Molecular principles of bidirectional signalling between membranes and small GTPases.

Most small GTPases actuate their functions on subcellular membranes, which are increasingly seen as integral components of small GTPase signalling. In this review, we used the highly studied regulation of Arf GTPases by their GEFs to categorize the molecular principles of membrane contributions to small GTPase signalling, which have been highlighted by integrated structural biology combining in vitro reconstitutions in artificial membranes and high-resolution structures. As an illustration of how this framework can be harnessed to better understand the cooperation between small GTPases, their regulators and membranes, we applied it to the activation of the small GTPase Rac1 by DOCK-ELMO, identifying novel contributions of membranes to Rac1 activation. We propose that these structure-based principles should be considered when interrogating the mechanisms whereby small GTPase systems ensure spatial and temporal control of cellular signalling on membranes.

Targeting non-alcoholic fatty liver disease: Design, X-ray co-crystal structure and synthesis of 'first-in-kind' inhibitors of serine/threonine kinase25.

We describe the synthesis of a series of 3-t-butyl 5-aminopyrazole p-substituted arylamides as inhibitors of serine-threonine25 (STK25), an enzyme implicated in the progression of non-alcoholic fatty liver disease (NAFLD). Appending a p-N-pyrrolidinosulphonamide group to the arylamide group led to a 'first-in kind' inhibitor with IC50 = 228 nM. A co-crystal structure with STK 25 revealed productive interactions which were also reproduced using molecular docking. A new series of triazolo dihydro oxazine carboxamides of 3-t-butyl 5-aminopyrazole was not active against STK25.

[New structures of mTORC1: Focus on Rag GTPases]. / Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag.

mTORC1 is a central player in cell growth, a process that is tightly regulated by the availability of nutrients and that controls various aspects of metabolism in the normal cell and in severe diseases such as cancers. mTORC1 is a large multiprotein complex, composed of the kinase subunit mTOR, of Ragulator, which attaches mTOR to the lysosome membrane, of the atypical Rag GTPases and the small GTPase RheB, whose nucleotide states directly dictate its localization to the lysosome and its kinase activity, and of RAPTOR, an adaptor that assembles the complex. The activity of the Rag GTPases is further controlled by the GATOR1 and folliculin complexes, which regulate their GTP/GDP conversion. Here, we review recent structures of important components of the mTORC1 machinery, determined by cryo-electron microscopy for the most part, which allow to reconstitute the architecture of active mTORC1 at near atomic resolution. Notably, we discuss how these structures shed new light on the roles of Rag GTPases and their regulators in mTORC1 regulation, and the perspectives that they open towards understanding the inner workings of mTORC1 on the lysosomal membrane.

TITLE: Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag. ABSTRACT: mTORC1 est un acteur central de la croissance cellulaire, un processus étroitement régulé par la disponibilité de nutriments et qui contrôle diverses étapes du métabolisme dans la cellule normale et au cours de maladies, comme les cancers. mTORC1 est un complexe multiprotéique de grande taille constitué de nombreuses sous-unités, parmi lesquelles deux types de GTPases, Rag et RheB, contrôlent directement sa localisation membranaire et son activité kinase. Dans cette revue, nous faisons le point sur une moisson de structures récentes, déterminées pour la plupart par cryo-microscopie électronique, qui sont en passe de reconstituer le puzzle de l'architecture de mTORC1. Nous discutons ce que ces structures révèlent sur le rôle des GTPases, et ce que leur connaissance ouvre comme perspectives pour comprendre comment mTORC1 fonctionne à la membrane du lysosome.

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing.

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.

Protein-membrane interactions in small GTPase signalling and pharmacology: perspectives from Arf GTPases studies.

Small GTPases, in association with their GEFs, GAPs and effectors, control major intracellular processes such as signal transduction, cytoskeletal dynamics and membrane trafficking. Accordingly, dysfunctions in their biochemical properties are associated with many diseases, including cancers, diabetes, infections, mental disorders and cardiac diseases, which makes them attractive targets for therapies. However, small GTPases signalling modules are not well-suited for classical inhibition strategies due to their mode of action that combines protein-protein and protein-membrane interactions. As a consequence, there is still no validated drug available on the market that target small GTPases, whether directly or through their regulators. Alternative inhibitory strategies are thus highly needed. Here we review recent studies that highlight the unique modalities of the interaction of small GTPases and their GEFs at the periphery of membranes, and discuss how they can be harnessed in drug discovery.

