Jason T. Snyder

A crystallographic view of interactions between Dbs and Cdc42: PH domain‐assisted guanine nucleotide exchange

Dbl‐related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl‐family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1·Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl‐family proteins will utilize their PH domains similarly to Dbs.

Crystal structure of Rac1 bound to its effector phospholipase C-β2

Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-β2 (PLC-β2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-β2 to optimize its orientation for substrate membranes. Gβγ also engages the PH domain to activate PLC-β2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-δ, the PH domain of PLC-β2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1–PLC-β2 complex reveals determinants that dictate selectivity of PLC-β isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-β2 abrogate its stimulation by Rac1 but not by Gβγ, allowing for functional dissection of this integral signaling node.

Auto-inhibition of the Dbl Family Protein Tim by an N-terminal Helical Motif

Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process.

Structural Determinants Underlying the Temperature-sensitive Nature of a Gα Mutant in Asymmetric Cell Division of Caenorhabditis elegans

Heterotrimeric G-proteins are integral to a conserved regulatory module that influences metazoan asymmetric cell division (ACD). In the Caenorhabditis elegans zygote, GOA-1 (Gαo) and GPA-16 (Gαi) are involved in generating forces that pull on astral microtubules and position the spindle asymmetrically. GPA-16 function has been analyzed in vivo owing notably to a temperature-sensitive allele gpa-16(it143), which, at the restrictive temperature, results in spindle orientation defects in early embryos. Here we identify the structural basis of gpa-16(it143), which encodes a point mutation (G202D) in the switch II region of GPA-16. Using Gαi1(G202D) as a model in biochemical analyses, we demonstrate that high temperature induces instability of the mutant Gα. At the permissive temperature, the mutant Gα was stable upon GTP binding, but switch II rearrangement was compromised, as were activation state-selective interactions with regulators involved in ACD, including GoLoco motifs, RGS proteins, and RIC-8. We solved the crystal structure of the mutant Gα bound to GDP, which indicates a unique switch II conformation as well as steric constraints that suggest activated GPA-16(it143) is destabilized relative to wild type. Spindle severing in gpa-16(it143) embryos revealed that pulling forces are symmetric and markedly diminished at the restrictive temperature. Interestingly, pulling forces are asymmetric and generally similar in magnitude to wild type at the permissive temperature despite defects in the structure of GPA-16(it143). These normal pulling forces in gpa-16(it143) embryos at the permissive temperature were attributable to GOA-1 function, underscoring a complex interplay of Gα subunit function in ACD.

Direct activation of purified phospholipase C epsilon by RhoA studied in reconstituted phospholipid vesicles

Phospholipase C-epsilon (PLC-epsilon) was shown recently to be a downstream effector of Rho GTPases, and we have used an in vitro phospholipid vesicle reconstitution system with purified proteins to show this regulation to be direct. This chapter describes high-level expression of a hexahistidine-tagged fragment of PLC-epsilon encompassing the catalytic core of the enzyme through the tandem RA domains by use of a recombinant baculovirus and High Five insect cells. The recombinant protein is purified to homogeneity using metal chelate affinity and size exclusion chromatography. The small GTPase RhoA also is expressed to high levels in a lipidated form after baculovirus expression in High Five cells and is purified to near homogeneity after detergent extraction and metal chelate affinity chromatography. The capacity of GTPgammaS-bound RhoA to stimulate the phospholipase activity of PLC-epsilon is assessed by reconstitution of the RhoA in mixed-detergent phospholipid micelles containing PtdIns(4,5)P2 substrate.

Regulation of PLCβ isoforms by Rac

Small GTPases function as molecular switches, which transduce cellular signals from upstream regulators to downstream effectors in a guanine nucleotide–dependent manner. Direct binding partners of small GTPases fall into four classes of both regulators and effectors that can be differentiated on the basis of the state of nucleotide required for binding. Here we describe a procedure for the rapid screening and quantitative assessment of direct interactions of the Rho family of small GTPases with effector molecules of the phospholipase Cβ class of enzymes using surface plasmon resonance technology. The experimental format described is also readily adaptable toward characterizing guanine nucleotide–dependent binding events of both small and heterotrimeric G proteins with various classes of GTPase regulatory proteins.

Structural Features of RhoGEFs

Publisher Summary This chapter surveys the structural features of RhoGEFs and highlights the key determinants responsible for dictating the activation of Rho GTPases. All GTPases cycle between two discrete states, an inactive guanosine diphosphate (GDP)-bound form, and an active guanosine triphosphate (GTP)-bound form. The removal of GDP nucleotide from an inactive GTPase allows subsequent loading of GTP, thereby triggering these “binary switches” to recognize downstream effectors. This critical process of GTPase activation is rigidly controlled by guanine nucleotide exchange factors (GEFs). Rho GTPases manage various critical cellular processes. Therefore, it is understood that these small GTPases, as well as their activators (RhoGEFs), promote oncogenesis when constitutively activated. Membership within the Dbl family of RhoGEFs is solely dependent upon the possession of an approximately 300 amino acid segment containing a Dbl homology (DH) domain directly adjacent to a pleckstrin homology (PH) domain. While PH domains exist in a multitude of signaling proteins, the DH domain is unique to these RhoGEFs, and accordingly constitutes the primary catalytic portion of a Dbl protein by supporting nucleotide exchange activity within a substrate Rho GTPase in vitro and in vivo.

CHAPTER 246 – Structural Features of RhoGEFs

This chapter surveys the structural features of RhoGEFs and highlights the key determinants responsible for dictating the activation of Rho GTPases. Dbl family proteins are the major recognized class of GEFs for the Rho family of small GTPases. Rho GTPases have risen to prominence since a large body of work over the last ten years has implicated these ∼25-kDa members of the Ras superfamily in controlling vital cellular functions, including organization of the actin cytoskeleton, progression through the cell cycle, and regulation of transcriptional activities. Given that Rho GTPases manage various critical cellular processes, it is not surprising that these small GTPases, as well as their activators (RhoGEFs), promote oncogenesis when constitutively activated. Membership within the Dbl family of RhoGEFs is solely dependent upon the possession of an -300 amino acid segment containing a Dbl homology (DH) domain directly adjacent to a pleckstrin homology (PH) domain. While PH domains exist in a multitude of signaling proteins, the DH domain is unique to these RhoGEFs, and accordingly constitutes the primary catalytic portion of a Dbl protein by supporting nucleotide exchange activity within a substrate Rho GTPase in vitro and in vivo . Recent biophysical investigations into the function of Dbl-family proteins have revealed substantial insight into the means by which these RhoGEFs catalyze the removal of bound nucleotide from Rho proteins. Specifically, an understanding at atomic resolution of the roles of the conserved DH and PH domains found within all Dbl-related proteins is now available.