Tierras de cultivo

Molecular modelling was used initially to investigate the potential ligand specificity effects of the observed variation. To do this a model of the interaction of the receptor's amino- terminal domain with that of its most extensively characterised chemokine ligand,

CCL2/MCP-1, was generated. The advantage of this was that both the tertiary structure of CCL2 and receptor interaction interfaces have previously been determined [Handel & Domaille, 1996; Lubkowski et al., 1997; Chakravarty et al., 1998; Paavola et al., 1998;

Jarnagin et al., 1999; Hemmerich et al., 1999; Lau et al., 2004]. In contrast, the structure of U51, like most human and viral chemokine receptors, has yet to be resolved resulting from the complexities of membrane association in the structural determination of GPCRs. In those cases where structural information is available for large protein binding GPCRs such as chemokine receptors, data on the amino-terminal domain is limited. It is often only present with a 5' truncation, if at all, which is believed to stem from either a disordered structure or the flexible nature of at least the immediate 5' end of this region of the protein [Wu et al., 2010; Park et al., 2012; Tan et al., 2013; Qin et al., 2015]. Indeed studies on the structures of N-terminal peptides of chemokine receptors complexed to

chemokines also suggest a predominantly irregular loop structure to the amino terminal domain of the receptor, at least in the bound state, which binds a hydrophobic groove along the surface of the chemokine ligand [Skelton et al., 1999; Mizoue et al., 1999; Ye et al., 2000; Love et al., 2012].

To investigate possible similar structures in U51 of HHV-6A, a selection of bioinformatic software programs, utilising a variety of different prediction methods, were employed to model the secondary structure of amino-terminal domain. These predicted the 5' end of the domain had a disordered structure with an alpha-helical structure towards the 3' end of the N-terminal domain, figure 5.3A-C. Additionally a helical wheel plot [Schiffer & Edmundson, 1967] indicated that if modelled as an alpha helical structure the acidic residues of the domain would be clustered on one side of the helix with basic residues found on opposite face figure 5.3D. Since it was not possible to model a random coil structure for the U51 N-terminal domain and there was some indication of an alpha helical structure at least at the 3' end. An alpha helical structure was used to initiate a model investigating potential interactions with the chemokine. Wild type and mutant receptors were modelled as alpha helices using HyperChem software, figure 5.5.

Figure 5.3 Secondary structure predictions of amino-terminal domain of U51A. (A) RaptorX web server [Kallberg et al., 2012] (B) JPred web server [Cole et al., 2008] (C) SOPMA web server [Geourjon & Deleage, 1995] (D) Helical wheel plot, red colouring indicates acidic residues and blue

colouring indicates basic residues.

Docking was performed with CCL2 since this ligand of U51 has crystal structures resolved in a number of forms, in addition to extensive functional characterisation of interaction sites of the chemokine [Handel & Domaille, 1996; Lubkowski et al., 1997; Chakravarty et al., 1998; Paavola et al., 1998; Jarnagin et al., 1999; Hemmerich et al., 1999; Lau et al., 2004]. These structures showed that like the majority of other chemokines CCL2 can form higher order oligomers. However, the obligate dimeric forms of CCL2 were unable to bind or activate CCR2 [Tan et al., 2012], implying the monomeric form is required for receptor interaction. Yet obligate monomeric mutants of CCL2 lacked the ability to recruit cells in an in vivo intraperitoneal recruitment assay [Proudfoot et al., 2003]. This Suggested that oligomerisation is essential for an aspect of chemokine function distinct from direct receptor binding. Together this data indicates that the dimerisation interface was unlikely to be involved in initial interactions with the N-terminus of chemokine receptors and therefore the modelling considered only the monomeric receptor binding interactions. A number of the surface features of CCL2 identified from the structural data have also been investigated via mutational studies to characterise the effect of specific regions in its known functions: dimerisation, GAG binding and receptor interactions [Chakravarty et al., 1998; Paavola et al., 1998; Jarnagin et al., 1999; Hemmerich et al., 1999; Lau et al., 2004]. With regards to the receptor binding determinants, Arg24, Lys49, Tyr13 and the ligands N- terminus have all been implicated to play a major role in interactions with the receptor. Of these Tyr13 and N-terminal truncations/mutants are predominantly linked to loss of receptor signalling or chemotactic activity while still retaining receptor binding abilities [Paavola et al., 1998; Jarnagin et al., 1999; Hemmerich et al., 1999], implying they are involved more in the secondary interactions with the extracellular loops of the chemokine receptor that determine receptor activation in the prevailing 'two-site' model. In contrast, Arg24 and Lys49 are strongly linked to loss or reduction in receptor binding affinity

Alpha helix (H)  Beta sheet (E)   Random coil (­) 

A B C

[Paavola et al., 1998; Jarnagin et al., 1999; Hemmerich et al., 1999], implying they are involved in the primary interactions with the amino-terminal domain of the chemokine receptor that are believed to determine the specificity of the interaction. Utilising this data allowed the search space used for the docking of the U51A N-terminal peptide to CCL2 to be narrowed down, concentrating around a hydrophobic groove running along the length of CCL2, figure 5.4A-B.

