions, conflicts and societal responses generated by the configuration of hydrosocial territories (see Boelens et al., 2016; Swyngedouw and Williams, 2016); how water politics are ingrained in such socio-natural and techno-political arrangements, enhancing or challenging unequal distribution of resources and decision-making power in water governance (Boelens, 2015; Swyngedouw, 2015). Therefore, hydrosocial territories (imagined, planned or materialized) have contested functions, values and meanings as they define processes of inclusion and exclusion, development and marginalization, and the distribution of benefits and burdens that affect different groups of people in distinct but often deeply unequal manners. Taking the co-production of “nature and society” in twentieth-century Spain as our entry point, we seek to elucidate the relationship among transformations in and of “hydrosocial territory,” the state, and the contested modernization, and to tease out the multiple power relationships that enroll, transform and distribute water. In doing so, we seek to excavate how nature becomes political and, through this, how environmental reconfiguration parallels ongoing state transformation (Swyngedouw, 2014; cf. Carroll, 2012; Perreault et al., 2015).

GH (growth hormone) binding to the GHR (GH receptor) triggers essential signalling pathways that promote growth and metabolic regulation. The sensitivity of the cells to GH is mainly controlled by the endocytosis of the receptor via βTrCP (β-transducin repeat-containing protein). In the present study, we show that βTrCP interacts directly via its WD40 domain with the UbE (ubiquitin-dependent endocytosis) motif in GHR, promoting GHR ubiquitination in vitro. NMR experiments demonstrated that the UbE motif is essentially unstructured, and, together with functional mapping of the UbE and βTrCP WD40 residues necessary for binding, led to a unique interaction model of βTrCP with GHR–UbE. This interaction is different from the conventional βTrCP–substrate interactions described to date. This interaction therefore represents a promising specific target to develop drugs that inhibit GHR endocytosis and increase GH sensitivity in cachexia patients.

Structure determination of protein-nucleic acid complexes remains a challenging task. Here we present a simple method for generating crystals of a CsrA-nucleic acid complex, guided entirely by results from nuclear magnetic resonances spectroscopy (NMR) spectroscopy. Using a construct that lacks thirteen non-essential C-terminal residues, efficient binding to DNA could be demonstrated. One CsrA dimer interacts with two DNA oligonucleotides, similar to previous findings with RNA. Furthermore, the NMR study of the CsrA-DNA complex was the basis for successfully homing in on conditions that were suitable for obtaining crystals of the CsrA-DNA complex. Our results may be useful for those cases where RNA in protein-nucleic acid complexes may be replaced by DNA.

International audience; The glycopeptide CcTx, isolated from the venom of the piscivorous cone snail Conus consors, belongs to the κA-family of conopeptides. These toxins elicit excitotoxic responses in the prey by acting on voltage-gated sodium channels. The structure of CcTx, a first in the κA-family, has been determined by high-resolution NMR spectroscopy together with the analysis of its O-glycan at Ser7. A new type of glycopeptide O-glycan core structure, here registered as core type 9, containing two terminal L-galactose units {α-L-Galp-(1→4)-α-D-GlcpNAc-(1→6)-[α-L-Galp-(1→2)-β-D-Galp-(1→3)-]α-D-GalpNAc-(1→O)}, is highlighted. A sequence comparison to other putative members of the κA-family suggests that O-linked glycosylation might be more common than previously thought. This observation alone underlines the requirement for more careful and in-depth investigations into this type of post-translational modification in conotoxins.

We summarize the use of NMR spectroscopy in the production and the screening of stability and foldedness of protein domains, and apply it to the RING domains of E3 ubiquitin-ligases. RING domains are involved in specific interactions with E2 ubiquitin-conjugating enzymes and thus play an essential role in the ubiquitination pathway. Protein production of the Zn(2+) containing and cysteine rich RING domains for molecular studies frequently turns out to be problematic. We compared the expression and solubility of 14 E3 RING/U-box domains fused to the N-terminal tags of His(6), His(6)-GB1, His(6)-Trx and His(6)-GST at small scale and analyzed, by NMR spectroscopy, their correct folding after purification. The addition of GST, Trx or GB1 to the N-terminal His(6) tag significantly improved both the expression and solubility of target proteins as compared to His(6) tag alone. More importantly most of the immobilized metal affinity chromatography (IMAC) purified proteins were largely unfolded as judged by analysis of the (1)H-(15)N HSQC spectra. We demonstrate that imidazole causes a concentration dependent decrease in stability of RING proteins ascribed to metal depletion and resulting in unfolding or precipitation. In contrast, using glutathione affinity chromatography, the His(6)-GST fused RING and U-box domains were purified as correctly folded proteins with high yields. Our data clearly demonstrate that IMAC should be avoided and that GST-fusion affinity chromatography is generally applicable for expression and purification of Zn(2+) containing proteins.

