P. Shing Ho

Halogen bonds as orthogonal molecular interactions to hydrogen bonds

Halogen bonds (X-bonds) are shown to be geometrically perpendicular to and energetically independent of hydrogen bonds (H-bonds) that share a common carbonyl oxygen acceptor. This orthogonal relationship is accommodated by the in-plane and out-of-plane electronegative potentials of the oxygen, which are differentially populated by H- and X-bonds. Furthermore, the local conformation of a peptide helps to define the geometry of the H-bond and thus the oxygen surface that is accessible for X-bonding. These electrostatic and steric forces conspire to impose a strong preference for the orthogonal geometry of X- and H-bonds. Thus, the optimum geometry of an X-bond can be predicted from the pattern of H-bonds in a folded protein, enabling X-bonds to be introduced to improve ligand affinities without disrupting these structurally important interactions. This concept of orthogonal molecular interactions can be exploited for the rational design of halogenated ligands as inhibitors and drugs, and in biomolecular engineering.

Halogen bonding (X-bonding): A biological perspective

The concept of the halogen bond (or X‐bond) has become recognized as contributing significantly to the specificity in recognition of a large class of halogenated compounds. The interaction is most easily understood as primarily an electrostatically driven molecular interaction, where an electropositive crown, or σ‐hole, serves as a Lewis acid to attract a variety of electron‐rich Lewis bases, in analogous fashion to a classic hydrogen bonding (H‐bond) interaction. We present here a broad overview of X‐bonds from the perspective of a biologist who may not be familiar with this recently rediscovered class of interactions and, consequently, may be interested in how they can be applied as a highly directional and specific component of the molecular toolbox. This overview includes a discussion for where X‐bonds are found in biomolecular structures, and how their structure–energy relationships are studied experimentally and modeled computationally. In total, our understanding of these basic concepts will allow X‐bonds to be incorporated into strategies for the rational design of new halogenated inhibitors against biomolecular targets or toward molecular engineering of new biological‐based materials.

Mass Spectrometric Approaches Using Electrospray Ionization Charge States and Hydrogen-Deuterium Exchange for Determining Protein Structures and Their Conformational Changes

Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.

A crystallographic map of the transition from B-DNA to A-DNA.

The transition between B- and A-DNA was first observed nearly 50 years ago. We have now mapped this transformation through a set of single-crystal structures of the sequence d(GGCGCC)2, with various intermediates being trapped by methylating or brominating the cytosine bases. The resulting pathway progresses through 13 conformational steps, with a composite structure that pairs A-nucleotides with complementary B-nucleotides serving as a distinct transition intermediate. The details of each step in the conversion of B- to A-DNA are thus revealed at the atomic level, placing intermediates for this and other sequences in the context of a common pathway.

Scalable Anisotropic Shape and Electrostatic Models for Biological Bromine Halogen Bonds.

Halogens are important substituents of many drugs and secondary metabolites, but the structural and thermodynamic properties of their interactions are not properly treated by current molecular modeling and docking methods that assign simple isotropic point charges to atoms. Halogen bonds, for example, are becoming widely recognized as important for conferring specificity in protein-ligand complexes but, to this point, are most accurately described quantum mechanically. Thus, there is a need to develop methods to both accurately and efficiently model the energies and geometries of halogen interactions in biomolecular complexes. We present here a set of potential energy functions that, based on fundamental physical properties of halogens, properly model the anisotropic structure-energy relationships observed for halogen interactions from crystallographic and calorimetric data, and from ab initio calculations for bromine halogen bonds in a biological context. These energy functions indicate that electrostatics alone cannot account for the very short-range distances of bromine halogen bonds but require a flattening of the effective van der Waals radius that can be modeled through an angular dependence of the steric repulsion term of the standard Lennard-Jones type potential. This same function that describes the aspherical shape of the bromine is subsequently applied to model the charge distribution across the surface of the halogen, resulting in a force field that uniquely treats both the shape and electrostatic charge parameters of halogens anisotropically. Finally, the electrostatic potential was shown to have a distance dependence that is consistent with a charge-dipole rather than a simple Coulombic type interaction. The resulting force field for biological halogen bonds (ffBXB) is shown to accurately model the geometry-energy relationships of bromine interactions to both anionic and neutral oxygen acceptors and is shown to be tunable by simply scaling the electrostatic component to account for effects of varying electron-withdrawing substituents (as reflected in their Hammett constants) on the degree of polarization of the bromine. This approach has broad applications to modeling the structure-energy relationships of halogen interactions, including the rational design of inhibitors against therapeutic targets.

Computational Tools To Model Halogen Bonds in Medicinal Chemistry.

