X-ray Crystal Structure of a Cyclic-PIP–DNA Complex in the Reverse-Binding Orientation

Abstract

Elucidation of the details of the associating mode is one of the major concerns for the precise design of DNA-binding molecules that are used for gene regulation. Pyrrole-imidazole polyamide (PIP) is a well-established synthetic DNA-binding molecule that has sequence-specificity for duplex DNA. By the design of the sequence of pyrrole, imidazole, and other synthetic units, PIP is bound to the target DNA sequence selectively. Here, we report the X-ray crystal structure of newly synthesized chiral cyclic PIP (cPIP) complexed with DNA at 1.5 Å resolution and reveal that cPIP binds in the reverse orientation in the DNA minor groove. Analysis of the crystal structure revealed that the positions of the hydrogen bonds between the bases and the pyrrole-imidazole moieties of cPIP were similar for both forward-and reverse-binding orientations and that the distortion of the B-form DNA structure caused by cPIP binding was also similar for both orientations. We further found that new hydrogen bonds formed between the amino groups on the γ-turn units and DNA at both ends of the cPIP molecule. Additionally, by comparing the reverse PIP orientation with the forward orientation, we could clarify that the cause of the preference toward the reverse orientation in the Sform cPIP as used in this study is the overall conformation of the cPIP-DNA complex, particularly the configuration of hydrogen bonds. These results thus provide an explanation for the different stereoselectivity of cPIP binding in the minor groove.

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

“…The binding of hairpin Py-Im 1 leads to minor groove widening as well as major groove compression of the DNA duplex in a similar manner as cyclic Py-Im molecules ( SI Appendix , Fig. S1 C ) ( 33 , 34 , 42 , 43 ). This high-resolution structure hairpin Py-Im–dsDNA complex was used as a starting model for building the downstream Py-Im–dsDNA region of the Pol II EC–Py-Im complex and paved the way for our structural studies of Py-Im–induced Pol II–arrested complexes.…”

“…Our structure shows the conserved base recognition pattern between Py-Im and DNA base pairs: Im/Py recognizes G/C and Py/Py recognizes A/T or T/A ( SI Appendix , Fig. S1 B ) ( 33 , 34 , 42 , 43 ). The binding of hairpin Py-Im 1 leads to minor groove widening as well as major groove compression of the DNA duplex in a similar manner as cyclic Py-Im molecules ( SI Appendix , Fig.…”

Pyrrole–Imidazole Polyamides – A Frontrunner in Nucleic Acid‐Based Small Molecule Drugs

“…Abe et al. [ 31 ] have confirmed the reverse orientation binding preference of chiral cPIP to the DNA minor groove through the X‐ray crystal structure of cPIP complexed with DNA at 1.5 Å resolution (Figure 2D,E). From the crystal structure, the positions of the hydrogen bonds between the bases and the cPIP moieties were similar for both forward‐ and reverse‐binding orientations.…”

“…Hence, the amino groups at the 𝛾-turn units could be positioned into PIPs to shift the orientation preference exclusively toward the reverse binding when it was difficult to target by the forward binding modes such as 5′-XXXG-3′ or 5′-GXG-3′. [28][29][30] Abe et al [31] have confirmed the reverse orientation binding preference of chiral cPIP to the DNA minor groove through the X-ray crystal structure of cPIP complexed with DNA at 1.5 Å resolution (Figure 2D,E). From the crystal structure, the positions of the hydrogen bonds between the bases and the cPIP moieties were similar for both forward-and reverse-binding orientations.…”

Anticancer Activities of DNA-Alkylating Pyrrole–Imidazole Polyamide Analogs Targeting RUNX Transcription Factors against p53-Mutated Pancreatic Cancer PANC-1 Cells

“…In some cases, however, they prefer binding in the reverse orientation . And several studies indicated that structural differences affect the orientation preferences of PIPs. − In order to find PIP-based RUNX inhibitors with high efficiency and low off-target effect, we prepared not only conjugate 1 but also three analogs of conjugate 1 (conjugates 2 – 4 ; Figures and S2–S5). Conjugate 2 recognizes RUNX-binding sequences in a forward binding orientation similar to that of conjugate 1 , but the positions of its γ-turn and Chb are point-symmetric with conjugate 1 .…”