Louise J. Campbell

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.

The Rac1 Polybasic Region Is Required for Interaction with Its Effector PRK1

Protein kinase C-related kinase 1 (PRK1 or PKN) is involved in regulation of the intermediate filaments of the actin cytoskeleton, as well as having effects on processes as diverse as mitotic timing and apoptosis. It is activated by interacting with the Rho family small G proteins and arachidonic acid or by caspase cleavage. We have previously shown that the HR1b of PRK1 binds exclusively to Rac1, whereas the HR1a domain binds to both Rac1 and RhoA. Here, we have determined the solution structure of the HR1b-Rac complex. We show that HR1b binds to the C-terminal end of the effector loop and switch 2 of Rac1. Comparison with the HR1a-RhoA structure shows that this part of the Rac1-HR1b interaction is homologous to one of the contact sites that HR1a makes with RhoA. The Rac1 used in this study included the C-terminal polybasic region, which is frequently omitted from structural studies, as well as the core G domain. The Rac1 C-terminal region reverses in direction to interact with residues in switch 2, and the polybasic region itself interacts with residues in HR1b. The interactions with HR1b do not prevent the polybasic region being available to contact the negatively charged membrane phospholipids, which is considered to be its primary role. This is the first structural demonstration that the C terminus of a G protein forms a novel recognition element for effector binding.

Solution structure and dynamics of the small GTPase RalB in its active conformation: significance for effector protein binding

The small G proteins RalA/B have a crucial function in the regulatory network that couples extracellular signals with appropriate cellular responses. RalA/B are an important component of the Ras signaling pathway and, in addition to their role in membrane trafficking, are implicated in the initiation and maintenance of tumorigenic transformation of human cells. RalA and RalB share 85% sequence identity and collaborate in supporting cancer cell proliferation but have markedly different effects. RalA is important in mediating proliferation, while depletion of RalB results in transformed cells undergoing apoptosis. Crystal structures of RalA in the free form and in complex with its effectors, Sec5 and Exo84, have been solved. Here we have determined the solution structure of free RalB bound to the GTP analogue GMPPNP to an RMSD of 0.6 A. We show that, while the overall architecture of RalB is very similar to the crystal structure of RalA, differences exist in the switch regions, which are sensitive to the bound nucleotide. Backbone 15N dynamics suggest that there are four regions of disorder in RalB: the P-loop, switch I, switch II, and the loop comprising residues 116-121, which has a single residue insertion compared to RalA. 31P NMR data and the structure of RalB.GMPPNP show that the switch regions predominantly adopt state 1 (Ras nomenclature) in the unbound form, which in Ras is not competent to bind effectors. In contrast, 31P NMR analysis of RalB.GTP reveals that conformations corresponding to states 1 and 2 are both sampled in solution and that addition of an effector protein only partially stabilizes state 2.

The RalB-RLIP76 complex reveals a novel mode of ral-effector interaction.

RLIP76 (RalBP1) is a multidomain protein that interacts with multiple small G protein families: Ral via a specific binding domain, and Rho and R-Ras via a GTPase activating domain. RLIP76 interacts with endocytosis proteins and has also been shown to behave as a membrane ATPase that transports chemotherapeutic agents from the cell. We have determined the structure of the Ral-binding domain of RLIP76 and show that it comprises a coiled-coil motif. The structure of the RLIP76-RalB complex reveals a novel mode of binding compared to the structures of RalA complexed with the exocyst components Sec5 and Exo84. RLIP76 interacts with both nucleotide-sensitive regions of RalB, and key residues in the interface have been identified using affinity measurements of RalB mutants. Sec5, Exo84, and RLIP76 bind Ral proteins competitively and with similar affinities in vitro.

