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The flagellar motor is a sophisticated rotary machine facilitating locomotion and signal transduction. Owing to its important role in bacterial behavior, its assembly and activity are tightly regulated. For example, chemotaxis relies on a sensory pathway coupling chemical information to rotational bias of the motor through phosphorylation of the motor switch protein CheY. Using a chemical proteomics approach, we identified a novel family of CheY-like (Cle) proteins in Caulobacter crescentus, which tune flagellar activity in response to binding of the second messenger c-di-GMP to a C-terminal extension. In their c-di-GMP bound conformation Cle proteins interact with the flagellar switch to control motor activity. We show that individual Cle proteins have adopted discrete cellular functions by interfering with chemotaxis and by promoting rapid surface attachment of motile cells. This study broadens the regulatory versatility of bacterial motors and unfolds mechanisms that tie motor activity to mechanical cues and bacterial surface adaptation.

In major histocompatibility complex (MHC) class I molecules, monomorphic β 2 -microglobulin (β 2 m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. β 2 M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed β 2 m and to understand the evolution of the interfaces between β 2 m and different HC. Using human β 2 m and the HLA–B*27:09 complex as a model system, a comparison of free and HC-bound β 2 m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of β 2 m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved β 2 m–HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that β 2 m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.

The bacterial second messenger cyclic diguanylate (c-di-GMP) regulates a wide range of cellular functions from biofilm formation to growth and survival. Targeting a second-messenger network is challenging because the system involves a multitude of components with often overlapping functions. Here, we present a strategy to intercept c-di-GMP signaling pathways by directly targeting the second messenger. For this, we developed a c-di-GMP–sequestering peptide (CSP) that was derived from a CheY-like c-di-GMP effector protein. CSP binds c-di-GMP with submicromolar affinity. The elucidation of the CSP·c-di-GMP complex structure by NMR identified a linear c-di-GMP–binding motif, in which a selfintercalated c-di-GMP dimer is tightly bound by a network of H bonds and π-stacking interactions involving arginine and aromatic residues. Structure-based mutagenesis yielded a variant with considerably higher, low-nanomolar affinity, which subsequently was shortened to 19 residues with almost uncompromised affinity. We demonstrate that endogenously expressed CSP intercepts c-di-GMP signaling and effectively inhibits biofilm formation in Pseudomonas aeruginosa, the most widely used model for serious biofilm-associated medical implications.

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β(2)-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.

Abstract In this study, three single nucleotide polymorphisms (SNPs) located within the promoter of the human interleukin (IL)-10 gene [rs1800896 (position: −1087G > A), rs1800871 (position: −824C > T) and rs1800872 (position: −597C > A)] were investigated in 84 rheumatoid arthritis (RA) patients and 95 age- and sex-matched healthy subjects using polymerase chain reaction-restriction fragment length polymorphism method. Production of IL-10 by peripheral blood lymphocytes from the RA patients and healthy subjects cultured in the presence of Concanavalin A (Con A) was determined by using enzyme-linked immunosorbent assay. The results show that the distribution of the IL-10 genotypes did not differ significantly between RA patients and healthy subjects (P > 0.05). However, a significant difference was observed in allele frequencies of −824CT, −824TT, −597CA, and −597AA between the RA patients and healthy volunteers (P = 0.04). The −1087A/−824T/−597A (ATA) haplotype, which comprises all mutant alleles, was associated with lower IL-10 production when compared with the other haplotypes. In contrast, the RA patients who did not display the ATA haplotype produced significantly higher levels of IL-10 when compared with those carrying either one (P = 0.012) or two (P = 0.005) ATA haplotypes. Our findings suggest that there is an association between SNPs in the promoter of the human IL-10 gene and susceptibility to RA.

In major histocompatibility complex (MHC) class I molecules, monomorphic [beta]^sub 2^-microglobulin ([beta]^sub 2^m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. [beta]^sub 2^M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed [beta]^sub 2^m and to understand the evolution of the interfaces between [beta]^sub 2^m and different HC. Using human [beta]^sub 2^m and the HLAâ[euro]\"B*27:09 complex as a model system, a comparison of free and HC-bound [beta]^sub 2^m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of [beta]^sub 2^m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved [beta]^sub 2^mâ[euro]\"HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that [beta]^sub 2^m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.[PUBLICATION ABSTRACT]

In major histocompatibility complex (MHC) class I molecules, monomorphic beta sub(2)-microglobulin ( beta sub(2)m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. beta sub(2)M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed beta sub(2)m and to understand the evolution of the interfaces between beta sub(2)m and different HC. Using human beta sub(2)m and the HLA-B*27:09 complex as a model system, a comparison of free and HC-bound beta sub(2)m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of beta sub(2)m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved beta sub(2)m-HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that beta sub(2)m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β2-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.

In this study, three single nucleotide polymorphisms (SNPs) located within the promoter of the human interleukin (IL)-10 gene [rs1800896 (position: -1087G > A), rs1800871 (position: -824C > T) and rs1800872 (position: -597C > A)] were investigated in 84 rheumatoid arthritis (RA) patients and 95 age- and sex-matched healthy subjects using polymerase chain reaction-restriction fragment length polymorphism method. Production of IL-10 by peripheral blood lymphocytes from the RA patients and healthy subjects cultured in the presence of Concanavalin A (Con A) was determined by using enzyme-linked immunosorbent assay. The results show that the distribution of the IL-10 genotypes did not differ significantly between RA patients and healthy subjects (P > 0.05). However, a significant difference was observed in allele frequencies of -824CT, -824TT, -597CA, and -597AA between the RA patients and healthy volunteers (P = 0.04). The -1087A/-824T/-597A (ATA) haplotype, which comprises all mutant alleles, was associated with lower IL-10 production when compared with the other haplotypes. In contrast, the RA patients who did not display the ATA haplotype produced significantly higher levels of IL-10 when compared with those carrying either one (P = 0.012) or two (P = 0.005) ATA haplotypes. Our findings suggest that there is an association between SNPs in the promoter of the human IL-10 gene and susceptibility to RA. [PUBLICATION ABSTRACT]

In this study, three single nucleotide polymorphisms (SNPs) located within the promoter of the human interleukin (IL)-10 gene [rs1800896 (position: -1087G>A), rs1800871 (position: -824C>T) and rs1800872 (position: -597C>A)] were investigated in 84 rheumatoid arthritis (RA) patients and 95 age- and sex-matched healthy subjects using polymerase chain reaction-restriction fragment length polymorphism method. Production of IL-10 by peripheral blood lymphocytes from the RA patients and healthy subjects cultured in the presence of Concanavalin A (Con A) was determined by using enzyme-linked immunosorbent assay. The results show that the distribution of the IL-10 genotypes did not differ significantly between RA patients and healthy subjects (P>0.05). However, a significant difference was observed in allele frequencies of -824CT, -824TT, -597CA, and -597AA between the RA patients and healthy volunteers (P=0.04). The -1087A/-824T/-597A (ATA) haplotype, which comprises all mutant alleles, was associated with lower IL-10 production when compared with the other haplotypes. In contrast, the RA patients who did not display the ATA haplotype produced significantly higher levels of IL-10 when compared with those carrying either one (P=0.012) or two (P=0.005) ATA haplotypes. Our findings suggest that there is an association between SNPs in the promoter of the human IL-10 gene and susceptibility to RA.