The bacterial iron sensor IdeR recognizes its DNA targets by indirect readout

ABSTRACTIdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium, and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. The results of our structural and mutational studies support a model wherein IdeR uses an indirect readout mechanism, identifying its targets via the sequence-specific DNA backbone structure rather than through direct contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins.

Context contribution to the intermolecular recognition of human ACE2-derived peptides by SARS-CoV-2 spike protein: implications for improving the peptide affinity but not altering the peptide specificity by optimizing indirect readout

Disrupting the intermolecular interaction of SARS-CoV-2 S protein with its cell surface receptor hACE2 is a therapeutic strategy against COVID-19. The protein context plays an essential role in hACE α1-helix recognition by viral S protein.

Distortion of double-stranded DNA structure by the binding of the restriction DNA glycosylase R.PabI

R.PabI is a restriction DNA glycosylase that recognizes the sequence 5′-GTAC-3′ and hydrolyses the N-glycosidic bond of adenine in the recognition sequence. R.PabI drastically bends and unwinds the recognition sequence of double-stranded DNA (dsDNA) and flips the adenine and guanine bases in the recognition sequence into the catalytic and recognition sites on the protein surface. In this study, we determined the crystal structure of the R.PabI-dsDNA complex in which the dsDNA is drastically bent by the binding of R.PabI but the base pairs are not unwound. This structure is predicted to be important for the indirect readout of the recognition sequence by R.PabI. In the complex structure, wedge loops of the R.PabI dimer are inserted into the minor groove of dsDNA to stabilize the deformed dsDNA structure. A base stacking is distorted between the two wedge-inserted regions. R.PabI is predicted to utilize the distorted base stacking for the detection of the recognition sequence.

Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease

ABSTRACTFilament or run-on oligomer formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms run-on oligomeric (ROO) filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the 3.5 Å cryo-electron microscopy structure of the ROO filament of SgrAI bound to a mimic of cleaved primary site DNA and Mg2+. Large conformational changes stabilize a second metal ion cofactor binding site within the catalytic pocket and facilitate assembling a higher-order enzyme form that is competent for rapid DNA cleavage. The structural changes illuminate the mechanistic origin of hyper-accelerated DNA cleavage activity within the filamentous SgrAI form. An analysis of the protein-DNA interface and the stacking of individual nucleotides reveals how indirect DNA readout within filamentous SgrAI enables recognition of substantially more nucleotide sequences than its low-activity form, thereby expanding DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI’s catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.

The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms

The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o -chlorophenol acetic acid, generating the phenol acetic acid product. It has been shown that CprK has ability to distinguish between the chlorinated CprA substrate and the de-halogenated end product, with an estimated an estimated 10 4 -fold difference in affinity. Using a green fluorescent protein GFP UV -based transcriptional reporter system, we establish that CprK can sense o -chlorophenol acetic acid at the nanomolar level, whereas phenol acetic acid leads to transcriptional activation only when approaching micromolar levels. A structure–activity relationship study, using a range of o -chlorophenol acetic-acid-related compounds and key CprK mutants, combined with p K a calculations on the effector binding site, suggests that the sensitive detection of chlorination is achieved through a combination of direct and indirect readout mechanisms. Both the physical presence of the bulky chloride substituent as well as the accompanying electronic effects lowering the inherent phenol p K a are required for high affinity. Indeed, transcriptional activation by CprK appears strictly dependent on establishing a phenolate–K133 salt bridge interaction, rather than on the presence of a halogen atom per se . As K133 is strictly conserved within the CprK family, our data suggest that physiological function and future applications in biosensing are probably restricted to phenolic compounds.