Dynamics of Ionic Interactions at Protein-Nucleic Acid Interfaces

Abstract

Molecular association of proteins with nucleic acids is required for many biological processes essential to life. Electrostatic interactions via ion pairs (salt bridges) of nucleic acid phosphates and protein side chains are crucial for proteins to bind to DNA or RNA. Counterions around the macromolecules are also key constituents for the thermodynamics of protein-nucleic acid association. Until recently, there had been only a limited amount of experiment-based information about how ions and ionic moieties behave in biological macromolecular processes. In the past decade, there has been significant progress in quantitative experimental research on ionic interactions with nucleic acids and their complexes with proteins. The highly negatively charged surfaces of DNA and RNA electrostatically attract and condense cations, creating a zone called the ion atmosphere. Recent experimental studies were able to examine and validate theoretical models on ions and their mobility and interactions with macromolecules. The ionic interactions are highly dynamic. The counterions rapidly diffuse within the ion atmosphere. Some of the ions are released from the ion atmosphere when proteins bind to nucleic acids, balancing the charge via intermolecular ion pairs of positively charged side chains and negatively charged backbone phosphates. Previously, the release of counterions had been implicated indirectly by the salt-concentration dependence of the association constant.Recently, direct detection of counterion release by NMR spectroscopy has become possible and enabled more accurate and quantitative analysis of the counterion release and its entropic impact on the thermodynamics of protein-nucleic acid association. Recent studies also revealed the dynamic nature of ion pairs of protein side chains and nucleic acid phosphates. These ion pairs undergo transitions between two major states. In one of the major states, the cation and the anion are in direct contact and form hydrogen bonds. In the other major state, the cation and the anion are separated by water. Transitions between these states rapidly occur on a picosecond to nanosecond time scale. When proteins interact with nucleic acids, interfacial arginine (Arg) and lysine (Lys) side chains exhibit considerably different behaviors. Arg side chains show a higher propensity to form rigid contacts with nucleotide bases, whereas Lys side chains tend to be more mobile at the molecular interfaces. The dynamic ionic interactions may facilitate adaptive molecular recognition and play both thermodynamic and kinetic roles in protein-nucleic acid interactions.

Figure 1

Key factors that influence the enthalpic (ΔH) and entropic (ΔS) terms of the binding free energy ΔG for formation of a protein–nucleic acid complex. The main factors of the entropic changes are (1) the hydrophobic effects that accompany a decrease in nonpolar surface areas exposed to the solvent, (2) rotational and translational restrictions of the interacting macromolecules, (3) conformational dynamics, and (4) counterion release. Empirical formulas for these entropic effects are given in ref (9). The structures of the MEF2A DNA-binding domain, a DNA duplex, and their complex (PDB 1EGW) are shown with surface electrostatic potentials.

Figure 2

Ion condensation around nucleic acid and counterion release upon protein–nucleic acid association. (A) Impact of ion condensation on ionic diffusion. Blue and red spheres represent cations and anions, respectively. The apparent diffusion coefficient, D_app, for cations is given by a population average of the diffusion coefficients D_f for the free ions and D_b for the bound ions within the ion atmosphere. The D_app data for NH₄⁺ ions measured at various concentrations of a 15-bp DNA duplex are shown. The diffusion coefficients D_f and D_b can be determined from D_app data at various concentrations of the nucleic acid. (B) NMR diffusion data showing the direct evidence of counterion release upon the Antp homeodomain–DNA association. Adapted from Pletka et al. with permission from Wiley-VCH.

Figure 3

Electrostatic interactions between protein and nucleic acids via contact ion pairs (CIPs). (A) Ion pairs of Lys/Arg side chain and phosphate groups forming hydrogen bonds in PDB 2HDD. (B) CIPs with basic side chains are predominant in intermolecular hydrogen bonds between protein side chain and phosphate moieties of protein–nucleic acid complexes. The table lists the total numbers of hydrogen bonds between protein side chain and phosphate moieties found in 3213 crystal structures of protein–DNA or −RNA complexes solved at a resolution <2.5 Å. (C) Spatial distribution of Lys N_ζ atoms forming a hydrogen bond to backbone phosphate in high-resolution (<2.0 Å) crystal structures. On the right-hand side, the probability density maps are shown separately for the Lys N_ζ atoms contacting O_P1 and for those contacting O_P2. As a guide to the eye, C_5′ and C_3′ atoms are also shown with the backbone torsion angles of α = −50° and ζ = −114°, which are in a typical range for B-form DNA. (D) Probability density maps of hydration of water oxygen atoms around backbone phosphate in high-resolution (<2.0 Å) crystal structures. Six peaks in the probability density are indicated with blue spheres and annotated as defined by Schneider et al. Each probability density map represents an enclosure at a 90% probability.

Figure 4

Dynamics of ion pairs of Lys side-chain NH₃⁺ groups and backbone phosphates. (A) Transitions between the contact ion pair (CIP) state and the solvent-separated ion pair (SIP) state observed in molecular dynamics (MD) simulations. (B) Probability distribution and free-energy landscape (i.e., potential of mean force [PMF]) as a function of the O···N distance obtained from MD trajectories. (C) Histogram of the O···N distances for 3038 Lys NH₃⁺–DNA phosphate ion pairs found in high-resolution (<2.0 Å) crystal structures. PMF obtained from this histogram is also presented. These data represent experimental evidence of SIP as a metastable state in electrostatic interactions. (D) NMR data supporting the CIP–SIP transitions. The ^h3J_NP coupling data show the presence of the CIP states. The S² and ³J_NζCγ data show good agreement with those obtained from MD trajectories, suggesting that these ion pairs are as dynamic as seen in the MD simulations.

Figure 5

Potential roles of CIP–SIP equilibria at protein–nucleic acid interfaces. See the main text for details.

Figure 6

Differences in properties of the cationic moieties of Arg and Lys side chains. See the main text for details.