4.3 Results
4.3.5 Dimer stabilisation through inter helical side chain interactions
All four systems were found to be in favour of forming a dimer. By measuring thexy-lateral distance between helices, the umbrella window which approximated the location of the free energy minima was isolated. These trajectories were used for subsequent analysis over the last 20 ns.
The NeuIV umbrella window whoseinterhelical distance was closest to 1.35 nm (Figure 4.16
(A)) was selected as the trajectory of the free energy minima. Bootstrap analysis suggested that the free energy values between 1.3 nm to 1.75 nm were statistical similar. Within this range,
∆G≈ −108 kJ mol−1was identified as an average free energy of self-association across this range. Residues directly in contact with the opposite helix and less than 10 Å (with the aim to identify residues immediately at the helix-helix interface) were identified using distance calculations in VMD (201) and are drawn in space fill representation in Figure 4.19 (A). Additional residues are still within shortinterhelical interaction distance, but do not form direct contacts at the forefront of the helix-helix interface. These additional residues can be identified from theinterhelical contact maps in Figure 4.14 (A). At the interface there are no small-xxx-small motifs. The I659xxxV663
motif was within the short-range cut offbut had rotated away from the interface. The foremost contacts are dominated by a collection of beta-branched side chain amino acids; phenylalanine, leucine and isoleucine, with side chain contacts spanning the length of both helices. Given that the umbrella potential had been applied between the centre-of-mass of each helix these beta-branched side chains help explain the location of the free energy minimum along the reaction coordinate. That is, the dominance of long side chains at the interfaces, increases the separation of the helices compared to what would be expected from a small-xxx-small motif.
The free energy minimum for NeuAGwas found within the range 0.9 to 1.19 nm. Bootstrap analysis
suggests that the free energy values within this region are statistically similar. Within this range
∆G ≈ −97 kJ mol−1was identified as an average free energy of self-association. The umbrella window whose trajectory approximated an averageinterhelical distance of 1.15 nm (Figure 4.16 (B)) was selected and is drawn Figure 4.19 (B). The value of the reaction coordinate at the free energy minimum is slightly less (helices are closer) than NeuIVas may be the result of shorter side
chain contacts. Although the A661xxxG665motif was within the short-range cut off, it was not in
directly at the interface can be seen in the contact map Figure 4.14 (B). As with the NeuIVdimer,
there were no small-xxx-small motifs present and there were side chain contacts which spanned the entire length of each helix.
The free energy andinterhelical side chain contacts for Neu*IVdiffer greatly from those of NeuIV.
The free energy minimum was∆G≈ −55kJ mol−1on average and fell within a broad energy well statistically similar between the distances 1.3 nm and 1.8 nm. In comparison, this is 53 kJ mol−1 less favourable than NeuIV. The free energy minima for Neu*IVand NeuIVwere found at a similar
interhelical distance (see Figure 4.16 (C)). Unlike the wildtype, interface contacts were only found toward the N terminal of each helix with only nine residues in total directly involved in helix-helix contacts (Figure 4.20 (A). Interestingly, theinterhelical packing of the I659xxxV663 motif was
captured at the final frame of the free energy minimum trajectory, which contradicts the atom-atom distance plot in Figure 4.14 (B). Careful observation of the trajectory confirmed that the I659xxxV663
motif deviated from a I659xxxV663to I659xxxV663helix-helix potential interaction,and therefore it
was not recorded as a more permanentinterhelical feature on the atom-atom distance plot. The rearrangement of the helices is most likely down to the interation between the hydrophilic side chain of the glutamic acids and the hydrophobic lipid core. Both glutamic acids acid side chains orientated towards the hydrophobic lipid core. At least one of the glutamic acid side chains is within the distance needed to form a hydrogen bond between the carboxylic group on its side chain with the oxygen on the backbone of L660(see Figure 4.17 (A)). A hydrogen bond would help elevate
the energetic penalty incurred from positioning the glutamic acid side chain within the lipid core. Additional side chain contacts can be seen from the contact map in Figure 4.14 (B), yet there were no side chains within a potentialinterhelical contact distance at the C terminal half of both helices.
The Neu*AG umbrella window whose interhelical distance distribution approximated 0.9 nm
(Figure 4.16 (D)) was selected as the best representational trajectory of the free energy minimum. The bootstrap analysis suggested that the free energy values between 0.77 nm to 1 nm were statistically similar. Therefore, within this region,∆G≈-58 kJ mol−1was the average free energy of association. The wildtype was in favour compared to the mutant by 39 kJ mol−1. The free energy of self-association between Neu*IVand Neu*AGwas considered to be statistically similar although
the minima for Neu*AGoccurs at a smallerinterhelical distance than for Neu*IV. Residues directly
A
B Å
Figure 4.14: Interhelical atom-atom contact plots of (A) NeuIV, and (B) Neu*IV. For clarity, distances beyond 10 Å were
A
B Å
Figure 4.15: Interhelical atom-atom contact plots of (A) NeuAG, and (B) Neu*AG. For clarity, distances beyond 10 Å were
not included.
a significant number of contacts between side chains along theinterhelical interface, spanning almost the entire length of both helices. The A661xxxG665motif has been preserved and the E664
residue buries its side chain within the helix-helix interface (see Figure 4.17 (B)). All other potential contacts between residues were recorded in the contact map Figure 4.15 (B).