The use of halogens in therapeutics dates back to the earliest days of medicine when seaweed was used as a source of iodine to treat goiters. The incorporation of halogens to improve the potency of drugs is now fairly standard in medicinal chemistry. In the past decade, halogens have been recognized as direct participants in defining the affinity of inhibitors through a noncovalent interaction called the halogen bond or X-bond. Incorporating X-bonding into structure-based drug design requires computational models for the anisotropic distribution of charge and the nonspherical shape of halogens, which lead to their highly directional geometries and stabilizing energies. We review here current successes and challenges in developing computational methods to introduce X-bonding into lead compound discovery and optimization during drug development. This fast-growing field will push further development of more accurate and efficient computational tools to accelerate the exploitation of halogens in medicinal chemistry.

The crystal structures of DNA Holliday junctions

Nearly 40 years ago, Holliday proposed a four-stranded complex or junction as the central intermediate in the general mechanism of genetic recombination. During the past two years, six single-crystal structures of such DNA junctions have been determined by three different research groups. These structures all essentially adopt the antiparallel stacked-X conformation, but can be classified into three distinct categories: RNA-DNA junctions; ACC trinucleotide junctions; and drug-induced junctions. Together, these structures provide insight into how local and distant interactions help to define the detailed and general physical features of Holliday junctions at the atomic level.

Caution! DNA Crossing: Crystal Structures of Holliday Junctions

A four-stranded DNA junction (Fig. 1a) was first proposed by Robin Holliday in 1964 as a structural intermediate in a mechanistic model to account for the means by which genetic information is exchanged in yeast (1). This mechanism for genetic exchange is now generally known as homologous recombination and the four-stranded intermediate as the Holliday junction. The general mechanism for recombination has undergone a number of revisions in detail, but the Holliday junction remains a key component in the process and in a growing number of analogous cellular mechanisms (reviewed in Refs. 2 and 3), including site-specific recombination (4), resolution of stalled replication forks (5, 6), DNA repair (7, 8), and phage integration (9). Consequently, the structural and dynamic properties of the Holliday junction have been the focus of intense study since the mid-1960s. As is often the case in science, a multitude of single crystal structures of Holliday junctions in various forms have been solved over a relatively short period of time but only after decades of disappointment. The first structures of junctions in complexes with recombination and DNA repair proteins were reported in 1997 and 1998 (10, 11), whereas junctions in RNA-DNA complexes (12, 13) and in DNA-only constructs (14, 15) emerged just as the twentieth century came to a close. In this review, we will focus on the structures of DNA-only junctions and their geometries, as defined by sequence and ion-dependent interactions.

The stacked-X DNA Holliday junction and protein recognition.

The crystal structure of the four‐stranded DNA Holliday junction has now been determined in the presence and absence of junction binding proteins, with the extended open‐X form of the junction seen in all protein complexes, but the more compact stacked‐X structure observed in free DNA. The structures of the stacked‐X junction were crystallized because of an unexpected sequence dependence on the stability of this structure. Inverted repeat sequences that contain the general motif NCC or ANC favor formation of stacked‐X junctions, with the junction cross‐over occurring between the first two positions of the trinucleotides. This review focuses on the sequence dependent structure of the stacked‐X junction and how it may play a role in structural recognition by a class of dimeric junction resolving enzymes that themselves show no direct sequence recognition. Copyright

Enthalpy-entropy compensation in biomolecular halogen bonds measured in DNA junctions.

Interest in noncovalent interactions involving halogens, particularly halogen bonds (X-bonds), has grown dramatically in the past decade, propelled by the use of X-bonding in molecular engineering and drug design. However, it is clear that a complete analysis of the structure-energy relationship must be established in biological systems to fully exploit X-bonds for biomolecular engineering. We present here the first comprehensive experimental study to correlate geometries with their stabilizing potentials for fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) X-bonds in a biological context. For these studies, we determine the single-crystal structures of DNA Holliday junctions containing halogenated uracil bases that compete X-bonds against classic hydrogen bonds (H-bonds), estimate the enthalpic energies of the competing interactions in the crystal system through crystallographic titrations, and compare the enthalpic and entropic energies of bromine and iodine X-bonds in solution by differential scanning calorimetry. The culmination of these studies demonstrates that enthalpic stabilization of X-bonds increases with increasing polarizability from F to Cl to Br to I, which is consistent with the σ-hole theory of X-bonding. Furthermore, an increase in the X-bonding potential is seen to direct the interaction toward a more ideal geometry. However, the entropic contributions to the total free energies must also be considered to determine how each halogen potentially contributes to the overall stability of the interaction. We find that bromine has the optimal balance between enthalpic and entropic energy components, resulting in the lowest free energy for X-bonding in this DNA system. The X-bond formed by iodine is more enthalpically stable, but this comes with an entropic cost, which we attribute to crowding effects. Thus, the overall free energy of an X-bonding interaction balances the stabilizing electrostatic effects of the σ-hole against the competing effects on the local structural dynamics of the system.