Thermogenomic Analysis of Left-Handed Z-DNA Propensities in Genomes.

The initial discovery of left-handed Z-DNA was met with great excitement as a dramatic alternative to the right-handed double-helical conformation of canonical B-DNA. In this chapter, we describe the workings of the program ZHUNT as a computational approach to mapping Z-DNA in genomic sequences using a rigorous thermodynamic model for the transition between the two conformations (the B-Z transition). The discussion starts with a brief summary of the structural properties that differentiate Z- from B-DNA, focusing on those properties that are particularly relevant to the B-Z transition and the junction that splices a left- to right-handed DNA duplex. We then derive the statistical mechanics (SM) analysis of the zipper model that describes the cooperative B-Z transition and show that this analysis very accurately simulates this behavior of naturally occurring sequences that are induced to undergo the B-Z transition through negative supercoiling. A description of the ZHUNT algorithm and its validation are presented, followed by how the program had been applied for genomic and phylogenomic analyses in the past and how a user can access the online version of the program. Finally, we present a new version of ZHUNT (called mZHUNT) that has been parameterized to analyze sequences that contain 5-methylcytosine bases and compare the results of the ZHUNT and mZHUNT analyses on native and methylated yeast chromosome 1.

Non-classical Non-covalent σ-Hole Interactions in Protein Structure and Function: Concepts for Potential Protein Engineering Applications.

The structures and associated functions of biological molecules are driven by noncovalent interactions, which have classically been dominated by the hydrogen bond (H-bond). Introduction of the σ-hole concept to describe the anisotropic distribution of electrostatic potential of covalently bonded elements from across the periodic table has opened a broad range of nonclassical noncovalent (ncNC) interactions for applications in chemistry and biochemistry. Here, we review how halogen bonds, chalcogen bonds and tetrel bonds, as they are found naturally or introduced synthetically, affect the structures, assemblies, and potential functions of peptides and proteins. This review intentionally focuses on examples that introduce or support principles of stability, assembly and catalysis that can potentially guide the design of new functional proteins. These three types of ncNC interactions have energies that are comparable to the H-bond and, therefore, are now significant concepts in molecular recognition and design. However, the recently described H-bond enhanced X-bond shows how synergism among ncNC interactions can be exploited as potential means to broaden the range of their applications to affect protein structures and functions.

A Reduced Generalized Force Field for Biological Halogen Bonds.

The halogen bond (or X-bond) is a noncovalent interaction that is increasingly recognized as an important design tool for engineering protein-ligand interactions and controlling the structures of proteins and nucleic acids. In the past decade, there have been significant efforts to characterize the structure-energy relationships of this interaction in macromolecules. Progress in the computational modeling of X-bonds in biological molecules, however, has lagged behind these experimental studies, with most molecular mechanics/dynamics-based simulation methods not properly treating the properties of the X-bond. We had previously derived a force field for biological X-bonds (ffBXB) based on a set of potential energy functions that describe the anisotropic electrostatic and shape properties of halogens participating in X-bonds. Although fairly accurate for reproducing the energies within biomolecular systems, including X-bonds engineered into a DNA junction, the ffBXB with its seven variable parameters was considered to be too unwieldy for general applications. In the current study, we have generalized the ffBXB by reducing the number of variables to just one for each halogen type and show that this remaining electrostatic variable can be estimated for any new halogenated molecule through a standard restricted electrostatic potential calculation of atomic charges. In addition, we have generalized the ffBXB for both nucleic acids and proteins. As a proof of principle, we have parameterized this reduced and more general ffBXB against the AMBER force field. The resulting parameter set was shown to accurately recapitulate the quantum mechanical landscape and experimental interaction energies of X-bonds incorporated into DNA junction and T4 lysozyme model systems. Thus, this reduced and generalized ffBXB is more readily adaptable for incorporation into classical molecular mechanics/dynamics algorithms, including those commonly used to design inhibitors against therapeutic targets in medicinal chemistry and materials in biomolecular engineering.

Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function.

