Structural studies on the full‐length LysR‐type regulator TsaR from Comamonas testosteroni T‐2 reveal a novel open conformation of the tetrameric LTTR fold

Abstract

SummaryLysR-type transcriptional regulators (LTTRs) constitute the largest family of regulators in prokaryotes. The full-length structures of the LTTR TsaR from Comamonas testosteroni T-2 and its complex with the natural inducer para-toluensulfonate have been characterized by X-ray diffraction. Both ligand-free and complexed forms reveal a dramatically different quaternary structure from that of CbnR from Ralstonia eutropha, or a putative LysR-type regulator from Pseudomonas aeruginosa, the only other determined full-length structures of tetrameric LTTRs. Although all three show a head-to-head tetrameric ring, TsaR displays an open conformation, whereas CbnR and PA01-PR present additional contacts in opposing C-terminal domains that close the ring. Such large differences may be due to a broader structural versatility than previously assumed or either, reflect the intrinsic flexibility of tetrameric LTTRs. On the grounds of the sliding dimer hypothesis of LTTR activation, we propose a structural model in which the closed structures could reflect the conformation of a ligand-free LTTR, whereas inducer binding would bring about local changes to disrupt the interface linking the two compact C-terminal domains. This could lead to a TsaR-like, open structure, where the pairs of recognition helices are closer to each other by more than 10 Å.

The β-Lactamase Gene Regulator AmpR Is a Tetramer That Recognizes and Binds the d-Ala-d-Ala Motif of Its Repressor UDP-N-acetylmuramic Acid (MurNAc)-pentapeptide

“…A transcriptional activator ligand for AphB has not been identified; however, the protein is known to respond to intracellular pH and oxygen concentrations (64). Unlike other full-length tetrameric LTTRs that have been structurally characterized (36,65,66), AphB adopts a distinctively flat shape, with a linear arrangement of its DNA-binding domains (64). Although AphB and a variant of the protein (N100E) both bind DNA in solution, AphB (N100E) crystallized in a conformation where the relative orientations of the ␣-helices in its DNA-binding domains suggest that this variant can bind a linear stretch of DNA, unlike in the wild-type crystal structure, where the DNA binding domains are not appropriately aligned (64).…”

“…The prevailing model of LTTR activation proposes that small conformational changes in the EBD of LTTR proteins translate to larger conformational changes in their linker helix and DNA binding domains to trigger transcription (63). TsaR, the only full-length LTTR crystallized with its native inducer ligand did not demonstrate significant conformational changes relative to the unliganded protein (36). In the case of A, hydrodynamic radii (R h ) determined for the AmpR⅐21-bp dsDNA complex (blue) and AmpR⅐21-bp dsDNA⅐UDP-MurNAc-pentapeptide complex (red) at various protein concentrations.…”

“…Although a number of crystal structures of full-length LTTRs have been determined, including one bound to its natural activator ligand (36), none have been determined in complex with a natural repressor molecule. Nor have any full-length LTTR complexes with DNA been structurally studied, thus the interactions with promoter elements and associated conformational changes that underpin the switch from gene repression to activation remain poorly understood.…”

Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators

“…A large number of bacterial gene regulators adopt this strategy and use tetramers to recognize two DNA sites. Some of the best-studied examples include the Lac repressor (LacI or LacR), the l repressor (lcI), members of the LysR family, and a few members of the TetR and IclR regulator families (Lewis et al 1996;Schumacher et al 2001;Molina-Henares et al 2006;Stayrook et al 2008;Monferrer et al 2010). Although this mode of recognition is widespread, there is no structural information on a tetrameric protein bound to a continuous DNA operator containing two or more binding sites, which has hindered our understanding of cooperative binding by tetrameric gene regulators and the mechanism of their activation.…”

Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain ofRhodobacter capsulatusMopB

“…MopB exhibits 32% sequence identity to the LTTR-like regulator ModE from Escherichia coli (Anderson et al, 1997). The crystal structures of ModE and a few other LTTRs have been solved, revealing the exact modular organization of the proteins as well as the conformational changes upon effector binding (Hall et al, 1999;Muraoka et al, 2003;Zaim & Kierzek, 2003;Monferrer et al, 2010). In the present study, we demonstrate that the isolated DNA-binding domain of MopB (MopB HTH ) retains full binding activity for the anfA promoter.…”