The binding interaction of the pYEEX peptides and the SrcSH2 domain

The pY residue is absolutely required for binding (see Section 1.3), this is supported by the lack o f binding interaction observed in both the ITC and nano flow ESI-M S experiments between the SrcSH2 domain and the YEEI peptide (Section 2.3). The specificity and requirement o f water mediation was investigated in the “two-pronged plug” model (Chapter 1) for the binding o f the specific peptide. Since the pY is necessary for binding, and the structural data on the SrcSH2/pYEEI complex shows that the Glu 4-2 residue is required to maintain the integrity o f the water network, only the pY 4-3 residue was varied. This would provide data on the role o f the pY 4-3 residue dictating specificity without disrupting the energetic contribution from the water molecules.

2.4.2.1.1 Calorimetric data supports the hypothesis that the mediating water

network is conserved with the mutant pYEEX peptides

For all the pYEEX/SrcSH2 (where X = He, Asp, Glu, Val or Trp, Table 2.4) interactions measured using ITC the K\y obs were observed as ranging within the same order o f magnitude (1 0^ M'^). Despite the differences in the pY 4-3 position residues the ATb obs remain similar e.g. changing the hydrophobic He (Æb obs = 1 0 .8 x 1 0^ M'*) to a charged Glu side chain (Kb obs = 4.87 x 10^ M'^). The thermodynamic data for these interactions (Table 2.4) suggest firstly, that the pY 4-3 residue confers little in terms o f specific contacts. Secondly, there is no significant loss in favourable entropy or change in enthalpy going from the free to the bound state o f the different complexes, which is consistent with no net change in the interactions and suggests no extra water incorporation or removal. The maintenance o f the Glu 4-1 and Glu 4-2 residues in all the pYEEX peptides appears to preserve the mediating water network (Figure 2.25a), which accounts at least in part, for the conserved level o f binding affinity.

The main contribution to the AG°obs for the pYEEX interactions is from the AH° derived from non-covalent bond formation (i.e. van der Waals and hydrophobic contact). This may be characteristic o f pTyr peptide binding interactions with SH2 domains (in aqueous conditions at 25 °C). It is observed in identical interactions with FynSH2 and the pYEEI peptides (Section 2.3.4). Furthermore, such a dominant AH° contribution has

Chapter 2: Water-mediated binding in Src Homology 2 domains

been found in other peptide/SH2 binding studies (Lemmon & Ladbury, 1994; Ladbury

et a l, 1995; Ladbury et a l, 1996; Charifson et a l, 1997; Renzoni et a l, 1997;

Bradshaw et a l, 1998). Upon binding to the SH2 domain, the burial o f the hydrophobic peptide surface area and the removal o f the water molecule solvation layers on the peptide surface will be similar for all the pYEEX peptides. Thus, the positive (favourable) contribution to the AG°obs is approximately equivalent for all the peptides. Differences between the energetics of the individual peptides should reflect only the differences in the pY +3 residue due to 1) the interactions the residue side chain has in the SH2 domain pocket and 2) the varying levels of loss o f the degrees o f freedom. For example, the He and Glu have similar carbon-length side chains thus the Glu will reproduce several o f the hydrophobic contacts in the pY +3 pocket, resulting in a favourable A //° contribution to binding. The charged functionality at the end o f the Glu side chain is likely to result in unfavourable interactions with the apolar residues in the pY +3 pocket. Thus, accounts, at least in part, for the overall lower affinity in the pY +3 Glu peptide. A further example is in the binding interaction between SrcSH2 and pYEEI (Xb = 10.8 X 10^ M '') and pYEEV (ATy = 6.2 x 10^ M'*), which show similar

affinities. Although, the He interaction has a favourable TAS°obs ( 1 4 ± 3.1 kJ mof^) contribution to binding, the Val has a more favourable TAS°obs (10.15 ± 1.7 kJ mol'*) contribution. The He is able to form several hydrophobic interactions with the pY +3 SH2 pocket (Waksman et a l, 1993), suggesting that He is tightly bound i.e. the side chain is restricted into a particular conformation. In contrast, the Val side chain will not be able to reproduce all the hydrophobic contacts in the pocket. Consequently, the Val side chain retains more o f its mobility in the pY +3 pocket (i.e. less restriction o f degrees o f the conformational freedom of the side chain).

The weakest affinity interactions were observed with X = Asp or Trp in the mutant series o f peptides investigated. The bulky nature o f the Trp residue means that there may be problems inserting this residue into the pY +3 pocket. The Trp is likely to be ‘pushed ou t’ at the surface o f the pocket. In this scenario, the Trp residue could still form interactions with the hydrophobic residues at the mouth o f the pY +3 pocket (favourable A//°) - this has been confirmed by molecular dynamics simulations (Dr. M. Zvelebil, unpublished data). Yet, such an interaction would distort the peptide backbone away from the peptide surface, disrupting some of the interactions (unfavourable A//°).