Figure 5.4 Search space parameters for ligand docking modelling. (A) Key interacting residues of CCL2 (B) Definition of search space parameters for amino-terminal domain docking. Subsequently, docking of the helical viral receptor N-terminal peptides to the defined CCL2 search space were modelled using the AutoDock Vina software [Trott & Olson, 2010]; albeit with the actual roles reversed using the software such that the 'receptor' in the docking model was played by the crystal structure of CCL2 and the role of the 'ligand' by the viral receptor N-terminal domain peptides. The interaction was initially modelled with the wild type strain U1102 receptor N-terminal peptide strictly constrained to the alpha helical conformation by limiting the rotation of the bonds in the peptide backbone, figures 5.5A-B. The optimal predicted binding mode from this docking suggested that the viral receptor N-terminal domain would be orientated in a manner that would allow for further interactions of the N-terminal domain of CCL2 with the receptor binding pocket created by the extracellular loops. Additionally it gave some indication that the glutamic acid residue at position 2 of the viral receptors N-terminal domain had the potential to be an interacting partner with functionally important arginine residue at position 24 on the chemokine ligand. However, the predicted binding affinity was low, possibly from the structural constraints put on the rotation of the bonds. Additionally as the current evidence suggests a strict helical structure is unlikely to represent the natural form the amino-terminal domain of the receptor. The docking procedure was performed again, but this time with removal of the parameters constraining bond rotation in the peptide

backbone, figure 5.5C-D. Again the optimal binding mode from this docking model predicted the receptor N-terminal peptide to be orientated in a manner which would be consistent with the 'two-site' model of chemokine receptor-ligand interaction, as well as

A

B

suggesting close association of the receptor's Glu2 residue and ligands Arg24 residue. This model had a much higher predicted binding affinity. Interestingly, this predicted binding model also suggested the glutamic acid residue at position 4 of the viral receptor peptide may also be interacting with Arg24 of the ligand, figure 5.5E. As well as placing the

tryptophan residue at position 10 of the viral peptide in an orientation facing away from the interactions with the chemokine ligand. In structural studies of other chemokine receptors, most notably CXCR1, a tryptophan residue in this orientation found

approximately half way along the length of the N-terminal domain has been implicated in anchoring the domain to the cell membrane, which would be accommodated in the U51 model [Szpakowska et al., 2012 & Park et al., 2011].

Figure 5.5 Modelling and predicted affinity of the interaction between the amino-terminal domain of HHV-6A strain U1102 U51 and the crystal structure of the chemokine ligand CCL2. (A) Ribbon structure representation of the N-terminus of U51 constrained in an alpha helical structure

docked to a space filling representation of CCL2. (B) Molecular structure representation of the N- terminus of U51 constrained in an alpha helical structure docked to a space filling representation

of CCL2. (C) Ribbon structure representation of the N-terminus of U51 with the torsional constraints on the peptide backbone removed, docked to a space filling representation of CCL2. (D) Molecular structure representation of the N-terminus of U51 with the torsional constraints on

the peptide backbone removed, docked to a space filling representation of CCL2. (E) As (D) but also indicating the position of the tryptophan residue at position 10.

A B

C D

Next, the model was used to predict effects of the substitutions observed in the amino- terminal domain of the CI-HHV-6A samples. To do this docking was then performed with a viral peptide containing the glutamic acid to glycine mutation at position 2 (ΔE2G)

encoded in all CI-HHV-6A U51 genes, figure 5.6A-B. While the overall orientation of the viral peptide was the same as that seen for the strain U1102 peptide, the ΔE2G change led to distinct conformational differences at the immediate 5' end of the peptide, and notably a predicted reduction in binding affinity, figure 5.5C-D vs 5.6A-B.

Figure 5.6 Modelling and predicted affinity of the interaction between the amino-terminal domain of the U51 from the CI-HHV-6A isolates and the crystal structure of the chemokine ligand

CCL2. (A) Ribbon structure representation of the N-terminus of U51 docked to a space filling representation of CCL2. (B) Molecular structure representation of the N-terminus of U51 docked

to a space filling representation of CCL2.

These models indicated that variation observed in this domain may alter the ligand binding dynamics of the receptor. Therefore the integrated U51 could have different function from that of some circulating viral strains. Notably, U51 from HHV-6A strain U1102 which has been utilised for all functional investigation to date. Based on these models, biological evidence for functional differences were next investigated.