1.3 1.4 XY plane distance (nm) 0 5 10 15 Count A 1.1 1.2 1.3 XY plane distance (nm) 0 5 10 Count B 1.2 1.3 1.4 1.5 XY plane distance (nm) 0 10 20 30 40 50 Count C 0.7 0.8 0.9 1 1.1 XY plane distance (nm) 0 10 20 30 Count D
Figure 4.16: Interhelical distance distributions calculated along thexy-dimension. Taken from umbrella simulation windows pertaining to the free energy of systems of(A)NeuIV,(B)NeuAG,(C)Neu*IV, and(D)Neu*AGfrom simulation
umbrella windows five, two, six, and zero, respectively.
All four global minimum trajectories were subjected to solvent accessible surface area analysis (SASA) to determine whether there was a significant change in solvent accessibility to each helix and to enable comparison with deuterium exchange experiments. Approximately, 14 nm2to 15 nm2 of each dimer was exposed to the solvent (see Table 4.1), indicative of the polar residues at either terminus: arganine; serine; threonine; lysine and alanine, are partaking in the lipid-water interface. All four SASA calculations compare well to percent deuterium exchange detection of the Neu
I659!
A!
B!
V663!
E664!
L660!
E664!
A661!
E664!
G665!
H· · ·C−→Figure 4.17: Final frame of the (A) Neu*IVI659xxxV663and (B) NeuAGA661xxxG665,interhelical motifs in addition to
E664.
and Neu* peptide backbone which suggested that Neu and Neu* peptides were almost completely shielded from exchange by being buried inside the lipid membrane (46). There was particular interest in whether the glutamic acid substitution was interacting with water at the lipid-water interface as polar residues have been shown to mediate the permeation of water into the bilayer hydrophobic core (202, 203, 204), potentially disrupting helical stability. The density profiles of both Neu*AGand Neu*IVsystems were calculated to determined whether there was a noticeable
overlap between water and glutamic acid residues in either helix-orientation. As seen in Figure 4.18 (A), the glutamic acid residues (blue and violet) in the Neu*AGdimer do not overlap with the water.
This was expected given that both residues are buried at the helix-helix interface. However, in the opposite helical orientation (Figure 4.18 (B)), one of the two glutamic acid residues in Neu*IV
overlaps with the water density calculations.
It should be noted that the concentration of proto-oncogenic Neu wild type and oncogenic Neu dimers in POPC bilayers do not yield a selectable orientation of oligomerisation, therefore direct comparison to the I659xxxV663and A661xxxG665orientations presented in this study are not possible.
Therefore,α-helical values were generated from the combined average of both I659xxxV663and
A661xxxG665simulations using the Gromacs g_helix tool, as summarised in Table 4.1 , theα-helical
composition of NeuIVwas 57%±1% and NeuAGwas 69%±1%. These results compare well to
experimental CD results of proto-oncogenic Neu and oncogenic Neu in POPC bilayers, which yield an averageα-helical composition of 67.5% and 63.2%, respectively (46).
0
2
4
6
8
10
Box (nm)
20
40
Density (kg m
-3)
POPC Water Protein GLU GLU A0
2
4
6
8
10
Box (nm)
20
40
Density (kg m
-3)
BFigure 4.18: Particle density normal to the bilayer of (A) Neu*AG, and (B) Neu*IV. The presence of water is indicated by
the red shaded segment.
The average dimer tilt angle at the global free energy minima is presented to enable comparisons with linear dichroism (LD) experimental measurements of tilt angle of proto-oncogenic Neu and oncogenic Neu in POPC bilayers (178). As summarised in Table 4.1, NeuIVand NeuAG, with
respect to the bilayer normal returned tilt angles of 45◦ ±3◦and 24◦±2◦, respectively, and the oncogenic form returned tilt angles of 54◦± 2◦ and 24◦± 4◦ for Neu*IV and Neu*AG dimer,
respectively. LD experiments indicated that oncogenic Neu tilts in excess of 30◦. The spectra of proto-oncogenic Neu suggested a tilt angle less than oncogenic Neu. As with theα-helical composition, a direct comparison to experimentation is not possible unless one considers an equal distribution of both orientations of oligomerisation across the population of Neu peptides. In such a case, the average dimer tilt angles in this study are 39◦and 34.5◦for oncogenic and proto-oncogenic Neu, respectively, which compares well to the experimental results.