The properties of NodD were affected by mere variation in length within its hinge region

Abstract

In Rhizobium leguminosarum bv. viciae, NodD, a member of the LysR-type transcriptional regulators, while auto-regulating, activates transcription of other nod genes in the presence of naringenin. A hinge region of NodD was previously identified in our laboratory as a functional region independent of its N-terminal DNA-binding and C-terminal regulatory domain. Further study was carried out to see the possible effect of the length variation in the hinge region on NodD's properties. To our surprise, as many as seven classes of phenotypes were observed. Class I is deficient of activating nodA transcription and abolishes auto-regulation; class II is able to activate nodA transcription independently of naringenin and abolishes auto-regulation; class III retains autoregulating but partial activating ability; class IV is able to activate transcription independently of naringenin and retains auto-regulation; in class V, nodA is transcribed constitutively but the transcription level is drastically down-regulated in the presence of naringenin; in class VI, nodA is transcribed constitutively with higher induction ratio; in class VII, nodA is transcribed constitutively with lower induction ratio. To learn more about the possible mechanism, circular permutation assays were done, which showed that the length variation of the hinge of NodD caused by mutation led to the change in bend angles of nod promoter. This finding should help to get an insight into how transcriptional regulation is mediated by NodD at the molecular level as well as to understand the regulatory system of this important family.

Chimeric LysR-Type Transcriptional Biosensors for Customizing Ligand Specificity Profiles toward Flavonoids

“…viciae (Accession NodD: WP_018068348) was investigated. 57 Here, it was observed that the NodD hinge domain does not interact with DNA or ligand molecules but that a wide range of regulatory phenotypes could be generated simply by altering the length of this hinge domain.…”

“…viciae (85′IAWDPIN-PAESD, 83% hinge domain identity, see Supplementary Figure S1). 57 Second, the newly identified NodD1 hinge domain was used to pinpoint the FdeR hinge domain (84′IAALPAFVPA-EST, 41% identity for the full amino acid sequences, see Supplementary Figure S2). These identified hinge domains are clearly located at the end of the helix-turn-helix DBD, quintessential of the LysR-family of TFs (see Supplementary Figure S1 and S2).…”

“…This is substantiated by the high similarity of the 47 basepair (bp) TFBS sequences of FdeR and NodD1 is observed (60%, see Figure b), in conjunction with the high amino acid similarity of the DBD of FdeR and NodD1 (61% DBD identity, 41% for the complete amino acid sequences, see Supplementary Figure S2). , In addition, both TFBSs share the common 25, 5, and 7 bp nod boxes and the pair of palindromic NodD consensus sequences, [AT-N 10 -GAT]-N 7 -[ATC-N 10 -AT] (see Figure b). ,, The chimeric biosensor variant, pChimNodD1-PfdeAR, was created by replacing the fdeR coding sequence in pSynFdeR with the nodD1 coding sequence, thus generating a chimeric TF-TFBS pair (see Figure a). No significant response was detected for any of three tested flavonoids (one-way ANOVA: F = 0.656, 0.067, and 0.163 with p -values = 0.773, 0.999, and 0.999 > 0.05 for naringenin, apigenin, and luteolin, respectively, see Supplementary Table S2).…”

“…Further, the longer hinge domain could aid in exposing activating sites that are otherwise less available to RNAP in this chimeric conformation. 57 Because the only difference between these three chimeric circuits lies in the hinge domain, it is evident that the hinge domain has an influence on both the response curve characteristics as well as on the ligand specificity profile. This substantiates the fact that the TF hinge domain is a compelling target for various biosensor engineering efforts to further fine-tune both the ligand specificity and the response curve.…”

Proline substitutions in a Mip-like peptidyl-prolyl cis-trans isomerase severely affect its structure, stability, shape and activity

“…The domain-connecting hinges in many proteins appears to be crucial for preserving their conformation, stability and function [21] , [22] , [23] , [24] , [25] . The domain-connecting flexible regions in the E. coli FKBP22 and the orthologous proteins are largely structured with a lengthy, protease-sensitive helix designated α3 [14] , [15] , [16] , [17] , [18] .…”