Suchergebnisse

13 páginas, 6 figuras, 3 tablas ; Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853–RR468 and EnvZ–OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH. ; The following funding is acknowledged: Spanish Government (Ministerio de Economia y Competitividad; Grant no. BIO2016-78571-P to Alberto Marina; grant No. BFU2016-78606-P to Patricia Casino; contract No. RYC-2014-16490 to Patricia Casino); Valencian Government Prometeo program (grant No. II/2014/029 to A.M.). C.M.-M. is the recipient of a Ph.D. fellowship from the Programa de Becas, Secretaría de Educación Superior, Ciencia, Tecnología e Innovación of Ecuador Government (2015-AR2Q9228). X-ray diffraction data collection was supported by Diamond Light Source block allocation group (BAG) Proposal MX14739 and MX20229 and Spanish Synchrotron Radiation Facility ALBA Proposal 2017072262 and 2018072901. ; Peer reviewed

16 páginas, 7 figuras, 2 tablas ; Microbial colonization of different environments is enabled to a great extent by the plasticity of their sensory mechanisms, among them, the two-component signal transduction systems (TCS). Here, an example of TCS plasticity is presented: the regulation of L-malate catabolism via malic enzyme by MaeRK in Lactobacillales. MaeKR belongs to the citrate family of TCS as the Escherichia coli DcuSR system. We show that the Lactobacillus casei histidine-kinase MaeK is defective in autophosphorylation activity as it lacks a functional catalytic and ATP binding domain. The cognate response regulator MaeR was poorly phosphorylated at its phosphoacceptor Asp in vitro. This phosphorylation, however, enhanced MaeR binding in vitro to its target sites and it was required for induction of regulated genes in vivo. Elucidation of the MaeR structure revealed that response regulator dimerization is accomplished by the swapping of α4-β5-α5 elements between two monomers, generating a phosphoacceptor competent conformation. Sequence and phylogenetic analyses showed that the MaeKR peculiarities are not exclusive to L. casei as they are shared by the rest of orthologous systems of Lactobacillales. Our results reveal MaeKR as a non-canonical TCS displaying distinctive features: a swapped response regulator and a sensor histidine kinase lacking ATP-dependent kinase activity. ; This study was financed by grants AGL2007–60975 and AGL2010–15679 from the former Spanish Ministry of Science and by grant ACOMP2012/137 from Generalitat Valenciana to V.M. and M.Z., by grants BIO2013–42619-P and BIO2016–78571-P from Spanish Ministry of Economy and Competitiveness and PrometeoII/2014/029 from the Valencian Government to A.M and by grant BFU2016–78606-P from the Spanish Ministry of Economy and Competitiveness to P.C. P.C. is the recipient of a Ramon y Cajal contract, and L.M.-R. is the recipient of a FPI fellow contract, both from the Ministry of Economy and Competitiveness. J.M.L. was the recipient of a Juan de la Cierva contract from the former Spanish Ministry of Science and Innovation. X-ray diffraction data collection was supported by Diamond Light Source block allocation group (BAG) Proposal MX14739 and Spanish Synchrotron Radiation Facility ALBA Proposal 2015071314 and 2016071762. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007–2013) under BioStruct-X (grant agreement N°283570). ; Peer reviewed

17 páginas, 7 figuras, 1 tabla ; The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3-. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a β5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic. ; Spanish Government (Ministry of Economy and Competitiveness) Grants [BIO2013–42619-P, BIO2016–78571-P to A. M, BFU2016–78606-P to P.C, BIO2016–77639-P to F.G.dP]; Valencian Government Grant Prometeo [II/2014/029 to A.M.]; Ministry of Economy and Competitiveness Ramón y Cajal contract (to P.C.); Diamond Light Source block allocation group (BAG) Proposal [MX10121]; Spanish Synchrotron Radiation Facility ALBA Proposal [2015071314]. Funding for open access charge: Spanish Government (Ministry of Economy and Competitiveness) Grants [BIO2013-42619-P, BIO2016-78571-P to A. M, BIO2016-77639-P to F.G.dP, BFU2016-78606-P to P.C.]; Valencian Government grant Prometeo [II/2014/029 to A.M.]. ; Peer reviewed

RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription. ; Spanish Government (Ministerio de Economía y Competitividad y Ministerio de Ciencia e Innovación) [BIO2016-78571-P, PID2019-108541GB-I00 to A.M., BFU2016-78606-P, PID2019-110630GB-I00 to P.C., BIO2016-77639-P to F.G.-dP.]; Valencian Government grant PROMETEO/2020/012 to A.M. P.C.

18 páginas, 7 figuras, 2 tablas ; RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription. ; Spanish Government (Ministerio de Econom´ıa y Competitividad y Ministerio de Ciencia e Innovacion) ´ [BIO2016-78571-P, PID2019-108541GB-I00 to A.M., BFU2016-78606-P, PID2019-110630GB-I00 to P.C., BIO2016-77639-P to F.G.-dP.]; Valencian Government grant PROMETEO/2020/012 to A.M. P.C. is recipient of a Ramon y Cajal contract from the Ministry of Economy and ´ Competitiveness. Funding for open access charge: Spanish government. ; Peer reviewed

15 páginas, 6 figuras, 1 tabla. ; Basement membranes are extracellular structures of epithelia and endothelia that have collagen IV scaffolds of triple α-chain helical protomers that associate end-to-end, forming networks. The molecular mechanisms by which the noncollagenous C-terminal domains of α-chains direct the selection and assembly of the α1α2α1 and α3α4α5 hetero-oligomers found in vivo remain obscure. Autoantibodies against the noncollagenous domains of the α3α4α5 hexamer or mutations therein cause Goodpasture's or Alport's syndromes, respectively. To gain further insight into oligomer-assembly mechanisms as well as into Goodpasture's and Alport's syndromes, crystal structures of non-collagenous domains produced by recombinant methods were determined. The spontaneous formation of canonical homohexamers (dimers of trimers) of these domains of the α1, α3 and α5 chains was shown and the components of the Goodpasture's disease epitopes were viewed. Crystal structures of the α2 and α4 non-collagenous domains generated by recombinant methods were also determined. These domains spontaneously form homo-oligomers that deviate from the canonical architectures since they have a higher number of subunits (dimers of tetramers and of hexamers, respectively). Six flexible structural motifs largely explain the architectural variations. These findings provide insight into noncollagenous domain folding, while supporting the in vivo operation of extrinsic mechanisms for restricting the self-assembly of noncollagenous domains. Intriguingly, Alport's syndrome missense mutations concentrate within the core that nucleates the folding of the noncollagenous domain, suggesting that this syndrome, when owing to missense changes, is a folding disorder that is potentially amenable to pharmacochaperone therapy. ; The following funding is acknowledged: Spanish Government (Ministerio de Economia y Competitividad; grant Nos. BIO2013-42619 and BIO2016-78571-P to Alberto Marina; grant Nos. SAF 2009-10703, IPT-2011-1527-010000 and RTC2014-2415-1 to Juan Saus; grant No. BFU2016-78606-P to Patricia Casino; grant No. BFU2014-58229-P to Vicente Rubio; contract No. RYC-2014-16490 to Patricia Casino); Valencian Government Prometeo program (grant No. II/2014/029 to Alberto Marina, Vicente Rubio and Juan Saus); National Institutes of Health (grant NIH-DK18381 to Billy G. Hudson); European Community's Seventh Framework Programme FP7/2007-2013 (BioStruct-X; grant No. 283570); and a visiting researcher grant from the program 'Atraccio´ de Talent' of the University of Valencia to Billy G. Hudson. ; Peer reviewed