Charles D. Blundell

Comparative pharmacology and computational modelling yield insights into allosteric modulation of human α7 nicotinic acetylcholine receptors

The human alpha7 nicotinic acetylcholine receptor (nAChR) subunit and its Caenorhabditis elegans homolog, ACR-16, can generate functional recombinant homomeric receptors when expressed in Xenopus laevis oocytes. Both nAChRs express robustly in the presence of the co-injected chaperone, RIC-3, and show striking differences in the actions of a type I positive allosteric modulator (PAM), ivermectin (IVM). Type I PAMs are characterised by an increase in amplitude only of the response to acetylcholine (ACh), whereas type II PAMs exhibit, in addition, changes in time-course/desensitization of the ACh response. The type I PAMs, ivermectin, 5-hydroxyindole (5-HI), NS-1738 and genistein and the type II PAM, PNU-120596, are all active on human alpha7 but are without PAM activity on ACR-16, where they attenuate the amplitude of the ACh response. We used the published structure of avermectin B1a to generate a model of IVM, which was then docked into the candidate transmembrane allosteric binding site on alpha7 and ACR-16 in an attempt to gain insights into the observed differences in IVM actions. The new pharmacological findings and computational approaches being developed may inform the design of novel PAM drugs targeting major neurological disorders.

Hyaluronan: The absence of amide-carboxylate hydrogen bonds and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure models

Contradictory descriptions for the aqueous solution conformation of the glycosaminoglycan hyaluronan (HA) exist in the literature. According to hydrodynamic and simulation data, HA molecules are stiffened by a rapidly interchanging network of transient hydrogen bonds at the local level and do not significantly associate at the global level. In marked contrast, models derived from NMR data suggest that the secondary structure involves persistent hydrogen bonds and that strong associations between chains can occur to form vast stable tertiary structures. These models require an extended 2-fold helical conformation of the HA chain and specific hydrogen bonds between amide and carboxylate groups. To test these descriptions, we have used 15N-labelled oligosaccharides and high-field NMR to measure pertinent properties of the acetamido group. The amide proton chemical shift perturbation and carboxylate group pK(a) value are inconsistent with a highly populated hydrogen bond between the amide and carboxylate groups. Amide proton temperature coefficients and chemical exchange rates confirm this conclusion. Comparison of oligomer properties with polymeric HA indicates that there is no discernible difference in amide proton environment between the centre of octasaccharides and the polymer, inconsistent with the formation of tertiary structures. A [1H-1H-15N] NOESY-HSQC (heteronuclear single-quantum correlation) spectrum recorded on an HA octasaccharide revealed that amide groups in the centre are in a trans orientation and that the average solution conformation is not an extended 2-fold helix. Therefore the two key aspects of the secondary and tertiary structure models are unlikely to be correct. Rather, these new NMR data agree with descriptions from hydrodynamic and simulations data.

Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan

TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His4) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pKa values of charged residues within the Link module were determined using NMR. These data indicated that His4 makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp89), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr12). This molecular network transmits the change in ionization state of His4 to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His45, is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.

A Refined Model for the TSG-6 LINK Module in Complex with Hyaluronan: use of Defined Oligosaccharides to Probe Structure and Function.

Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-6 during inflammation and ovulation. Results: NMR spectroscopy on TSG-6 in the presence of defined sugars provided restraints that allowed modeling of a refined hyaluronan/TSG-6 complex. Conclusion: TSG-6 binding causes bending of hyaluronan that explains its condensation of this polysaccharide. Significance: This provides novel structural insights into protein-hyaluronan interactions. Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.

Quantification of free ligand conformational preferences by NMR and their relationship to the bioactive conformation.

Graphical abstract

Investigating the molecular basis for the virulence of Escherichia coli K5 by nuclear magnetic resonance analysis of the capsule polysaccharide.

The capsular polysaccharide of Escherichia coli K5 has been hypothesised to promote virulence through its molecular mimicry of host heparan sulphate. To test this hypothesis, we have produced pure oligosaccharides from K5 capsular polysaccharide and investigated their conformational properties with ultra-high-field nuclear magnetic resonance (NMR) (900 MHz). Ultra-high-field affords a significant resolution enhancement over previous studies and allowed a full-atomic assignment of the K5 hexasaccharide for the first time. All carbohydrate rings adopt a 4C1 conformation, the amide sidechains have a trans orientation and the hydroxymethyl group is freely exposed to bulk solvent. Initial models of the glycosidic linkage conformation based upon simple interpretation of NOE cross-peaks suggests that the β1→4 linkage adopts a 3D geometry of φ ≈ 60°, ψ ≈ 0° and the α1→4 linkage prefers φ ≈ –30°, ψ ≈ –30° (φ and ψ being defined by dihedral angles involving linkage protons). In this conformation the overall molecular geometries of K5 polysaccharide, heparan sulphate and even fully-sulphated heparin are remarkably similar. These results substantiate the hypothesis that the K5 capsular polysaccharide confers virulence to E. coli K5 by being a 3D molecular mimetic of host heparan sulphate, helping it to evade detection by the mammalian immune system.

