Alternative modes of binding of proteins with tandem SH2 domains

Abstract

The issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2~SH2! domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85~including just the two SH2 domains and the inter-SH2 region!, both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein.

Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110α and are disrupted in oncogenic p85 mutants

“…The relaxation delays for the T 2 (1/R2) measurements were 0, 8,16,24,32,40,48,56,64,80,96,120,160, and 248 ms. The NOE and no NOE experiments were run in an interleaved fashion with recycle delays of 5 s, 128 transients per increment, as 1,024 by 90 complex points in proton and nitrogen, respectively.…”

“…We and others have shown that the iSH2 domain-ABD interface is structurally rigid and does not regulate p110 activity (23)(24)(25). In contrast, the Nterminal SH2 (nSH2) domain of p85 is required for inhibition of p110␣.…”

Computational Models of Tandem Src Homology 2 Domain Interactions and Application to Phosphoinositide 3-Kinase

“…Interactions between the tandem SH2 domains of PI3K p85 regulatory subunit and its bisphosphorylated binding site in PDGF ␤-receptor were analyzed in detail, and the cooperativity of the SH2 domains in forming a high avidity ring complex was evaluated in terms of the concentration factor, . Analysis of SPR and ITC measurements, which differ with respect to peptide configuration (immobilized versus soluble) and species concentrations (nanomolar versus micromolar), yielded a consistent order of magnitude estimate of ϭ ϳ10 M. Significantly lower values do not yield the effective K D values reported for tandem SH2 binding to Tyr(P) 740 /Tyr(P) 751 (16,18), nor do they give the extent of inhibition observed in competition binding assays (17). Significantly higher values promote ring formation even when one of the components is in micromolar excess, in clear disagreement with ITC measurements (18).…”

“…O'Brien and colleagues (18) performed such experiments with fulllength p85, injecting increasing amounts of bisphosphorylated Tyr(P) 740 /Tyr(P) 751 peptide into the calorimeter; the net energy change required to maintain the system at constant temperature with each injection was plotted as a function of the increasing molar ratio of peptide/p85. Two distinct changes in the heat/injection were observed, one starting at a molar ratio of Ϸ0.5 and another, more dramatic reduction induced at a molar ratio of Ϸ1.0; at a molar ratio of 2.0, the heat released was near 0, indicating saturation of the SH2 domains.…”

“…4). Based on a concentration of 10 M p85 in the calorimeter initially and given that 1.5 nmol of peptide was introduced per injection, achieving a molar ratio of 2.0 after 16 injections of 15 l each (18), the total concentrations of p85 and peptide after each injection were determined. Thus, the total peptide concentration increases from 1.2 M after the first injection up to 16.7 M at the end; the p85 is diluted in the process, with a final concentration of 8.3 M. Based on those concentrations, and using the same default K D values from Fig.…”