Chapter 2: Water-mediated binding in Src Homology 2 domains

To retain a similar binding affinity there must be some significant interaction between the peptide and SH2 domain. The remainder of the peptide could stay close enough to the SH2 domain to retain some o f the protein-peptide and water mediated contacts (favourable AH°). There would however, be some strain and disruption on the mediating water network and probably prevention o f the formation o f some o f the water-water H- bonding (unfavourable

In the case o f the pYEED peptide, a similar case o f distortion could occur, though for different reasons. At the bottom of the pY +3 pocket on the SrcSH2 domain there are some hydrophilic residues, it is possible that the Glu residue in the pYEEE peptide reaches these and forms some level o f ionic/electrostatic interaction (thus compensating for the unfavourable energetics o f placing a charged moiety in a largely hydrophobic pocket). The carbon chain o f the Asp residue is shorter than the Glu residue and would be less likely to reach the hydrophihc groups to form contact. The charged COO group o f the Asp is more likely to ‘sit’ further up in the pocket, surrounded by the highly unfavourable environment o f hydrophobic groups. Thus, the COO o f Asp is probably pushed out o f the pocket into the interface and surrounding solvent. Again, such an interaction would distort the peptide backbone causing a disruption (but not total removal) o f the water network, and weakening the H-bond contacts as they are stretched. Moreover, the negative charge from the COO functional group may have an electrostatic effect on the water molecules (interacting with the pY +1 and +2 Glu residues) drawing them away from their preferred binding orientation, causing disruption o f the H-bonded water molecule network. This is reflected in the ITC data by the weaker ATb value and by the reduction in the favourable A//° and increase in favourable TA5° contribution to binding.

Energy minimisation data for the LckSH2/pYEED peptide complex indicated a possible rearrangement o f water positions. This rearrangement redistributed the H-bonding network compensating for the inclusion o f an extra water molecule into the mouth of the pY 4-3 cavity (Renzoni et a l , 1997). Whilst the data obtained in this work does not support the notion o f the incorporation o f extra water molecules into the SrcSH2/pYEED interaction, the distortion o f the water network could occur - resulting in the rearrangement and redistribution o f the H-bonds.

Chapter 2: Water-mediated binding in Src Homology 2 domains

2.4.2.1.2 Determination of the number of interacting water molecules

In the absence o f structural data, the nanoflow ESI-MS technique can yield information about the water content o f a system, however, it cannot determine anything regarding location or the extent o f interaction. Nanoflow ESI-MS experiments with the pYEEX peptides and the SrcSH2 domain show a clear difference in the distribution o f peaks corresponding to bound water molecules between the free and peptide bound SrcSH2 species. The apo SrcSH2 domain has the highest probability o f being detected as a one water-bound species (Figure 2.12A and Figure 2.14). However, there is significantly high probability o f detecting a two or three water-bound species - corresponding to ‘less tightly’ interacting water molecules on the apo domain. W hen X = I, E or V in the pYEEX/SrcSH2 interactions, complex species with three water molecules bound appear to be the most probable species (Figure 2.12B and Figure 2.14). Such a propensity for a three-water-bound complex species is consistent with the number o f water molecules seen in pYEEI/SrcSH2 structure (Figure 2.24 & 2.25a) and the predictions by the GRID analysis (Chung et a l, 1998).

W hen X = D or W, the most probable water-bound species observed with nano flow ESI-MS is a one water-bound species, the second highest probable species is one where three water molecules are retained within the complex. The anomaly in the Asp and Trp peptides (compared to expectations from structural information and the other pYEEX peptides) is hypothesised in this work to be due to the greater possibility o f disruption of the water network. This is potentially due to the inappropriate insertion o f the pY 4-3 residue in the hydrophobic pocket and distortion o f the peptide backbone. The likely distortion o f the peptide backbone in both these peptides is supported by observations using molecular dynamics simulations (Dr. Marketa Zvelebil, personal communication). Such a de-stabilised water network would be less able to withstand the desolvation and evaporation process and survive until detection in the mass spectrometer. Thus, a lower percentage o f three-water bound species would be expected. The single-water bound species could be indicative o f the strength o f a particular water-binding site in the interface. This may correspond to the same water that is observed in the apo structure for which some o f the same non-covalent bonds are observed in the complex. The high level o f probability for a three-bound-water species in three out the five peptide complexes (and being the second highest probable species in the other two) supports the suggestion that the pY 4-3 amino acid residue does not interact with any water

Chapter 2: Water-mediated binding in Src Homology 2 domains

molecules, since there little overall disruption in water content. Furthermore, the propensity for a three-bound-water species is in good agreement with the numbers o f water molecules observed in mediating the pYEEI motif (Figure 2.24).

Whilst the nanoflow ESI-MS technique cannot unequivocally designate the observed water molecules to the protein-peptide binding-site - this is a good assumption based on several factors:

1) Using the GRID program to predict the location o f the water binding-sites in the complex (Chung et a l , 1998): The sites with the lowest potential energy are found in the peptide-protein interface - coinciding with the observed binding-sites o f the mediating water molecules (Waksman et a l, 1993).

2) Increasing the cone voltage used in the ESI-MS experiments, in order to destabilise the complex species: The higher the cone voltage, the higher probability that the water-free complex species becomes destabilised, such that a smaller population survives through to detection. The water-bound species however, reveals a greater percentage (relative to the water-free species) survives under high-energy conditions (see Figure 2.22). Thus, the water-bound complexes are more stable than the water- free species’ supporting the notion that the water molecules are in the interface acting together to enhance binding (Chung et a l, 1999).

A recent study reported that the substitution o f the Glu in position pY +2 (with an Ala or Gly) resulted in the greatest loss in binding affinity as compared to the other residues C-terminal to the pTyr in the pYEEI motif (Bradshaw & Waksman, 1999). Serial substitutions were made systematically at positions pY +1 through to pY 4-3, and the binding interactions were probed using calorimetry. These results combined with those from this study suggest that the removal o f the pY +2 residue caused the most disruption o f the water network. Thus, the pY +2 residue may be o f greater influence on the specificity o f the system, due to its effects on the water network, than the pY 4-3 residue.