Stereospecific Effects of Oxygen‐to‐Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein–DNA Affinity

Abstract

Oxygen-to-sulfur substitutions in DNA phosphate often enhance affinity for DNA-binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both OP1 and OP2 atoms is due to an entropic gain associated with enhanced ion-pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the OP1 or OP2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain-DNA complex. By crystallography, we obtained the structural information on the RP and SP diastereomers of the phosphoromonothioate and their interaction with Lys57. By fluorescence-based assays, we found significant affinity enhancement upon sulfur substitution of the OP2 atom. By NMR spectroscopy, we found significant mobilization of the Lys57 side-chain NH3+ group upon sulfur substitution of the OP2 atom. These data provide further mechanistic insight into the affinity enhancement by oxygen-to-sulfur substitution in DNA phosphate.

Structural basis for the recognition of sulfur in phosphorothioated DNA

“…Here, in contrast to the previous postulation that the salt-bridge between the negatively charged phosphorothioated sulfur and the positively charged amino group of lysine/arginine provides primary interaction between PT-nucleotide and protein 35 , we reveal a highly conserved non-polar sulfur-recognizing surface cavity in SBD by determining the crystal structure of ScoMcrA−SBD in complex with PT-DNA at 1.70 Å resolution (Protein Data Bank (PDB) accession number 5ZMO [ https://www.rcsb.org/structure/5ZMO ]). This cavity is surrounded by a hydrophobic wall consisting of the methylene groups from H116, R117, Y164 and the methyl group from A168, and the bottom of the cavity is formed by the pyrolidine group of P165.…”

A Unique and Simple Approach to Improve Sensitivity in 15N-NMR Relaxation Measurements for NH3+ Groups: Application to a Protein-DNA Complex

“…The complex of the 15 N-labeled Antp homeodomain and unlabeled 15-bp DNA was prepared as described in our previous papers [ 21 , 25 , 44 ]. The DNA phosphate group at the K46 interaction site was dithioated in the chemical synthesis, as previously described [ 14 , 25 ].…”

“…Recently, there has been significant progress in NMR methods for investigating the dynamics of charged side chains of proteins [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. In particular, NMR methods for Lys side-chain NH 3 + groups have proven to be extremely useful for investigating the dynamics of hydrogen bonding and/or ion pairing [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”

Mobility of Histidine Side Chains Analyzed with 15N NMR Relaxation and Cross-Correlation Data: Insight into Zinc-Finger–DNA Interactions

“…Recently, we found that some lysine (Lys) and arginine (Arg) side chains interacting with DNA phosphates are highly mobile due to dynamic equilibria between the contact ion-pair (CIP) state and the solvent-separated ion-pair (SIP) state. [2][3][4][5][6] This high mobility of intermolecular ion pairs should reduce entropic loss upon molecular association. In contrast, Arg side chains that interact with DNA bases were found to become rigid upon binding.…”