Modified DNA bases functionally distinguish the taxonomic forms of life-5-methylcytosine separates prokaryotes from eukaryotes and 5-hydroxymethylcytosine (5hmC) invertebrates from vertebrates. We demonstrate here that mouse endonuclease G (mEndoG) shows specificity for both 5hmC and Holliday junctions. The enzyme has higher affinity (>50-fold) for junctions over duplex DNAs. A 5hmC-modification shifts the position of the cut site and increases the rate of DNA cleavage in modified versus unmodified junctions. The crystal structure of mEndoG shows that a cysteine (Cys69) is positioned to recognize 5hmC through a thiol-hydroxyl hydrogen bond. Although this Cys is conserved from worms to mammals, a two amino acid deletion in the vertebrate relative to the invertebrate sequence unwinds an α-helix, placing the thiol of Cys69 into the mEndoG active site. Mutations of Cys69 with alanine or serine show 5hmC-specificity that mirrors the hydrogen bonding potential of the side chain (C-H < S-H < O-H). A second orthogonal DNA binding site identified in the mEndoG structure accommodates a second arm of a junction. Thus, the specificity of mEndoG for 5hmC and junctions derives from structural adaptations that distinguish the vertebrate from the invertebrate enzyme, thereby thereby supporting a role for 5hmC in recombination processes.

Increasing Enzyme Stability and Activity through Hydrogen Bond-Enhanced Halogen Bonds.

The construction of more stable proteins is important in biomolecular engineering, particularly in the design of biologics-based therapeutics. We show here that replacing the tyrosine at position 18 (Y18) of T4 lysozyme with the unnatural amino acid m-chlorotyrosine ( mClY) increases both the thermal stability (increasing the melting temperature by ∼1 °C and the melting enthalpy by 3 kcal/mol) and the enzymatic activity at elevated temperatures (15% higher than that of the parent enzyme at 40 °C) of this classic enzyme. The chlorine of mClY forms a halogen bond (XB) to the carbonyl oxygen of the peptide bond at glycine 28 (G28) in a tight loop near the active site. In this case, the XB potential of the typically weak XB donor Cl is shown from quantum chemical calculations to be significantly enhanced by polarization via an intramolecular hydrogen bond (HB) from the adjacent hydroxyl substituent of the tyrosyl side chain, resulting in a distinctive synergistic HB-enhanced XB (or HeX-B for short) interaction. The larger halogens (bromine and iodine) are not well accommodated within this same loop and, consequently, do not exhibit the effects on protein stability or function associated with the HeX-B interaction. Thus, we have for the first time demonstrated that an XB can be engineered to stabilize and increase the activity of an enzyme, with the increased stabilizing potential of the HeX-B further extending the application of halogenated amino acids in the design of more stable protein therapeutics.

Structure of the Holliday junction: applications beyond recombination.

The Holliday junction (HJ) is an essential element in recombination and related mechanisms. The structure of this four-stranded DNA assembly, which is now well-defined alone and in complex with proteins, has led to its applications in areas well outside of molecular recombination, including nanotechnology and biophysics. This minireview explores some interesting recent research on the HJ, as it has been adapted to design regular two- or three-dimensional lattices for crystal engineering, and more complex systems through DNA origami. In addition, the sequence dependence of the structure is discussed in terms how it can be applied to characterize the geometries and energies of various noncovalent interactions, including halogen bonds in oxidatively damaged (halogenated) bases and hydrogen bonds associated with the epigenetic 5-hydroxylmethylcytosine base.

Sulfur as an Acceptor to Bromine in Biomolecular Halogen Bonds.

The halogen bond (X-bond) has become an important design element in chemistry, including medicinal chemistry and biomolecular engineering. Although oxygen is the most prevalent and best characterized X-bond acceptor in biomolecules, the interaction is seen with nitrogen, sulfur, and aromatic systems as well. In this study, we characterize the structure and thermodynamics of a Br···S X-bond between a 5-bromouracil base and a phosphorothioate in a model DNA junction. The single-crystal structure of the junction shows the geometry of the Br···S to be variable, while calorimetric studies show that the anionic S acceptor is comparable to or slightly more stable than the analogous O acceptor, with a -3.5 kcal/mol difference in ΔΔH25°C and -0.4 kcal/mol ΔΔG25°C (including an entropic penalty ΔΔS25°C of -10 cal/(mol K)). Thus sulfur is shown to be a favorable acceptor for bromine X-bonds, extending the application of this interaction for the design of inhibitors and biological materials.

