The N terminus of Myxococcus xanthus CarA repressor is an autonomously folding domain that mediates physical and functional interactions with both operator DNA and antirepressor protein

Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.     

Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA

In the bacterium Myxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of the crt carotenoid biosynthesis genes. The majority of crt genes are clustered in the crtEBDC operon, which is repressed in the dark by CarA. Genetic data suggest that, in the light, CarS is synthesized and achieves activation of the crtEBDC operon by removing the repressive action of CarA. As CarS contains no known DNA-binding motif, the relief of CarA-mediated repression was postulated to result from a direct interaction between these two proteins. Use of the yeast two-hybrid system demonstrated direct interaction between CarA and CarS. The two-hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization. Direct evidence for CarS anti-repressor action was provided in vitro. A glutathione S-transferase (GST)-CarA protein fusion was shown to bind specifically to a palindromic operator sequence within the crtEBDC promoter. CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS. CarS is therefore an anti-repressor.     

Structural basis for operator and antirepressor recognition by Myxococcus xanthus CarA repressor

Blue light induces carotenogenesis in Myxococcus xanthus. The carB operon encodes all but one of the structural genes involved, and its expression is regulated by the CarA-CarS repressor-antirepressor pair. In the dark, CarA-operator binding represses carB. CarS, produced on illumination, interacts physically with CarA to dismantle the CarA-operator complex and activate carB. Both operator and CarS bind to the autonomously folded N-terminal domain of CarA, CarA(Nter), which in excess represses carB. Here, we report the NMR structure of CarA(Nter), and map residues that interact with operator and CarS by NMR chemical shift perturbations, and in vivo and in vitro analyses of site-directed mutants. We show CarA(Nter) adopts the winged-helix topology of MerR-family DNA-binding domains, and conserves the majority of the helix-turn-helix and wing contacts with DNA. Tellingly, helix alpha2 in CarA, a key element in operator DNA recognition, is also critical for interaction with CarS, implying that the CarA-CarS protein-protein and the CarA-operator protein-DNA interfaces overlap. Thus, binding of CarA to operator and to antirepressor are mutually exclusive, and CarA may discern structural features in the acidic CarS protein that resemble operator DNA. Repressor inactivation by occluding the DNA-binding region may be a recurrent mechanism of action for acidic antirepressors.     

1H, 13C and 15N backbone and side chain resonance assignments of a Myxococcus xanthus anti-repressor with no known sequence homologues

The CarS antirepressor activates a photo-inducible promoter in Myxococcus xanthus by physically interacting with the CarA repressor and eliminating the latter’s binding to operator DNA. Interestingly, interactions with both CarS and operator are crucially dependent on the DNA recognition helix of the CarA winged-helix DNA-binding domain. The CarA–CarS and the CarA-operator interfaces therefore overlap, and CarS may have structural features that mimic operator DNA. CarS has no known sequence homologues and its Gly and Pro contents are unusually high. Here, we report ¹H, ¹³C and ¹⁵N backbone and side chain assignments of CarS1, an 86-residue truncated yet fully functional variant of CarS. Secondary structural elements inferred from these data differ from those predicted from sequence.     

Coenzyme B12-dependent and independent photoregulation of carotenogenesis across Myxococcales

Light-induced carotenogenesis in Myxococcus xanthus is controlled by the B₁₂-based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B₁₂-independent CarH paralogue CarA and various other proteins, some eukaryotic-like. Whether other myxobacteria conserve these pathways and undergo photoregulated carotenogenesis is unknown. Here, comparative analyses across 27 Myxococcales genomes identified carotenogenic genes, albeit arranged differently, with carH often in their genomic vicinity, in all three Myxococcales suborders. However, CarA and its associated factors were found exclusively in suborder Cystobacterineae, with carA-carH invariably in tandem in a syntenic carotenogenic operon, except for Cystobacter/Melittangium, which lack CarA but retain all other factors. We experimentally show B₁₂-mediated photoregulated carotenogenesis in representative myxobacteria, and a remarkably plastic CarH operator design and DNA binding across Myxococcales. Unlike the two characterized CarH from other phyla, which are tetrameric, Cystobacter CarH (the first myxobacterial homologue amenable to analysis in vitro) is a dimer that combines direct CarH-like B₁₂-based photoregulation with CarA-like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B₁₂-dependent photoreceptors. It further establishes the B₁₂-dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B₁₂-independent circuit.     

The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences.