Structural and functional diversity of hyaluronan-binding proteins.

Many diverse biological functions have been attributed to hyaluronan (HA); such as an involvement in the physiological processes of development, ovulation, and wound repair; and in various diseases such as coronary atherosclerosis, inflammatory bowel disease, and cancer. This diversity of roles may seem surprising for such a simple polysaccharide, and while free HA forms solutions that provide space-filling, lubricating, and filtering functions, the wider functional diversity of HA is in fact likely to be generated by its interactions with specific HA-binding proteins. These “hyaladherins,” of which there are increasing numbers, differ in their tissue expression, cellular localisation, affinity, and regulation. HA is an unbranched glycosaminoglycan (GAG) comprised entirely of a repeating disaccharide of D-glucuronic acid and N- acetyl-D-glucosamine. Unlike other GAGs, it is not sulphated at any position and is not covalently attached to a core protein. Molecules of HA are generally of very high molecular mass, ranging from about 105 to 107 Da, although they can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions. HA is found in the extracellular matrix of all tissues in adult vertebrates, with particularly high concentrations being present in skin, synovial fluid and the eye vitreous.

Using Molecular Dynamics Simulations To Provide New Insights into Protein Structure on the Nanosecond Timescale: Comparison with Experimental Data and Biological Inferences for the Hyaluronan-Binding Link Module of TSG-6.

Link module domains play an essential role in extracellular matrix assembly and remodeling by binding to the flexible glycosaminoglycan hyaluronan. A high-resolution NMR-structure of the Link module from the protein product of tumor necrosis factor-stimulated gene-6 (Link_TSG6) has been determined, but a fuller appreciation of protein dynamics may be necessary to understand its hyaluronan-binding. Therefore, we have performed a 0.25 μs MD simulation, starting from the lowest-energy NMR-derived solution structure of Link_TSG6, with explicit water and ions, using the CHARMM22 protein force field. The simulation was as good a fit to the NMR data as the ensemble from simulated annealing, except in the β5-β6 loop. Furthermore, analysis revealed that secondary structure elements extended further than previously reported and underwent fast picosecond time scale dynamics, whereas nanosecond dynamics was found in certain loops. In particular, surface side chains proposed to interact with glycosaminoglycans were predicted to be highly mobile and be directed away from the protein surface. Furthermore, the hyaluronan-binding β4-β5 loop remained in a closed conformation, favoring an allosteric interaction mechanism. This enhanced view of the Link module provides general insight into protein dynamics and may be helpful for understanding the dynamic molecular basis of tissue assembly, remodeling, and disease processes.

GETTING TO GRIPS WITH HA-PROTEIN INTERACTIONS

ABSTRACT The interactions between HA and proteins are most commonly mediated by a domain termed a Link module. The Link module from human TSG-6, produced by expression in E. coli, has been used previously to determine its tertiary structure and identify the position of the HA-binding site by NMR spectroscopy in solution [1,2]. In addition, isothermal titration calorimetry (ITC) has been used to characterize the thermodynamics of this HA-protein interaction [2]. Microtitre plate assays have shown that the binding of the TSG-6 Link module to HA has a pH-dependency that is distinct from that of other hyaladherins; with maximal binding at pH 6.0 and a dramatic loss of function with increasing pH [3]. The NMR/ITC studies were carried out under low salt conditions (~2 mM NaCl), whereas the microtitre plate assays were performed in 100 mM NaCl. Here we show that the interaction of the TSG-6 Link module with HA8 is salt-strength dependent, involving the formation of 1 or 2 salt bridges. However, the structure of the Link module, its folding and the position of the HA-binding surface are the same in the absence and presence of NaCl. Therefore, results from the microtitre plate assays and NMR/ITC studies are comparable. The unusual pH-dependency of the HA interaction is probably mediated by the change of ionization state of one or more histidine residues, the pKa values of which are relatively unaffected by salt.

Chapter 16:Hyaluronan-Binding Proteins in Inflammation

Tony Day joined the Immunochemistry Unit in September 1984 to do a 10-month research project on factor H, under the supervision of Bob Sim, for the final year of his chemistry degree at the University of Oxford. He continued working on factor H for his DPhil (1985–1987)– Tony was Bobs second PhD st...