Recent technological advances in NMR spectroscopy have alleviated the size limitations for the determination of biomolecular structures in solution. At the same time, novel NMR parameters such as residual dipolar couplings are providing greater accuracy. As this review shows, the structures of protein-protein and protein-nucleic acid complexes up to 50 kDa can now be accurately determined. Although de novo structure determination still requires considerable effort, information on interaction surfaces from chemical shift perturbations is much easier to obtain. Advances in modelling and data-driven docking procedures allow this information to be used for determining approximate structures of biomolecular complexes. As a result, a wealth of information has become available on the way in which proteins interact with other biomolecules. Of particular interest is the fact that these NMR-based methods can be applied to weak and transient protein-protein complexes that are difficult to study by other structural methods.

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

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SummaryTet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

Heterogeneous mixtures of collagen fragments can be used as nutrition supplement or as key ingredients for ointments with therapeutic relevance in wound healing. Some mixtures of collagen fragments are referred to as collagen hydrolysates owing to the production process with hydrolytic enzymes. Since the precise composition of collagen hydrolysates is generally unknown, it is of interest to analyze samples containing various collagen fragments with appropriate biophysical methods. Any product optimization without a profound knowledge concerning the size and the molecular weight distribution of its components is nearly impossible. It turned out that a combination of AFM methods with NMR techniques is exceptionally suited to examine the size range and the aggregation behavior of the collagen fragments in the hydrolysates of fish, jellyfish, chicken, porcine and bovine collagen. Supported by molecular modeling calculations, the AFM and NMR experiments provide a detailed knowledge about the composition of collagen hydrolysates and collagen ointments. Furthermore, the data allow a correlation between the size of the fragments and their potential bioactivity.

Control of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of structural proteomics on our understanding of the molecular basis of gene expression. While most atomic structures were obtained by X-ray crystallography, the impact of solution NMR and cryo-electron microscopy is far from being negligible. Here, we summarize some highlights and illustrate the importance of specific technologies on the structural biology of protein-protein or protein/DNA transcription complexes: structure/function analysis of components the eukaryotic basal and activated transcription machinery with focus on the TFIID and TFIIH multi-subunit complexes as well as transcription regulators such as members of the nuclear hormone receptor families. We also discuss molecular aspects of promoter recognition and epigenetic control of gene expression. © 2011 Elsevier Inc., This work forms part of SPINE2-complexes Contract No. LSHG-CT-2006-031220, funded by the European Commission under the Integrated Programme ‘Quality of Life and Management of Living Resources’. This work was also supported by grants from the EC FP7 IS project P-CUBE (to I.B.), from the Spanish ‘Ministerio de Ciencia e Innovación’ (Grants BFU2008-02372/BMC and CSD2006-00023) and the ‘Generalitat de Catalunya’ (Grant 2009SGR-1309) (to A.B. and M.C.), from the Netherlands Organisation for Scientific Research, Division of Chemical Sciences, NWO-CW (to R.B.) and from the Agence Nationale de la Recherche, the Association de la Recherche sur le Cancer and the Institut National du Cancer (to J.C., P.S, D.M. and A.P.).

