Christopher M. Read

Design and structure of stapled peptides binding to estrogen receptors

Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 degrees C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.

The Structure of the Hmg Box and its Interaction with DNA

A number of eukaryotic DNA binding domains are now known which recognise DNA by insertion of an α-helix (pre-existing or induced), into the major groove of the DNA, e.g. the homeobox, zinc finger and bZIP/bHLH motifs. The HMG box differs from these domains in three important respects: (1) the principle contacts are to the minor groove, (2) a considerable bend is introduced into the DNA on binding and (3) there is wide variation in the DNA sequence specificity between members of the HMG box family. From this it is clear that new principles will be revealed by understanding the nature of the interaction between the HMG box and DNA. However, the structure of an HMG box bound to DNA is not presently available.

The Interface between Catalytic and Hemopexin Domains in Matrix Metalloproteinase-1 Conceals a Collagen Binding Exosite

Background: The precise role of the hemopexin domain of matrix metalloproteinase-1 (MMP-1) in collagenolysis is unknown. Results: The hemopexin domain collagen binding site is on β-propeller blades 1 and 2, and includes a Phe that is buried in the interface with the catalytic domain in the MMP-1 crystal structure. Conclusion: Domain dislocation is required for exosite exposure. Significance: MMP-1 may undergo significant domain rearrangements during collagenolysis. Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. The two components interact with 1:1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe301, Val319, and Asp338 in collagen binding. Intriguingly, Phe301 is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity.

Solution structure and backbone dynamics of the DNA binding domain of mouse Sox-5

The fold of the murine Sox‐5 (mSox‐5) HMG box in free solution has been determined by multidimensional NMR using 15N‐labeled protein and has been found to adopt the characteristic twisted L‐shape made up of two wings: the major wing comprising helix 1 (F10–F25) and helix 2 (N32–A43), the minor wing comprising helix 3 (P51–Y67) in weak antiparallel association with the N‐terminal extended segment. 15N relaxation measurements show considerable mobility (reduced order parameter, S2) in the minor wing that increases toward the amino and carboxy termini of the chain. The mobility of residues C‐terminal to Q62 is significantly greater than the equivalent residues of non‐sequence‐specific boxes, and these residues show a weaker association with the extended N‐terminal segment than in non‐sequence boxes. Comparison with previously determined structures of HMG boxes both in free solution and complexed with DNA shows close similarity in the packing of the hydrophobic cores and the relative disposition of the three helices. Only in hSRY/DNA does the arrangement of aromatic sidechains differ significantly from that of mSox‐5, and only in rHMG1 box 1 bound to cisplatinated DNA does helix 1 have no kink. Helix 3 in mSox‐5 is terminated by P68, a conserved residue in DNA sequence‐specific HMG boxes, which results in the chain turning through ∼90°.

Defining the thermodynamics of protein/DNA complexes and their components using micro-calorimetry

Understanding the forces driving formation of protein/DNA complexes requires measurement of the Gibbs energy of association, DeltaG, and its component enthalpic, DeltaH, and entropic, DeltaS, contributions. Isothermal titration calorimetry provides the enthalpy (heat) of the binding reaction and an estimate of the association constant, if not too high. Repeating the ITC experiment at several temperatures yields DeltaC ( p ), the change in heat capacity, an important quantity permitting extrapolation of enthalpies and entropies to temperatures outside the experimental range. Binding constants, i.e. Gibbs energies, are best obtained by optical methods such as fluorescence at temperatures where the components are maximally folded. Since DNA-binding domains are often partially unfolded at physiological temperatures, the ITC-observed enthalpy of binding may need to be corrected for the negative contribution from protein refolding. This correction is obtained by differential scanning calorimetric melting of the free DNA-binding domain. Corrected enthalpies are finally combined with accurate Gibbs energies to yield the entropy factor (TDeltaS) at various temperatures. Gibbs energies can be separated into electrostatic and non-electrostatic contributions from the ionic strength dependence of the binding constant.

Conformation and dynamics of human urotensin II and urotensin related peptide in aqueous solution

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K8Y9 or F6W7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F6W7K8 or W7K8Y9. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

Enthalpy–entropy compensation: the role of solvation

Structural modifications to interacting systems frequently lead to changes in both the enthalpy (heat) and entropy of the process that compensate each other, so that the Gibbs free energy is little changed: a major barrier to the development of lead compounds in drug discovery. The conventional explanation for such enthalpy–entropy compensation (EEC) is that tighter contacts lead to a more negative enthalpy but increased molecular constraints, i.e., a compensating conformational entropy reduction. Changes in solvation can also contribute to EEC but this contribution is infrequently discussed. We review long-established and recent cases of EEC and conclude that the large fluctuations in enthalpy and entropy observed are too great to be a result of only conformational changes and must result, to a considerable degree, from variations in the amounts of water immobilized or released on forming complexes. Two systems exhibiting EEC show a correlation between calorimetric entropies and local mobilities, interpreted to mean conformational control of the binding entropy/free energy. However, a substantial contribution from solvation gives the same effect, as a consequence of a structural link between the amount of bound water and the protein flexibility. Only by assuming substantial changes in solvation—an intrinsically compensatory process—can a more complete understanding of EEC be obtained. Faced with such large, and compensating, changes in the enthalpies and entropies of binding, the best approach to engineering elevated affinities must be through the addition of ionic links, as they generate increased entropy without affecting the enthalpy.

Can Simulations and Modeling Decipher NMR Data for Conformational Equilibria? Arginine-Vasopressin.

Arginine vasopressin (AVP) has been suggested by molecular-dynamics (MD) simulations to exist as a mixture of conformations in solution. The (1)H and (13)C NMR chemical shifts of AVP in solution have been calculated for this conformational ensemble of ring conformations (identified from a 23 μs molecular-dynamics simulation). The relative free energies of these conformations were calculated using classical metadynamics simulations in explicit water. Chemical shifts for representative conformations were calculated using density-functional theory. Comparison with experiment and analysis of the results suggests that the (1)H chemical shifts are most useful for assigning equilibrium concentrations of the conformations in this case. (13)C chemical shifts distinguish less clearly between conformations, and the distances calculated from the nuclear Overhauser effect do not allow the conformations to be assigned clearly. The (1)H chemical shifts can be reproduced with a standard error of less than 0.24 ppm (<2.2 ppm for (13)C). The combined experimental and theoretical results suggest that AVP exists in an equilibrium of approximately 70% saddlelike and 30% clinched open conformations. Both newly introduced statistical metrics designed to judge the significance of the results and Smith and Goodmans DP4 probabilities are presented.