Determining selectivity of phosphoinositide-binding domains

Abstract

The burgeoning of phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily than others. With a focus on the methods used in our laboratory, we describe here the considerations that need to be taken into account when establishing-and quantitating-the specific binding of a protein or domain to phosphoinositides in membranes. We also discuss briefly a few examples in which no clear consensus has yet been reached as to the specificity of a given domain or protein because of discrepancies between different commonly used approaches.

Figure 1

Representative dot-blot results for the PH domains from PLCδ₁, pleckstrin (N-terminal) and human dynamin-1. Methods are described in the text. The phosphoinositide spotted at each position is labeled. Whereas the PLCδ₁ PH domain is specific for PtdIns(4,5)P₂ (labeled 45P₂), the pleckstrin and dynamin PH domains are quite promiscuous, and appear to bind similarly to all phosphoinositides. Data are from [20] or are unpublished. Note for the dynamin-1 PH domain that centrifugation experiments show that PtdIns3P and PtdIns4P bind ~10-fold more weakly than PtdIns(3,4)P₂ and PtdIns(4,5)P₂ [51].

Figure 2

Representative data for analysis of phosphoinositide binding by vesicle centrifugation. Using the approach described in the text, binding to vesicles containing 3% (mole/mole) PtdIns(4,5)P₂ is compared for monomeric and dimeric forms of the dynamin-1 PH domain. Dimerization is induced by fusion to GST. Fits to the equation described in Section 2.3.2.2. are plotted. According to this analysis, the monomeric PH domain binds vesicles with a K value of approximately 479 M⁻¹ (K_D ~ 65 µM if 1:1 stoichiometry is assumed), while the largely dimeric GST fusion gives a K value of 1626 M⁻¹, corresponding to an estimated K_D in the 18 µM range. Data are taken from [51].

Figure 3

SPR analysis of phosphoinositide binding by the β-propeller proteins [57] Atg18p (from S. cerevisiae) and Dm3 (from D. melanogaster), using approaches described in the text. Experiments were performed in HBS-N containing 0.5 mM MgCl₂. Atg18p experiments employed a bacterially-expressed GST fusion protein, which leads to enhanced binding affinities due to dimerization (see [57]). Dm3 experiments utilized monomeric protein produced in Sf9 cells infected with recombinant baculovirus. (A), individual sensorgrams are shown for a representative set of experiments. Each is labeled with the protein concentration used. Steady state response levels for each sensorgram were assessed and plotted against the relevant protein concentration in (B) to give binding curves that can be fit for K_D determination as described in the text. The data shown in (B) illustrate the clear tendency of Atg18p to bind specifically to PtdIns(3,5)P₂, but not PtdIns(4,5)P₂ or PtdIns3P (contrary to the suggestions of [70]). Dm3 binds much more significantly to PtdIns3P, but does not bind detectably to PtdIns(4,5)P₂, PtdIns(3,4)P₂, or PtdIns4P.