The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state. To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites. Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences. Sequence alignment with 186-like repressors predicts an N-terminal helix-turn-helix (HTH) motif, albeit with poor homology to a large master set of such motifs. The domain structure of CI was investigated by linker insertion mutagenesis and limited proteolysis. CI consists of an N-terminal domain, which weakly dimerizes and binds both A and B type sequences, and a C-terminal domain, which associates to octamers but is unable to bind DNA. A fusion protein consisting of the 186 N-terminal domain and the phage lambda oligomerization domain binds A and B type sequences more efficiently than the isolated 186 CI N-terminal domain, hence the 186 C-terminal domain likely mediates oligomerization and cooperativity. Site-directed mutation of the putative 186 HTH motif eliminates binding to both A and B type sites, supporting the idea that binding to the two distinct DNA sequences is mediated by a variant HTH motif.     

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B(betabeta).

P26olf from olfactory tissue of frog, which may be involved in olfactory transduction or adaptation, is a Ca2+-binding protein with 217 amino acids. The p26olf molecule contains two homologous parts consisting of the N-terminal half with amino acids 1-109 and the C-terminal half with amino acids 110-217. Each half resembles S100 protein with about 100 amino acids and contains two helix-loop-helix Ca2+-binding structural motifs known as EF-hands: a normal EF-hand at the C-terminus and a pseudo EF-hand at the N-terminus. Multiple alignment of the two S100-like domains of p26olf with 18 S100 proteins indicated that the C-terminal putative EF-hand of each domain contains a four-residue insertion when compared with the typical EF-hand motifs in the S100 protein, while the N-terminal EF-hand is homologous to its pseudo EF-hand. We constructed a three-dimensional model of the p26olf molecule based on results of the multiple alignment and NMR structures of dimeric S100B(betabeta) in the Ca2+-free state. The predicted structure of the p26olf single polypeptide chain satisfactorily adopts a folding pattern remarkably similar to dimeric S100B(betabeta). Each domain of p26olf consists of a unicornate-type four-helix bundle and they interact with each other in an antiparallel manner forming an X-type four-helix bundle between the two domains. The two S100-like domains of p26olf are linked by a loop with no steric hindrance, suggesting that this loop might play an important role in the function of p26olf. The circular dichroism spectral data support the predicted structure of p26olf and indicate that Ca2+-dependent conformational changes occur. Since the C-terminal putative EF-hand of each domain fully keeps the helix-loop-helix motif having a longer Ca2+-binding loop, regardless of the four-residue insertion, we propose that it is a new, novel EF-hand, although it is unclear whether this EF-hand binds Ca2+. P26olf is a new member of the S100 protein family.     

Hydrodynamic and functional analysis of HIV-1 Vif oligomerization

HIV-1 Vif is an accessory protein that induces the proteasomal degradation of the host restriction factor, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G). The N-terminal half of Vif binds to APOBEC3G, and the C-terminal half binds to subunits of a cullin 5-based ubiquitin ligase. This Vif-directed ubiquitin ligase induces the degradation of APOBEC3G (a cytidine deaminase) and thereby protects the viral genome from mutation. A conserved PPLP motif near the C-terminus of Vif is essential for Vif function and is also involved in Vif oligomerization. However, the mechanism and functional significance of Vif oligomerization is unclear. We employed analytical ultracentrifugation to examine the oligomeric properties of Vif in solution. Contrary to previous reports, we find that Vif oligomerization does not require the conserved PPLP motif. Instead, our data suggest a more complex mechanism involving interactions among the HCCH motif, the BC box, and downstream residues in Vif. Mutation of residues near the PPLP motif (S165 and V166) affected the oligomeric properties of Vif and weakened the ability of Vif to bind and induce the degradation of APOBEC3G. We propose that Vif oligomerization may represent a mechanism for regulating interactions with APOBEC3G.     

细菌GntR家族转录调控因子的研究进展

GntR家族转录调控因子是细菌中分布最为广泛的一类螺旋–转角–螺旋(helix-turn-helix,HTH)转录调控因子,此家族转录调控因子包含两个功能域,分别是N端的DNA结合结构域和C端的效应物结合结构域/寡聚化作用结构域。DNA结合结构域的氨基酸序列是非常保守的,但效应物结合结构域/寡聚化作用结构域的氨基酸序列却存在很大的差异性。目前许多GntR家族的转录调控因子已经被鉴定,这些转录因子调控细菌许多不同的细胞过程,如运动性、葡萄糖代谢、细菌的耐药性、病原细菌的致病力等。本文主要阐述了GntR家族转录调控因子的发现、二级结构、生物学功能、调控模式等方面的研究进展,旨在为研究者全面、深入地了解GntR家族转录调控因子的功能及作用机理提供帮助。