Rationalising sequence selection by ligand assemblies in the DNA minor groove : the case for thiazotropsin A

DNA-sequence and structure dependence on the formation of minor groove complexes at 5′-XCTAGY-3′ by the short lexitropsin thiazotropsin A are explored based on NMR spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD) and qualitative molecular modeling. The structure and solution behaviour of the complexes are similar whether X = A, T, C or G and Z = T, A, I or C, CCTAGI being thermodynamically the most favoured (ΔG = -11.1 ± 0.1 kcal.mol-1). Binding site selectivity observed by NMR for ACTAGT in the presence of TCTAGA when both accessible sequences are concatenated in a 15-mer DNA duplex construct is consistent with thermodynamic parameters (ΙΔGΙACTAGT > ΙΔGΙTCTAGA) measured separately for the binding sites and with predictions from modeling studies. Steric bulk in the minor groove for Y = G causes unfavourable ligand-DNA interactions reflected in lower Gibbs free energy of binding (ΔG = -8.5 ± 0.01 kcal.mol-1). ITC and CD data establish that thiazotropsin A binds the ODNs with binding constants between 106 and 108 M-1 and reveal that binding is driven enthalpically through hydrogen bond formation and van der Waals interactions. The consequences of these findings are considered with respect to ligand self-association and the energetics responsible for driving DNA recognition by small molecule DNA minor groove binder

Temperature-sensitive protein–DNA dimerizers

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a "switch"-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry

Quantification of Solvent Contribution to the Stability of Noncovalent Complexes

We introduce an indirect approach to estimate the solvation contributions to the thermodynamics of noncovalent complex formation through molecular dynamics simulation. This estimation is demonstrated by potential of mean force and entropy calculations on the binding process between β-cyclodextrin (host) and four drug molecules puerarin, daidzin, daidzein, and nabumetone (guest) in explicit water, followed by a stepwise extraction of individual enthalpy (ΔH) and entropy (ΔS) terms from the total free energy. Detailed analysis on the energetics of the host−guest complexation demonstrates that flexibility of the binding partners and solvation-related ΔH and ΔS need to be included explicitly for accurate estimation of the binding thermodynamics. From this, and our previous work on the solvent dependency of binding energies (Zhang et al. J. Phys. Chem. B 2012, 116, 12684−12693), it follows that calculations neglecting host or guest flexibility, or those employing implicit solvent, will not be able to systematically predict binding free energies. The approach presented here can be readily adopted for obtaining a deeper understanding of the mechanisms governing noncovalent associations in solution

Energetics: the fundamental thermodynamic parameters of molecular complexation via electrostatic interactions in water

textThe thermodynamic parameters that govern the molecular recognition phenomena continue to be a significant area of interest. As a molecular complex forms through non-bonded interactions, quantification of the fundamental enthalpy and entropy changes that occur, offer a more comprehensive understanding of the process. The research presented here specifically explores the energetics of host-guest complexes that form through electrostatic interactions in water at neutral pH. Chapter 1 provides an introduction to molecular recognition and the binding forces that promote binding. Chapter 2 details extensive studies involving phosphate binding to two metalloreceptors; entropy changes are the dominant driving force. In Chapter 3 we report studies focused on determining the thermodynamic origin of cooperative binding; we report the presence of negative cooperativity having entropy as its origin. Chapter 4 is comprised of extensive investigations into the formation of 1:1 host/guest complexes as well as higher ordered complexes. The predominance of either complex results in different thermodynamic profiles.Chemistry and BiochemistryChemistr

Structural and biochemical insights of CypA and AIF interaction

The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370-394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases

On the energy components governing molecular recognition in the framework of continuum approaches

Molecular recognition is a process that brings together several biological macromolecules to form a complex and one of the most important characteristics of the process is the binding free energy. Various approaches exist to model the binding free energy, provided the knowledge of the 3D structures of bound and unbound molecules. Among them, continuum approaches are quite appealing due to their computational efficiency while at the same time providing predictions with reasonable accuracy. Here we review recent developments in the field emphasizing on the importance of adopting adequate description of physical processes taking place upon the binding. In particular, we focus on the efforts aiming at capturing some of the atomistic details of the binding phenomena into the continuum framework. When possible, the energy components are reviewed independently of each other. However, it is pointed out that rigorous approaches should consider all energy contributions on the same footage. The two major schemes for utilizing the individual energy components to predict binding affinity are outlined as well