Despite recent advances in the modeling of protein-protein complexes by docking, additional information is often required to identify the best solutions. For this purpose, NMR data deliver valuable restraints that can be used in the sampling and/or the scoring stage, like in the data-driven docking approach HADDOCK that can make use of NMR chemical shift perturbation (CSP) data to define the binding site on each protein and drive the docking. We show here that a quantitative use of chemical shifts (CS) in the scoring stage can help to resolve ambiguities. A quantitative CS-RMSD score based on 1Hα,13Cα, and 15N chemical shifts ranks the best solutions always at the top, as demonstrated on a small benchmark of four complexes. It is implemented in a new docking protocol, CS-HADDOCK, which combines CSP data as ambiguous interaction restraints in the sampling stage with the CS-RMSD score in the final scoring stage. This combination of qualitative and quantitative use of chemical shifts increases the reliability of data-driven docking for the structure determination of complexes from limited NMR data. Proteins 2011; © 2011 Wiley-Liss, Inc.

Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2-E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2-E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2-E3 interactions of the human ubiquitination system.

14 páginas, 5 figuras, 2 tablas -- PAGS nros. 4973-4986, Kid, the toxin of the parD (kis, kid) maintenance system of plasmid R1, is an endoribonuclease that preferentially cleaves RNA at the 5′ of A in the core sequence 5′-UA(A/C)-3′. A model of the Kid toxin interacting with the uncleavable mimetic 5′-AdUACA-3′ is available. To evaluate this model, a significant collection of mutants in some of the key residues proposed to be involved in RNA binding (T46, A55, T69 and R85) or RNA cleavage (R73, D75 and H17) were analysed by mass spectrometry in RNA binding and cleavage assays. A pair of substrates, 5′-AUACA-3′, and its uncleavable mimetic 5′-AdUACA-3′, used to establish the model and structure of the Kid–RNA complex, were used in both the RNA cleavage and binding assays. A second RNA substrate, 5′-UUACU-3′ efficiently cleaved by Kid both in vivo and in vitro, was also used in the cleavage assays. Compared with the wild-type protein, mutations in the residues of the catalytic site abolished RNA cleavage without substantially altering RNA binding. Mutations in residues proposed to be involved in RNA binding show reduced binding efficiency and a corresponding decrease in RNA cleavage efficiency. The cleavage profiles of the different mutants were similar with the two substrates used, but RNA cleavage required much lower protein concentrations when the 5′-UUACU-3′ substrate was used. Protein synthesis and growth assays are consistent with there being a correlation between the RNase activity of Kid and its inhibitory potential. These results give important support to the available models of Kid RNase and the Kid–RNA complex, JRDO was supported by Project BFU2005-03911 from the Spanish Ministry of Education and Science (MEC, Spain), BFU 2008-01566/BMC and CSD2008-00013 from the Ministry of Science and Innovation (MICIIN, Spain) and by a networking project of the CM (COMBACT, Comunidad de Madrid, Spain). EDN acknowledges the contribution of a predoctoral fellowship (BFI05.35) from the Basque Country Government, Spain and of a short term EMBO fellowship (ASTF No: 159-06) to visit and work at the Biomolecular Mass Spectrometry and Proteomics group at Utrecht University, the Netherlands. The technical assistance of Alicia Rodriguez-Bernabé and discussions with Marc Lemonnier, Ana María Hernandez-Arriaga and Juan López-Villarejo, are kindly acknowledged. RB, AJRH, and MBK acknowledge support from the Netherlands Organization for Chemical Research (NWO/CW) and the Center for Biomedical Genetics. RHH vdH was supported by a VENI fellowship (700.54.402) from The Netherlands Organization for Scientific Research (NWO). This work in Utrecht was also supported by the Netherlands Proteomics Centre

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2–E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5BUb thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.

The structures of a dimeric mutant of the Lac repressor DNA-binding domain complexed with the auxiliary operators O2 and O3 have been determined using NMR spectroscopy and compared to the structures of the previously determined Lac–O1 and Lac–nonoperator complexes. Structural analysis of the Lac–O1 and Lac–O2 complexes shows highly similar structures with very similar numbers of specific and nonspecific contacts, in agreement with similar affinities for these two operators. The left monomer of the Lac repressor in the Lac–O3 complex retains most of these specific contacts. However, in the right half-site of the O3 operator, there is a significant loss of protein–DNA contacts, explaining the low affinity of the Lac repressor for the O3 operator. The binding mode in the right half-site resembles that of the nonspecific complex. In contrast to the Lac–nonoperator DNA complex where no hinge helices are formed, the stability of the hinge helices in the weak Lac–O3 complex is the same as in the Lac–O1 and Lac–O2 complexes, as judged from the results of hydrogen/deuterium experiments.