Relationships between hydrogen bonds and halogen bonds in biological systems.

The recent recognition that halogen bonding (XB) plays important roles in the recognition and assembly of biological molecules has led to new approaches in medicinal chemistry and biomolecular engineering. When designing XBs into strategies for rational drug design or into a biomolecule to affect its structure and function, we must consider the relationship between this interaction and the more ubiquitous hydrogen bond (HB). In this review, we explore these relationships by asking whether and how XBs can replace, compete against or behave independently of HBs in various biological systems. The complex relationships between the two interactions inform us of the challenges we face in fully utilizing XBs to control the affinity and recognition of inhibitors against their therapeutic targets, and to control the structure and function of proteins, nucleic acids and other biomolecular scaffolds.

Structure-Energy Relationships of Halogen Bonds in Proteins.

The structures and stabilities of proteins are defined by a series of weak noncovalent electrostatic, van der Waals, and hydrogen bond (HB) interactions. In this study, we have designed and engineered halogen bonds (XBs) site-specifically to study their structure-energy relationship in a model protein, T4 lysozyme. The evidence for XBs is the displacement of the aromatic side chain toward an oxygen acceptor, at distances that are equal to or less than the sums of their respective van der Waals radii, when the hydroxyl substituent of the wild-type tyrosine is replaced by a halogen. In addition, thermal melting studies show that the iodine XB rescues the stabilization energy from an otherwise destabilizing substitution (at an equivalent noninteracting site), indicating that the interaction is also present in solution. Quantum chemical calculations show that the XB complements an HB at this site and that solvent structure must also be considered in trying to design molecular interactions such as XBs into biological systems. A bromine substitution also shows displacement of the side chain, but the distances and geometries do not indicate formation of an XB. Thus, we have dissected the contributions from various noncovalent interactions of halogens introduced into proteins, to drive the application of XBs, particularly in biomolecular design.

Effect of Hydroxymethylcytosine on the Structure and Stability of Holliday Junctions.

5-Hydroxymethylcytosine (5hmC) is an epigenetic marker that has recently been shown to promote homologous recombination (HR). In this study, we determine the effects of 5hmC on the structure, thermodynamics, and conformational dynamics of the Holliday junction (the four-stranded DNA intermediate associated with HR) in its native stacked-X form. The hydroxymethyl and the control methyl substituents are placed in the context of an amphimorphic GxCC trinucleotide core sequence (where xC is C, 5hmC, or the methylated 5mC), which is part of a sequence also recognized by endonuclease G to promote HR. The hydroxymethyl group of the 5hmC junction adopts two distinct rotational conformations, with an in-base-plane form being dominant over the competing out-of-plane rotamer that has typically been seen in duplex structures. The in-plane rotamer is seen to be stabilized by a more stable intramolecular hydrogen bond to the junction backbone. Stabilizing hydrogen bonds (H-bonds) formed by the hydroxyl substituent in 5hmC or from a bridging water in the 5mC structure provide approximately 1.5-2 kcal/mol per interaction of stability to the junction, which is mostly offset by entropy compensation, thereby leaving the overall stability of the G5hmCC and G5mCC constructs similar to that of the GCC core. Thus, both methyl and hydroxymethyl modifications are accommodated without disrupting the structure or stability of the Holliday junction. Both 5hmC and 5mC are shown to open the structure to make the junction core more accessible. The overall consequences of incorporating 5hmC into a DNA junction are thus discussed in the context of the specificity in protein recognition of the hydroxymethyl substituent through direct and indirect readout mechanisms.

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.

A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity.

HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

Biomolecular halogen bonds.

