Primary and Secondary Modes of DNA Recognition by the NarL Two-Component Response Regulator^,

Abstract

NarL is a model response regulator for bacterial two-component signal transduction. The NarL C-terminal domain DNA binding domain alone (NarL(C)) contains all essential DNA binding determinants of the full-length NarL transcription factor. In the full-length NarL protein, the N-terminal regulatory domain must be phosphorylated to release the DNA binding determinants; however, the first NarL(C)-DNA cocrystal structure showed that dimerization of NarL(C) on DNA occurs in a manner independent of the regulatory domain [Maris, A. E., et al. (2002) Nat. Struct. Biol. 9, 771-778]. Dimerization via the NarL(C) C-terminal helix conferred high-affinity recognition of the tail-to-tail promoter site arrangement. Here, two new cocrystal structures are presented of NarL(C) complexed with additional 20mer oligonucleotides representative of other high-affinity tail-to-tail NarL binding sites found in upstream promoter regions. DNA structural recognition properties are described, such as backbone flexibility and groove width, that facilitate NarL(C) dimerization and high-affinity recognition. Lys 188 on the recognition helix accommodates DNA sequence variation between the three different cocomplexes by providing flexible specificity, recognizing the DNA major groove floor directly and/or via bridging waters. The highly conserved Val 189, which enforced significant DNA base distortion in the first cocrystal structure, enforces similar distortions in the two new cocrystal structures. Recognition also is conserved for Lys 192, which hydrogen bonds to guanines at regions of high DNA helical writhe. DNA affinity measurements for model NarL binding sites, including those that did not cocrystallize, suggest a framework for explaining the diversity of heptamer site arrangement and orientation.

Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation

“…3C). This DNA-binding domain dimer configuration is consistent with all of the reported crystal structures of helix-turn-helix DNAbinding domains when bound to their target DNA sequences (26)(27)(28)(29). The DNA-binding domain dimer, with its twofold rotational symmetry, suggests that VraR would bind with the highest affinity to a palindromic DNA sequence.…”

“…E. coli NarL binds with highest affinity to a 7-2-7 tail-to-tail palindromic sequence, where each NarL DNA-binding domain recognizes one heptameric consensus site. Weaker DNA-binding activity is observed for NarL when the heptamer consensus sequence is arranged as a head-tohead palindrome, as tandem repeats or where only a single consensus heptamer is present (27).…”

Cooperative Binding of Phosphorylated DevR to Upstream Sites Is Necessary and Sufficient for Activation of the Rv3134c- devRS Operon in Mycobacterium tuberculosis : Implication in the Induction of DevR Target Genes

“…Like DevR, phosphorylated but not unphosphorylated NarL interacted specifically with its cognate DNA motif (17). Phosphorylation of NarL releases the DNA binding determinants and may also facilitate its oligomerization (16). With respect to DevR, we know now that phosphorylated protein binds first to the D site (Ϫ63.5 bp) and cooperatively recruits another molecule of DevRϳP to the secondary binding P site (Ϫ42.5 bp) that overlaps the promoter Ϫ35 element of the hypoxia-inducible Rv3134c promoter, T H (Fig.…”

Bacterial Two-Component Systems: Structures and Signaling Mechanisms

“…The second and third helices (8 and 9) form a helix-turn-helix motif that interacts with the major groove of DNA [41,42]. Helix 9 is the recognition helix that inserts into major groove of DNA with side chains interacting with DNA bases.…”