Katrina A. Evetts

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.

Structure of the Sterile α Motif (SAM) Domain of the Saccharomyces cerevisiae Mitogen-activated Protein Kinase Pathway-modulating Protein STE50 and Analysis of Its Interaction with the STE11 SAM

The sterile α motif (SAM) is a 65-70-amino acid domain found in over 300 proteins that are involved in either signal transduction or transcriptional activation and repression. SAM domains have been shown to mediate both homodimerization and heterodimerization and in some cases oligomerization. Here, we present the solution structure of the SAM domain of the Saccharomyces cerevisiae protein, Ste50p. Ste50p functions as a modulator of the mitogen-activated protein kinase (MAPK) cascades in S. cerevisiae, which control mating, pseudohyphal growth, and osmo-tolerance. This is the first example of the structure of a SAM domain from a MAPK module protein. We have studied the associative behavior of Ste50p SAM in solution and shown that it is monomeric. We have examined the SAM domain from Ste11p, the MAPK kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association Kd of ∼0.5 mm. We have also analyzed the interaction of Ste50p SAM with Ste11p SAM and the effects of mutations at Val-37, Asp-38, Pro-71, Leu-73, Leu-75, and Met-99 of STE50 on the heterodimerization properties of Ste50p SAM. We have found that L73A and L75A abrogate the Ste50p interaction with Ste11p, and we compare these data with the known interaction sites defined for other SAM domain interactions.

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.

Structure of the SAM domain of the S.cerevisiae MAPK pathway modulating protein STE50 and analysis of its interaction with STE11 SAM

The sterile α motif (SAM) is a 65-70-amino acid domain found in over 300 proteins that are involved in either signal transduction or transcriptional activation and repression. SAM domains have been shown to mediate both homodimerization and heterodimerization and in some cases oligomerization. Here, we present the solution structure of the SAM domain of the Saccharomyces cerevisiae protein, Ste50p. Ste50p functions as a modulator of the mitogen-activated protein kinase (MAPK) cascades in S. cerevisiae, which control mating, pseudohyphal growth, and osmo-tolerance. This is the first example of the structure of a SAM domain from a MAPK module protein. We have studied the associative behavior of Ste50p SAM in solution and shown that it is monomeric. We have examined the SAM domain from Ste11p, the MAPK kinase kinase that associates with Ste50p in vivo, and shown that it forms dimers with a self-association Kd of ∼0.5 mm. We have also analyzed the interaction of Ste50p SAM with Ste11p SAM and the effects of mutations at Val-37, Asp-38, Pro-71, Leu-73, Leu-75, and Met-99 of STE50 on the heterodimerization properties of Ste50p SAM. We have found that L73A and L75A abrogate the Ste50p interaction with Ste11p, and we compare these data with the known interaction sites defined for other SAM domain interactions.

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.

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.

1H, 13C and 15N resonance assignments for Binder of Arl2, BART.

We report 1H, 13C and 15N resonance assignments for Binder of Arl Two (BART), an effector of the small G protein Arl2. The BMRB accession code is 15914.