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2–E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5BUb thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCGt/aTa/tAATT) and, alone or fused to the protein oligomerization domain of phage λ CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::λCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::λCI and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8/10) to the H-NS binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr109, Arg113, Thr114 and Ala116) belonging to the same protein loop and Glu101. The DNA target is recognized by virtue of its sequence and of a TpA step which confers a structural irregularity to the B-DNA duplex.

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCGt/aTa/tAATT) and, alone or fused to the protein oligomerization domain of phage λ CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::λCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::λCI and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8/10) to the H-NS binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr109, Arg113, Thr114 and Ala116) belonging to the same protein loop and Glu101. The DNA target is recognized by virtue of its sequence and of a TpA step which confers a structural irregularity to the B-DNA duplex.

NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D20, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.

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Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-π interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 “methyl-methyl” switch in the histone code dynamically regulates TFIID-promoter association.

Epac1 is a cAMP-responsive exchange factor for the small G-protein Rap. It consists of a regulatory region containing a cyclic nucleotide binding (CNB) domain and a catalytic region that activates Rap. In the absence of cAMP, access of Rap to the catalytic site is blocked by the regulatory region. We analyzed the conformational states of the CNB domain in the absence and in the presence of cAMP and cAMP analogues by NMR spectroscopy, resulting in the first direct insights into the activation mechanism of Epac. We prove that the CNB domain exists in equilibrium between the inactive and the active conformation, which is shifted by binding of cAMP. cAMP binding results in conformational changes in both the ligand binding pocket and the outer helical segments. We used two different cAMP antagonists that block these successive changes to elucidate the steps of this process. Highlighting the role of dynamics, the superactivator 8-pCPT-2'-O-Me-cAMP induces similar conformational changes as cAMP but causes different internal mobility. The results reveal the critical elements of the CNB domain of Epac required for activation and highlight the role of dynamics in this process.

In glycoanalysis protocols, N-glycans from glycoproteins are most frequently released with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F (PNGase F). As the enzyme is an amidase, it cleaves the NH−CO linkage between the Asn side chain and the Asn-bound GlcNAc residue. Usually, the enzyme has a low activity, or is not active at all, on native glycoproteins. A typical example is native bovine pancreatic ribonuclease B (RNase B) with oligomannose-type N-glycans at Asn-34. However, native RNase BS, generated by subtilisin digestion of native RNase B, which comprises amino acid residues 21–124 of RNase B, is sensitive to PNGase F digestion. The same holds for carboxymethylated RNase B (RNase Bcm). In this study, NMR spectroscopy and molecular modeling have been used to explain the differences in PNGase F activity for native RNase B, native RNase BS, and RNase Bcm. NMR analysis combined with literature data clearly indicated that the N-glycan at Asn-34 is more mobile in RNase BS than in RNase B. MD simulations showed that the region around Asn-34 in RNase B is not very flexible, whereby the α-helix of the amino acid residues 1–20 has a stabilizing effect. In RNase BS, the α-helix formed by amino acid residues 23–32 is significantly more flexible. Using these data, the possibilities for complex formation of both RNase B and RNase BS with PNGase F were studied, and a model for the RNase BS−PNGase F complex is proposed.

Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-π interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 “methyl-methyl” switch in the histone code dynamically regulates TFIID-promoter association.

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon.

Human ERCC1/XPF is a structure-specific endonuclease involved in multiple DNA repair pathways. We present the solution structure of the non-catalytic ERCC1 central domain. Although this domain shows structural homology with the catalytically active XPF nuclease domain, functional investigation reveals a completely distinct function for the ERCC1 central domain by performing interactions with both XPA and single-stranded DNA. These interactions are non-competitive and can occur simultaneously through distinct interaction surfaces. Interestingly, the XPA binding by ERCC1 and the catalytic function of XPF are dependent on a structurally homologous region of the two proteins. Although these regions are strictly conserved in each protein family, amino acid composition and surface characteristics are distinct. We discuss the possibility that after XPF gene duplication, the redundant ERCC1 central domain acquired novel functions, thereby increasing the fidelity of eukaryotic DNA repair.

