1. You are analyzing an SH2-phophotyrosine peptide interaction and find that the

Question

1. You are analyzing an SH2-phophotyrosine peptide interaction and find that the Kd for the unphosphorylated peptide is 1 mM, while the Kd for the phosphorylated form is 100 nM. This peptide is found on the C-terminal domain of a receptor tyrosine kinase that is expressed at low levels. What is the approximate concentration range for the SH2-containing protein in the cell?

2. Protein interaction modules, such as SH2 domains or PDZ domains, often recognize a key physical feature in their cognate peptides, such as a phosphotyrosine side chain (SH2) or a free C-terminus (PDZ). How do individual members of the domain family establish distinct specificities?

Explanation / Answer

Answer 1

Presumably, the change in affinity that occurs upon phosphorylation of the target peptide should result in a significant change in the fraction of receptor that is bound by the cognate SH2 protein. Thus, for example, the concentration of the SH2 protein cannot be so high such that a significant fraction of the receptor is bound even when unphosphorylated (here, 50% of receptor would be bound if the SH2 protein was at 1000 µM). We can estimate a reasonable range of concentrations that would fit these criteria by using the equation for fraction bound, which applies when the receptor concentration is far lower than the Kd: fraction of receptor bound = [SH2] / (Kd + [SH2]).

Using this equation, we can plug in different concentrations for SH2 protein and calculate the fraction of receptor bound when the receptor is not phosphorylated or is phosphorylated.

Based on this table, we estimate that a reasonable upper boundary for the SH2 protein concentration is likely to be <100 µM, since at 100 µM, ~10% of the inactive (unphosphorylated) receptor will be bound. Levels higher than this will likely lead to excessive basal signaling. We can also estimate that a lower boundary for the SH2 protein concentration might be >0.1 µM, since at this concentration, the maximal fraction receptor bound would be 50%; any lower concentration might therefore limit the magnitude of an activated signal that could be transmitted. Thus we estimate that the concentration of the SH2 protein likely falls between 0.1 and 100 µM. As you can see from the table, at 1 µM SH2 protein, receptor phosphorylation would change fraction receptor bound from 0.1 to 90%, which would result in a significant dynamic range for induced signaling.

Answer 2

These types of protein modules often recognize peptide sequences of 7–10 residues, with only one or two of these residues serving as the hallmark recognition element for the entire family. Thus individual members of the domain family can achieve distinct specificities by recognizing different preferred flanking sequences within the motif. Often, such domains will structurally contain a central conserved recognition pocket, with more distinct specificity pockets flanking it.

Concentration
of SH2
protein (µM Unphosphorylated
fraction receptor
bound Kd = 1000 µM Phosphorylated
fraction receptor
bound Kd = 0.1 µM Change (n-fold)
upon
phosphorylation 1000 0.500 0.9999 2 100 0.091 0.999 11 10 0.010 0.990 100 1 0.001 0.909 910 .1 0.0001 0.500 5001 .01 0.00001 0.091 9091

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