Conserved sequence repeats of IQGAP1 mediate binding to Ezrin

Abstract

Mammalian IQGAP proteins all feature multiple ∼50 amino acid sequence repeats near their N-termini, and little is known about the function of these "Repeats". We have expressed and purified the Repeats from human IQGAP1 to identify binding partners. We used mass spectrometry to identify 42 mouse kidney proteins that associate with the IQGAP1 Repeats including the ERM proteins ezrin, radixin, and moesin. ERM proteins have an N-terminal FERM domain (4.1, ezrin, radixin, moesin) through which they bind to protein targets and phosphatidylinositol 4,5-bisphosphate (PIP2) and a C-terminal actin-binding domain and function to link the actin cytoskeleton to distinct locations on the cell cortex. Isothermal titration calorimetry (ITC) reveals that the IQGAP1 Repeats directly bind to the ezrin FERM domain, while no binding is seen for full-length "autoinhibited" ezrin or a version of full-length ezrin intended to mimic the activated protein. ITC also indicates that the ezrin FERM domain binds to the Repeats from IQGAP2 but not the Repeats from IQGAP3. We conclude that IQGAP1 and IQGAP2 are positioned at the cell cortex by ERM proteins. We propose that the IQGAP3 Repeats may likewise bind to FERM domains for signaling purposes.

Figure 1. IQGAP1 domain organization and procedural overview

(A) IQGAP1 schematic and purified 1Repeats protein. CHD, calponin homology domain; WW, tryptophan-rich polyproline-binding domain; IQ, isoleucine- and glutamine-containing motifs; GRD, RasGAP-related domain; CTD, RasGAP C-terminal domain (B) Procedure flow chart. (C) 2D-gel of proteins pulled-down by control and 6xHis-Repeats.

Figure 2. IQGAP1 Repeats bind the Ezrin FERM domain

(A) Schematic diagram of full length Ezrin (flEz), Ezrin T567D (EzT567D), Ezrin FERM domain (EzFERM) and Ezrin C-ERMAD (EzC) constructs. (B–D) ITC experiments of 1Repeats with flEz, EzT567D, EzFERM and EzC, respectively. The 1Repeats were the titrant and ezrin proteins were in the calorimeter cell. (F) EzFERM titrated into EzC at 30°C using an ITC₂₀₀. B–F, Each panel shows the raw ITC data (top) and the integrated heats (bottom) after subtraction of any heats arising from the injection of titrant into buffer.

Figure 3. Representative ITC data for IQGAP Repeats and EzFERM

Titrations were conducted at 23°C using an ITC₂₀₀. Repeats were the titrant and EzFERM was in the cell. (A) Schematic diagram of Repeats fragment constructs used in this study. (B) 1Repeats. (C) 2Repeats. (D) 3Repeats. B–D, Each panel shows the raw ITC data (top) and the integrated heats (bottom) after subtraction of any heats arising from injection of titrant into buffer alone.