The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: interfaces differ between the complexes.
IQGAP1 contains a domain related to the catalytic portion of the GTPase-activating proteins (GAPs) for the Ras small G proteins, yet it has no RasGAP activity and binds to the Rho family small G proteins Cdc42 and Rac1. It is thought that IQGAP1 is an effector of Rac1 and Cdc42, regulating cell-cell adhesion through the E-cadherin-catenin complex, which controls formation and maintenance of adherens junctions. This study investigates the binding interfaces of the Rac1-IQGAP1 and Cdc42-IQGAP1 complexes. We mutated Rac1 and Cdc42 and measured the effects of mutations on their affinity for IQGAP1. We have identified similarities and differences in the relative importance of residues used by Rac1 and Cdc42 to bind IQGAP1. Furthermore, the residues involved in the complexes formed with IQGAP1 differ from those formed with other effector proteins and GAPs. Relatively few mutations in switch I of Cdc42 or Rac1 affect IQGAP1 binding; only mutations in residues 32 and 36 significantly decrease affinity for IQGAP1. Switch II mutations also affect binding to IQGAP1 although the effects differ between Rac1 and Cdc42; mutation of either Asp-63, Arg-68, or Leu-70 abrogate Rac1 binding, whereas no switch II mutations affect Cdc42 binding to IQGAP1. The Rho family "insert loop" does not contribute to the binding affinity of Rac1/Cdc42 for IQGAP1. We also present thermodynamic data pertaining to the Rac1/Cdc42-RhoGAP complexes. Switch II contributes a large portion of the total binding energy to these complexes, whereas switch I mutations also affect binding. In addition we identify "cold spots" in the Rac1/Cdc42-RhoGAP/IQGAP1 interfaces. Competition data reveal that the binding sites for IQGAP1 and RhoGAP on the small G proteins overlap only partially. Overall, the data presented here suggest that, despite their 71% identity, Cdc42 and Rac1 appear to have only partially overlapping binding sites on IQGAP1, and each uses different determinants to achieve high affinity binding.
Owen
Darerca
D
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom. Electronic address: do@bioc.cam.ac.uk.
Campbell
Louise J
LJ
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom.
Littlefield
Keily
K
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom.
Evetts
Katrina A
KA
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom.
Li
Zhigang
Z
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Msssachusetts 02115.
Sacks
David B
DB
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Msssachusetts 02115.
Lowe
Peter N
PN
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom.
Mott
Helen R
HR
Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom. Electronic address: hrm28@bioc.cam.ac.uk.
D000595:Amino Acid Sequence; D001665:Binding Sites; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013816:Thermodynamics; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*
D020690:GTPase-Activating Proteins; C101203:IQ motif containing GTPase activating protein 1; D050505:Mutant Proteins; D020703:ras GTPase-Activating Proteins; C085379:rho GTPase-activating protein; D006160:Guanosine Triphosphate; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein