Cálculos de refractividad y factor de curvatura de la tierra

I used surface plasmon resonance (SPR) to analyze SH2 domain binding. SPR requires only small amounts of protein sample. Moreover, it is an ideal technique for quantitative competition assays. I immobilized phosphorylated C-tail on a CM5 chip as described in Section 4.7.6, and singly tested binding of individual SH2 domains to the C-tail protein. We measured binding by the SH2 domains from Grb2, PLCγ (N-terminal), PLCγ (C-terminal), and PI3K. Table 4.1 summarizes

the binding affinity derived from the SPR data.

Table 4.1 Summary of dissociation constant of SH2 domains binding to phosphorylated EGFR C- tail

116 SH2 domain Kd,app (µM) Grb2 0.64 ± 0.18 PLCγ1-N 18.4 ± 1.2 PLCγ1-C 0.63 ± 0.12 PLCγ1-NC 0.28 ± 0.06 PIK3R-N 18.0 ± 1.8 PIK3R-C 15.0 ± 1.3 PLCγ1-C (10 µM Grb2) 88.4 ± 18.4 Grb2 (5 µM PLCγ1-C) 142 ± 30

The first result evident from these data is that the Kd values obtained for SH2 domain binding to

the EGFR C-tail are not dramatically smaller than the micromolar range values measured with short phosphopeptides. Although the Grb2 SH2 domain showed a sub-micromolar Kd, this is

consistent with values obtained in peptide experiments (Marengere et al. 1994). The finding that the PI3K SH2 domains exhibit relatively low binding affinities (18.0 and 15.0 µM for the N-terminal and C-terminal SH2 domain, respectively) is also consistent with peptide studie (Zhou 1993). By contrast, my SPR data for the PLCγ SH2 domains was quite surprising. The C-terminal SH2

domain from PLCγ1 binds much more tightly to phosphorylated EGFR C-tail than does the PLCγ

N-terminal SH2 domain (Kd for N-SH2: 18.4 µM, Kd for C-SH2: 0.63 µM). It is generally reported

that the N-terminal SH2 domain of PLCγ binds to receptors, while the C-terminal SH2 interacts

with a phosphotyrosine residue intra-molecularly (although the opposite result is also reported sporadically in the literature) (Wahl et al. 1990; Kim et al. 1991; Gresset et al. 2010). My SPR data, however, clearly argue that the full-length EGFR C-tail protein preferentially binds to the C- terminal SH2 domain of PLCγ1. I did run mass spectrometry of my samples to rule out the

possibility that the N-terminal and C-terminal SH2 domain samples were accidentally swapped during the experiments.

Interesting, when I titrated the Grb2 and PLCγ1-C SH2 domains to a high concentration, I

could detect additional binding events, although with a much lower binding affinity. I was able to fit the binding curves to a model that consists of multiple binding sites. For both the Grb2 and PLCγ1-C SH2 domains, the additional binding shows a Kd value of approximately 200 µM.

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Estimation from the measured Bmax value suggests that four molecules of each SH2 domain bind

to the phosphorylated EGFR C-tail through this low affinity binding, whereas one molecule of SH2 domain binds with a sub-micromolar affinity. Although our SPR analysis cannot unequivocally determine the binding stoichiometry, and the caveat that this binding might have a non-specific component cannot be dismissed, these results do suggest that multiple SH2 domains can bind to a single phosphorylated EGFR C-tail, and possibly with distinct affinities. This also provides an explanation for the ‘rebinding’ of SH2 domains observed in single-molecule experiments of full- length receptor (Oh et al. 2012).

In order to test whether binding of one SH2 domain to the EGFR C-tail affects binding of a second, I performed a competition assay using Grb2 and PLCγ1-C SH2 domains, both of which

showed sub-micromolar Kds in my prior SPR experiments. I titrated one SH2 domain onto a

surface bearing the phosphorylated EGFR C-tail in the presence of a high concentration (5- 10 µM) of the other. Since Grb2 and PLCγ1-C SH2 domains are though to bind to different

phosphotyrosine sites (pY1068 and pY992 respectively), if these two SH2 domains bind independently to the C-tail, it is to be expected that the presence of an excess of another SH2 domain should not affect the binding curve – and that it will appear similar to that measured for binding of a single SH2 domain. Quite surprisingly, my competition assay results suggest the opposite. In the presence of 10 µM Grb2 SH2 domain, the PLCγ1 C-SH2 domain appears to bind

phosphorylated EGFR C-tail with a very low affinity, yielding a Kd value of 88.4 µM (Figure 4.8).

Similarly, the Kd measured for binding of the Grb2 SH2 domain is increased from 0.64 µM when

studied alone to a Kd of 142 µM when the experiment is performed in the presence of 5 µM

PLCγ1-C SH2 domain. These data clearly argue that the Grb2 and PLCγ1-C SH2 domains

compete for binding to the phosphorylated EGFR C-tail. Since their Kd values are similar for their

respective sites, it seems very unlikely that simple competition for the same set of sites would cause the presence of 5-10 µM competitor to elevate Kds for the Grb2 and PLCγ1-C SH2

domains to the 100 µM range. Rather, given that the Grb2 and PLCγ1-C SH2 domains also have

distinct binding sites on the C-tail, it seems possible that binding of one SH2 domain to its specific site on the C-tail alters the C-tail conformation (or conformational dynamics) in a way that

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disfavors binding of the other SH2 domain in a form of negative cooperativity – contrary to the positive cooperativity suggested for Grb2 and Cbl (Sigismund et al., 2013).

Although this phenomenon requires more investigation, including studies of HX of the phosphorylated C-tail (or EGFR-ICR) upon SH2 domain binding, the apparent competition between the Grb2 and PLCγ1 SH2 domains suggests that the activated EGFR might be biased

towards particular pathways – such that a Grb2-bound receptor is diminished in its ability to activated PLCγ signaling and vice versa. Indeed, competition between Grb2 and PLCγ signaling

has been reported in other contexts – notably in the case of FGFR2 signaling (Timsah et al. Ladbury, 2014), although through competition for a different type of binding site in this case.

Figure 4.8 SPR competition binding assays of Grb2 and PLCg-1C SH2 domains. Left: Binding

curve of Grb2 SH2 domain binding to immobilized phosphorylated C-tail (Kd = 142 µM). Right:

Binding curve of PLCg-1C SH2 domain binding to phosphorylated C-tail (Kd = 88.4 µM).