Direct Observation of the Ion-Pair Dynamics at a Protein–DNA Interface by NMR Spectroscopy

Abstract

Ion pairing is one of the most fundamental chemical interactions and is essential for molecular recognition by biological macromolecules. From an experimental standpoint, very little is known to date about ion-pair dynamics in biological macromolecular systems. Absorption, infrared, and Raman spectroscopic methods were previously used to characterize dynamic properties of ion pairs, but these methods can be applied only to small compounds. Here, using NMR 15N relaxation and hydrogen-bond scalar 15N-31P J-couplings (h3JNP), we have investigated the dynamics of the ion pairs between lysine side-chain NH3+ amino groups and DNA phosphate groups at the molecular interface of the HoxD9 homeodomain-DNA complex. We have determined the order parameters and the correlation times for C-N bond rotation and reorientation of the lysine NH3+ groups. Our data indicate that the NH3+ groups in the intermolecular ion pairs are highly dynamic at the protein-DNA interface, which should lower the entropic costs for protein-DNA association. Judging from the C-N bond-rotation correlation times along with experimental and quantum-chemically derived h3JNP hydrogen-bond scalar couplings, it seems that breakage of hydrogen bonds in the ion pairs occurs on a sub-nanosecond timescale. Interestingly, the oxygen-to-sulfur substitution in a DNA phosphate group was found to enhance the mobility of the NH3+ group in the intermolecular ion pair. This can partially account for the affinity enhancement of the protein-DNA association by the oxygen-to-sulfur substitution, which is a previously observed but poorly understood phenomenon.

Entropy redistribution controls allostery in a metalloregulatory protein

“…The increased flexibility is distributed throughout the core of the dimer. Because the formation of specific contacts with the DNA might be expected to restrict side-chain flexibility (7), the latter result is surprising but not unprecedented for protein-DNA complexes (36,37), and is consistent with prior molecular dynamics simulations (30). Moreover, the overall increase in internal mobility suggests that the negative linkage of Zn and DNA binding might be explained on the basis of fast internal motions, because these occupy opposite corners of the coupled equilibrium scheme (Fig.…”

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

“…Therefore, the intermolecular ion pair with DNA can be added or removed from the ZF-DNA interfaces by the T23K or K79T mutation. We confirmed it by using an NMR method that was recently developed in our laboratory (36) for measuring scalar coupling between lysine side-chain 15 N and DNA 31 P nuclei across a hydrogen bond (Fig. S1).…”

Phosphoribosylphosphate and phosphoribosylnicotinate pairing with phosphoribosylamine at the origin of the RNA world

“…Ionic amino-phosphate interactions are widespread in biology. One of the most common examples is the interaction of the ε-amino group of lysines of histone tails and transcription factors with the phosphate of DNA backbone (Erler et al, 2014;Anderson et al, 2013). More similar to the structure presented in Fig.…”

“…The final conformation of the amino-phosphate ionic interaction more likely depends on the global structure of the extended double-chain PRP-PRA polymers. The subsequent discussion is not dependent on the details of the bond conformation, and only assumes that the amino and phosphate groups of PRA and PRP interact by contact ionic pairing (Anderson et al, 2013).…”