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. 2021 Nov 19;22(22):12493. doi: 10.3390/ijms222212493

Figure 2.

Figure 2

Biochemical and structural view into the RHO GTPase-GDI interactions. (A) Kinetics of association of 2 µM GDI proteins (GDI1, GDI2 and GDI3ΔN20) with 0.2 µM mdGDP-bound RHO GTPases (twelve different proteins) was only monitored for RAC1, RAC3, RHOG and RHOA using stopped-flow fluorimetry. GDI2 but not GDI1 and GDI3 associated with RAC2. No binding was observed (n. b. o.) for the other GTPases. Obtained kobs values are the average of four to six independent fluorescence measurements, consisting of 1000 data points each (mean ± S.D.). Kinetic data are shown in Figure S2. (B) Individual rate constants were determined, under the same conditions as shown in Figure 1A–C, for the interaction of GDI1 with RAC1, RAC3, RHOG and RHOA, and GDI2 with RAC1, RAC2 and RAC3, respectively. Kinetic data were derived from the average of four to six independent measurements (mean ±S.D.). Kinetic data are shown in Figure S3. (C) An interaction matrix of the GDI proteins with twelve RHO family GTPases is generated to determine the frequency of contacts in respective structures. (see Table S2; for more detail see Figure S4). It comprises the amino acid sequence alignments of the RHO proteins (lower left panel) and the GDIs (upper right panel), respectively. Each element corresponds to a possible interaction of RHO residues (row; RAC numbering) and GDI residues (column; GDI1 numbering). The number of actual contact sites between RHO and GDI proteins (with distances of 4 Å or less) were calculated and are indicated with numbers for matrix elements between 1 and 9. (D) A detailed view into the structure (PDB code: 1HH4) of GDP-bound RAC1GG (grey ribbon) in complex with GDI1 (surface representation) revealed that the basic HVR (blue) is sandwiched between a series of acidic residues of GDI1 supplied by NTA (purple) and GGBD (orange). (E) Schematic diagrams of the domain organizations of GDI1 and RAC1GG illustrate their detailed boundaries. Amino acid sequence alignments of the N-terminal arm (NTA; 25 amino acids) and the C-terminal six residues of the GDI proteins (boxed) highlight negatively and positively charged residues (red and blue). Colors are the same in (D).