The structure of binder of Arl2 (BART) reveals a novel G protein binding domain: Implications for function

The ADP-ribosylation factor-like (Arl) family of small G proteins are involved in the regulation of diverse cellular processes. Arl2 does not appear to be membrane localized and has been implicated as a regulator of microtubule dynamics. The downstream effector for Arl2, Binder of Arl 2 (BART) has no known function but, together with Arl2, can enter mitochondria and bind the adenine nucleotide transporter. We have solved the solution structure of BART and show that it forms a novel fold composed of six α-helices that form three interlocking “L” shapes. Analysis of the backbone dynamics reveals that the protein is highly anisotropic and that the loops between the central helices are dynamic. The regions involved in the binding of Arl2 were mapped onto the surface of BART and are found to localize to these loop regions. BART has faces of differing charge and structural elements, which may explain how it can interact with other proteins.

1H, 13C, and 15N resonance assignments for the small G protein RalB in its active conformation

We report 1H, 15N and 13C resonance assignments for the small G protein RalB in its active conformation. Backbone amide dynamics parameters for a majority of residues have also been obtained. The BMRB accession code is 15320.

The Structure of the RLIP76 RhoGAP-Ral Binding Domain Dyad: Fixed Position of the Domains Leads to Dual Engagement of Small G Proteins at the Membrane.

Summary RLIP76 is an effector for Ral small GTPases, which in turn lie downstream of the master regulator Ras. Evidence is growing that Ral and RLIP76 play a role in tumorigenesis, invasion, and metastasis. RLIP76 contains both a RhoGAP domain and a Ral binding domain (GBD) and is, therefore, a node between Ras and Rho family signaling. The structure of the RhoGAP-GBD dyad reveals that the RLIP76 RhoGAP domain adopts a canonical RhoGAP domain structure and that the linker between the two RLIP76 domains is structured, fixing the orientation of the two domains and allowing RLIP76 to interact with Rho-family GTPases and Ral simultaneously. However, the juxtaposed domains do not influence each other functionally, suggesting that the RLIP76-Ral interaction controls cellular localization and that the fixed orientation of the two domains orientates the RhoGAP domain with respect to the membrane, allowing it to be perfectly poised to engage its target G proteins.

1H, 13C and 15N resonance assignments for the active conformation of the small G protein RalB in complex with its effector RLIP76

We report here the 1H, 15N and 13C resonance assignments for the small G protein RalB bound to the GTP analogue, GMPPNP and complexed with the Ral binding domain of its downstream effector RLIP76. The BMRB accession code is 15525.

Resonance assignments for the RLIP76 Ral binding domain in its free form and in complex with the small G protein RalB

We report 1H and 15N resonance assignments for the free Ral binding domain of RLIP76 (393–446) and the 1H, 15N and 13C resonance assignments for the RLIP76 Ral binding domain in complex with the active conformation of RalB. The BMRB accession code for free RLIP76 is 15524 and in complex with RalB is 15525.

Thermodynamic mapping of effector protein interfaces with RalA and RalB.

RalA and RalB are members of the Ras family of small G proteins and are activated downstream of Ras via RalGEFs. The RalGEF-Ral axis represents one of the major effector pathways controlled by Ras and as such is an important pharmacological target. RalA and RalB are approximately 80% identical at the amino acid level; despite this, they have distinct roles both in normal cells and in the disease state. We have used our structure of RalB-RLIP76 to guide an analysis of Ral-effector interaction interfaces, creating panels of mutant proteins to probe the energetics of these interactions. The data provide a physical mechanism that underpins the effector selective mutations commonly employed to dissect Ral G protein function. Comparing the energetic landscape of the RalB-RLIP76 and RalB-Sec5 complexes reveals mutations in RalB that lead to differential binding of the two effector proteins. A panel of RLIP76 mutants was used to probe the interaction between RLIP76 and RalA and -B. Despite 100% sequence identity in the RalA and -B contact residues with RLIP76, differences still exist in the energetic profiles of the two complexes. Therefore, we have revealed properties that may account for some of the functional separation observed with RalA and RalB at the cellular level. Our mutations, in both the Ral isoforms and RLIP76, provide new tools that can be employed to parse the complex biology of Ral G protein signaling networks. The combination of these thermodynamic and structural data can also guide efforts to ablate RalA and -B activity with small molecules and peptides.