Functional Specificity of a Protein-DNA Complex Mediated by Two Arginines Bound to the Minor Groove

Abstract

A bacteriophage Ø29 transcriptional regulator, protein p4, interacts with its DNA target by employing two mechanisms: by direct readout of the chemical signatures of only one DNA base and by inducing local modification on the topology of short A tracts (indirect readout). p4 binds as a dimer to targets consisting of imperfect inverted repeats. Here we used molecular dynamic simulation to define interactions of a cluster of 12 positively charged amino acids of p4 with DNA and biochemical assays with modified DNA targets and mutated proteins to quantify the contribution of residues in the nucleoprotein complex. Our results show the implication of Arg54, with non-base-specific interaction in the central A tract, in p4 binding affinity. Despite being chemically equivalent and in identical protein monomers, the two Arg54 residues differed in their interactions with DNA. We discuss an indirect-readout mechanism for p4-DNA recognition mediated by dissimilar interaction of arginines penetrating the minor groove and the inherent properties of the A tract. Our findings extend the current understanding of protein-DNA recognition and contribute to the relevance of the sequence-dependent conformational malleability of the DNA, shedding light on the role of arginines in binding affinity. Characterization of mutant p4R54A shows that the residue is required for the activity of the protein as a transcriptional regulator. Specific interactions between proteins and DNA are fundamental for the regulation of key biological processes, such as transcription, replication, and recombination. Understanding the mechanisms used by regulatory proteins to discern their target sequence within the DNA genome requires the consideration of the properties of all interactions with their cognate binding sites. Structural and biological studies of contacts between proteins and DNA have led to the conclusion that sequence-specific DNA recognition involves both direct and indirect readout of the target sequences (10,20,23). In addition to amino acid-base specific interactions (direct readout), the affinity of a protein for its DNA target involves indirect readout of the target, where sequence-dependent variation leads to recognition of aspects of DNA such as topology of major or minor grooves, local geometry of backbone phosphates, flexibility or malleability, intrinsic curvature, or water-mediated hydrogen bonds (18,42,54,57). Indirect readout explains some aspects leading to stability and the affinity of several prokaryotic transcriptional regulators for their target sequences. Among those, the Escherichia coli catabolite activator protein (CAP) is selective for a pyrimidine-purine step involved in sequence effects on the energy of kink formation required for bending the DNA around the protein (14, 24, 34). The trp repressor recognizes its operator sequence indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface by water-mediated contacts (3, 36). Bacteriophage 434 repressor...

DNA recognition by Escherichia coli CbpA protein requires a conserved arginine–minor-groove interaction

“…It is well-established that the minor groove is narrower at AT-tracts than other DNA sequences ( 20 ). Proteins that target AT-tracts frequently recognize this change in DNA conformation ( 21 ). We reasoned that CbpA may recognize DNA in this way.…”

The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS‐domain

“…The involvement of an arginine residue in the shape readout of a narrow minor groove seems to be a common phenomenon for many TFs (Rohs et al ., , ). Interestingly, this type of protein–DNA interaction occurs in prokaryotes as well as in eukaryotes and also in completely unrelated DNA‐binding protein families (Rohs et al ., ), with an increasing number of representatives being described in recent years (Mendieta et al ., ; Quade et al ., ; Alanazi et al ., ; Porrua et al ., ; Stevenson et al ., ; Chintakayala et al ., ).…”

The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS-domain

“…The involvement of an arginine residue in the shape readout of a narrow minor groove seems to be a common phenomenon for many transcription factors (Rohs et al, 2009; Rohs et al, 2010). Interestingly, this type of protein-DNA interaction occurs in prokaryotes as well as in eukaryotes and also in completely unrelated DNA-binding protein families (Rohs et al, 2009) with an increasing number of representatives being described in recent years (Mendieta et al, 2012; Quade et al, 2012; Alanazi et al, 2013; Porrua et al, 2013; Stevenson et al, 2013; Chintakayala et al, 2015).…”