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon.

BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensing receptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which are in close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides, is investigated through the introduction of several amino acid substitutions at these positions. NMR spectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was not significantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorption spectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In the Y21 mutants, FADH¥-W¥ radical pairs are transiently formed on a ps time scale and recombine to the ground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wild type AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant, all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves by intersystem crossing to the triplet state. Our results indicate that two competing light-driven electrontransfer pathways are available in BLUF domains: one productive pathway that involves electron transfer from the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathway from the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of the signaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from the conserved tyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.

The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here,we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid2-Kis2 tetramer up to Kis2-Kid2-Kis2-Kid2-Kis2 decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid2-Kis2-Kid2 heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the 1H 15N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF2-MazE2-MazF2 complex. Furthermore, we demonstrate that Kid2-MazE2 tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed.

The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here,we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid2-Kis2 tetramer up to Kis2-Kid2-Kis2-Kid2-Kis2 decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid2-Kis2-Kid2 heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the 1H 15N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF2-MazE2-MazF2 complex. Furthermore, we demonstrate that Kid2-MazE2 tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed.

We present a method that significantly enhances the robustness of (automated) NMR structure determination by allowing the NOE data corresponding to unassigned NMR resonances to be used directly in the calculations. The unassigned resonances are represented by additional atoms or groups of atoms that have no interaction with the regular protein atoms except through distance restraints. These so-called "proxy" residues can be used to generate NOE-based distance restraints in a similar fashion as for the assigned part of the protein. If sufficient NOE information is available, the restraints are expected to place the proxies at positions close to the correct atoms for the unassigned resonance, which can facilitate subsequent assignment. Convergence can be further improved by supplying additional information about the possible identities of the unassigned resonances. We have implemented this approach in the widely used automated assignment and structure calculation protocols ARIA and CANDID. We find that it significantly increases the robustness of structure calculations with regard to missing assignments and yields structures of higher quality. Our approach is still able to find correctly folded structures with up to 30% randomly missing resonance assignments, and even when only backbone and beta resonances are present! This should be of significant value to NMR-based structural proteomics initiatives.

Intrinsic flexibility of DNA has hampered the devel- opment of efficient proteinDNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) (C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731-1737) to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy mini- mization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressorDNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we cor- rectly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre- bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA con- formation. Our two-stage docking method is thus able to successfully predict proteinDNA com- plexes from unbound constituents using non- structural experimental data to drive the docking.

Proteins frequently contain unstructured regions apart from a functionally important and wellconserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, nuclear magnetic resonance (NMR), and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd.

Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3′,5′-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.

The AppA BLUF domain is a blue light photoreceptor containing flavin. Conserved glutamine 63 is necessary for the photocycle of the protein, and its side chain has been proposed to flip in response to blue light illumination. Recently published crystal structures of AppA WT and the AppA mutant C20S describe contradictory conclusions regarding the orientation of the conserved glutamine 63 side chain in the dark. Here, we present evidence from NMR spectroscopy confirming light-induced flipping of the glutamine side chain to form a strong hydrogen bond between the glutamine 63 side chain carbonyl group and the tyrosine 21 side chain hydroxyl proton in the light-induced state. Our conclusions are consistent with published data from UV/vis absorbance and FTIR spectroscopy, as well as the crystal structure of AppA WT.

Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors. In this paper, we report on the solution structure of CSPsg4, a chemosensory protein from the desert locust Schistocerca gregaria, which is expressed in the antennae and other chemosensory organs. The 3D NMR structure revealed an overall fold consisting of six α-helices, spanning residues 13−18, 20−31, 40−54, 62−78, 80−90, and 97−103, connected by loops which in some cases show dihedral angles typical of β-turns. As in the only other chemosensory protein whose structure has been solved so far, namely, CSP from the moth Mamestra brassicae, four helices are arranged to form a V-shaped motif; another helix runs across the two V's, and the last one is packed against the external face. Analysis of the tertiary structure evidenced multiple hydrophobic cavities which could be involved in ligand binding. In fact, incubation of the protein with a natural ligand, namely, oleamide, produced substantial changes to the NMR spectra, suggesting extensive conformational transitions upon ligand binding.