David K. Worthylake

Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.

The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.

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.

Structural basis for the selective activation of Rho GTPases by Dbl exchange factors.

Activation of Rho-family GTPases involves the removal of bound GDP and the subsequent loading of GTP, all catalyzed by guanine nucleotide exchange factors (GEFs) of the Dbl-family. Despite high sequence conservation among Rho GTPases, Dbl proteins possess a wide spectrum of discriminatory potentials for Rho-family members. To rationalize this specificity, we have determined crystal structures of the conserved, catalytic fragments (Dbl and pleckstrin homology domains) of the exchange factors intersectin and Dbs in complex with their cognate GTPases, Cdc42 and RhoA, respectively. Structure-based mutagenesis of intersectin and Dbs reveals the key determinants responsible for promoting exchange activity in Cdc42, Rac1 and RhoA. These findings provide critical insight into the structural features necessary for the proper pairing of Dbl-exchange factors with Rho GTPases and now allow for the detailed manipulation of signaling pathways mediated by these oncoproteins in vivo.

ATP Hydrolysis in Eg5 Kinesin Involves a Catalytic Two-water Mechanism

Motor proteins couple steps in ATP binding and hydrolysis to conformational switching both in and remote from the active site. In our kinesin·AMPPPNP crystal structure, closure of the active site results in structural transformations appropriate for microtubule binding and organizes an orthosteric two-water cluster. We conclude that a proton is shared between the lytic water, positioned for γ-phosphate attack, and a second water that serves as a general base. To our knowledge, this is the first experimental detection of the catalytic base for any ATPase. Deprotonation of the second water by switch residues likely triggers subsequent large scale structural rearrangements. Therefore, the catalytic base is responsible for initiating nucleophilic attack of ATP and for relaying the positive charge over long distances to initiate mechanotransduction. Coordination of switch movements via sequential proton transfer along paired water clusters may be universal for nucleotide triphosphatases with conserved active sites, such as myosins and G-proteins.

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.

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors

Rho GTPases are activated by a family of guanine nucleotide exchange factors (GEFs) known as Dbl family proteins. The structural basis for how GEFs recognize and activate Rho GTPases is presently ill defined. Here, we utilized the crystal structure of the DH/PH domains of the Rac-specific GEF Tiam1 in complex with Rac1 to determine the structural elements of Rac1 that regulate the specificity of this interaction. We show that residues in the Rac1 β2–β3 region are critical for Tiam1 recognition. Additionally, we determined that a single Rac1-to-Cdc42 mutation (W56F) was sufficient to abolish Rac1 sensitivity to Tiam1 and allow recognition by the Cdc42-specific DH/PH domains of Intersectin while not impairing Rac1 downstream activities. Our findings identified unique GEF specificity determinants in Rac1 and provide important insights into the mechanism of DH/PH selection of GTPase targets.

Allosteric Drug Discrimination Is Coupled to Mechanochemical Changes in the Kinesin-5 Motor Core

Essential in mitosis, the human Kinesin-5 protein is a target for >80 classes of allosteric compounds that bind to a surface-exposed site formed by the L5 loop. Not established is why there are differing efficacies in drug inhibition. Here we compare the ligand-bound states of two L5-directed inhibitors against 15 Kinesin-5 mutants by ATPase assays and IR spectroscopy. Biochemical kinetics uncovers functional differences between individual residues at the N or C termini of the L5 loop. Infrared evaluation of solution structures and multivariate analysis of the vibrational spectra reveal that mutation and/or ligand binding not only can remodel the allosteric binding surface but also can transmit long range effects. Changes in L5-localized 310 helix and disordered content, regardless of substitution or drug potency, are experimentally detected. Principal component analysis couples these local structural events to two types of rearrangements in β-sheet hydrogen bonding. These transformations in β-sheet contacts are correlated with inhibitory drug response and are corroborated by wild type Kinesin-5 crystal structures. Despite considerable evolutionary divergence, our data directly support a theorized conserved element for long distance mechanochemical coupling in kinesin, myosin, and F1-ATPase. These findings also suggest that these relatively rapid IR approaches can provide structural biomarkers for clinical determination of drug sensitivity and drug efficacy in nucleotide triphosphatases.

Crystal structure of the GTPase-activating protein-related domain from IQGAP1.

IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic “arginine finger” seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099–1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42·GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.

Regulation of ROCKII by localization to membrane compartments and binding to DynaminI

ROCKII kinase activity is known to be regulated by Rho GTPase binding; however, the context-specific regulation of ROCKII is not clearly understood. We pursued the C-terminal PH domain as a candidate domain for regulating ROCKII function. A proteomics-based screen identified potential ROCKII signaling partners, a large number of which were associated with membrane dynamics. We used subcellular fractionation to demonstrate that ROCKII is localized to both the plasma membrane and internal endosomal membrane fractions, and then used microscopy to show that the C-terminal PH domain can localize to internal or peripheral membrane compartments, depending on the cellular context. Co-immunoprecipitation demonstrated that Dynamin1 is a novel ROCKII binding partner. Furthermore, blocking Dynamin function with a dominant negative mutant mimicked the effect of inhibiting ROCK activity on the actin cytoskeleton. Our data suggest that ROCKII is regulated by localization to specific membrane compartments and its novel binding partner, Dynamin1.

Multipart Chaperone-Effector Recognition in the Type III Secretion System of Chlamydia trachomatis

Background: The type III secretion (T3S) chaperone Scc4 modulates Chlamydia RNA polymerase holoenzyme activity and is also required for secretion of the gatekeeper CopN. Results: Interactions between the Scc4 and Scc1 chaperones and CopN are characterized. Conclusion: Scc4 forms a ternary complex with Scc1 and CopN to promote CopN secretion during infection. Significance: Scc4 is an important link between the T3S system and transcription. Secretion of effector proteins into the eukaryotic host cell is required for Chlamydia trachomatis virulence. In the infection process, Scc1 and Scc4, two chaperones of the type III secretion (T3S) system, facilitate secretion of the important effector and plug protein, CopN, but little is known about the details of this event. Here we use biochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, and genetic analyses to characterize this trimolecular event. We find that Scc4 complexes with Scc1 and CopN in situ at the late developmental cycle of C. trachomatis. We show that Scc4 and Scc1 undergo dynamic interactions as part of the unique bacterial developmental cycle. Using alanine substitutions, we identify several amino acid residues in Scc4 that are critical for the Scc4-Scc1 interaction, which is required for forming the Scc4·Scc1·CopN ternary complex. These results, combined with our previous findings that Scc4 plays a role in transcription (Rao, X., Deighan, P., Hua, Z., Hu, X., Wang, J., Luo, M., Wang, J., Liang, Y., Zhong, G., Hochschild, A., and Shen, L. (2009) Genes Dev. 23, 1818–1829), reveal that the T3S process is linked to bacterial transcriptional events, all of which are mediated by Scc4 and its interacting proteins. A model describing how the T3S process may affect gene expression is proposed.