Halogens are atypical elements in biology, but are common as substituents in ligands, including thyroid hormones and inhibitors, which bind specifically to proteins and nucleic acids. The short-range, stabilizing interactions of halogens - now seen as relatively common in biology - conform generally to halogen bonds characterized in small molecule systems and as described by the σ-hole model. The unique properties of biomolecular halogen bonds (BXBs), particularly in their geometric and energetic relationship to classic hydrogen bonds, make them potentially powerful tools for inhibitor design and molecular engineering. This chapter reviews the current research on BXBs, focusing on experimental studies on their structure-energy relationships, how these studies inform the development of computational methods to model BXBs, and considers how BXBs can be applied to the rational design of more effective inhibitors against therapeutic targets and of new biological-based materials.

Force Field Model of Periodic Trends in Biomolecular Halogen Bonds.

The study of the noncovalent interaction now defined as a halogen bond (X-bond) has become one of the fastest growing areas in experimental and theoretical chemistry--its applications as a design tool are highly extensive. The significance of the interaction in biology has only recently been recognized, but has now become important in medicinal chemistry. We had previously derived a set of empirical potential energy functions to model the structure-energy relationships for bromines in biomolecular X-bonds (BXBs). Here, we have extended this force field for BXBs (ffBXB) to the halogens (Cl, Br, and I) that are commonly seen to form stable X-bonds. The ffBXB calculated energies show a remarkable one-to-one linear relationship to explicit BXB energies determined from an experimental DNA junction system, thereby validating the approach and the model. The resulting parameters allow us to interpret the stabilizing effects of BXBs in terms of well-defined physical properties of the halogen atoms, including their size, shape, and charge, showing periodic trends that are predictable along the Group VII column of elements. Consequently, we have established the ffBXB as an accurate computational tool that can be applied, for example, for the design of new therapeutic compounds against clinically important targets and new biomolecular-based materials.

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.

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.

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.

A rare nucleotide base tautomer in the structure of an asymmetric DNA junction.

The single-crystal structure of a DNA Holliday junction assembled from four unique sequences shows a structure that conforms to the general features of models derived from similar constructs in solution. The structure is a compact stacked-X form junction with two sets of stacked B-DNA-type arms that coaxially stack to form semicontinuous duplexes interrupted only by the crossing of the junction. These semicontinuous helices are related by a right-handed rotation angle of 56.5 degrees, which is nearly identical to the 60 degree angle in the solution model but differs from the more shallow value of approximately 40 degrees for previous crystal structures of symmetric junctions that self-assemble from single identical inverted-repeat sequences. This supports the model in which the unique set of intramolecular interactions at the trinucleotide core of the crossing strands, which are not present in the current asymmetric junction, affects both the stability and geometry of the symmetric junctions. An unexpected result, however, is that a highly wobbled A.T base pair, which is ascribed here to a rare enol tautomer form of the thymine, was observed at the end of a CCCC/GGGG sequence within the stacked B-DNA arms of this 1.9 A resolution structure. We suggest that the junction itself is not responsible for this unusual conformation but served as a vehicle for the study of this CG-rich sequence as a B-DNA duplex, mimicking the form that would be present in a replication complex. The existence of this unusual base lends credence to and defines a sequence context for the "rare tautomer hypothesis" as a mechanism for inducing transition mutations during DNA replication.

Thermogenomics: thermodynamic-based approaches to genomic analyses of DNA structure.

The postgenomic era is all about learning about function by comparing genomic sequences within and between organisms. This review describes an approach that applies detailed thermodynamic information, as opposed to sequence motif searches, to analyze genomes (thermogenomics) for the occurrence of sequences with the potential to form left-handed Z-DNA and those that bind the eukaryotic nuclear factor I (NFI) transcriptional regulators. Such thermogenomic strategies allow us to address the questions of whether Z-DNA forming sequences can potentially function in regulating transcription of eukaryotic genes and how such function may emerge relative to other GC-rich elements, such as NFI recognition sites, to become a transcriptional coactivator.