Structural Organization and Dynamics of Homodimeric Cytohesin Family Arf GTPase Exchange Factors in Solution and on Membranes.

Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed.

Publisher Correction: PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

In the version of this article originally published, several co-authors had incorrect affiliation footnote numbers listed in the author list. Tatiana Cañeque and Angelica Mariani should each have affiliation numbers 3, 4 and 5, and Emmanuelle Charafe-Jauffret should have number 6. Additionally, there was an extra space in the name of co-author Robert P. St.Onge. These errors have been corrected in the HTML and PDF versions of the paper and the Supplementary Information PDF.

PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein-membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2-Bragsin complex and structure-activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein-membrane interactions without disruption.

Multiple interactions between an Arf/GEF complex and charged lipids determine activation kinetics on the membrane.

Lipidated small GTPases and their regulators need to bind to membranes to propagate actions in the cell, but an integrated understanding of how the lipid bilayer exerts its effect has remained elusive. Here we focused on ADP ribosylation factor (Arf) GTPases, which orchestrate a variety of regulatory functions in lipid and membrane trafficking, and their activation by the guanine-nucleotide exchange factor (GEF) Brag2, which controls integrin endocytosis and cell adhesion and is impaired in cancer and developmental diseases. Biochemical and structural data are available that showed the exceptional efficiency of Arf activation by Brag2 on membranes. We determined the high-resolution crystal structure of unbound Brag2 containing the GEF (Sec7) and membrane-binding (pleckstrin homology) domains, revealing that it has a constitutively active conformation. We used this structure to analyze the interaction of uncomplexed Brag2 and of the myristoylated Arf1/Brag2 complex with a phosphatidylinositol bisphosphate (PIP2) -containing lipid bilayer, using coarse-grained molecular dynamics. These simulations revealed that the system forms a close-packed, oriented interaction with the membrane, in which multiple PIP2 lipids bind the canonical lipid-binding site and unique peripheral sites of the PH domain, the Arf GTPase and, unexpectedly, the Sec7 domain. We cross-validated these predictions by reconstituting the binding and kinetics of Arf and Brag2 in artificial membranes. Our coarse-grained structural model thus suggests that the high efficiency of Brag2 requires interaction with multiple lipids and a well-defined orientation on the membrane, resulting in a local PIP2 enrichment, which has the potential to signal toward the Arf pathway.

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles.

Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.

Chlamydia Hijacks ARF GTPases To Coordinate Microtubule Posttranslational Modifications and Golgi Complex Positioning.

The intracellular bacterium Chlamydia trachomatis develops in a parasitic compartment called the inclusion. Posttranslationally modified microtubules encase the inclusion, controlling the positioning of Golgi complex fragments around the inclusion. The molecular mechanisms by which Chlamydia coopts the host cytoskeleton and the Golgi complex to sustain its infectious compartment are unknown. Here, using a genetically modified Chlamydia strain, we discovered that both posttranslationally modified microtubules and Golgi complex positioning around the inclusion are controlled by the chlamydial inclusion protein CT813/CTL0184/InaC and host ARF GTPases. CT813 recruits ARF1 and ARF4 to the inclusion membrane, where they induce posttranslationally modified microtubules. Similarly, both ARF isoforms are required for the repositioning of Golgi complex fragments around the inclusion. We demonstrate that CT813 directly recruits ARF GTPases on the inclusion membrane and plays a pivotal role in their activation. Together, these results reveal that Chlamydia uses CT813 to hijack ARF GTPases to couple posttranslationally modified microtubules and Golgi complex repositioning at the inclusion.IMPORTANCEChlamydia trachomatis is an important cause of morbidity and a significant economic burden in the world. However, how Chlamydia develops its intracellular compartment, the so-called inclusion, is poorly understood. Using genetically engineered Chlamydia mutants, we discovered that the effector protein CT813 recruits and activates host ADP-ribosylation factor 1 (ARF1) and ARF4 to regulate microtubules. In this context, CT813 acts as a molecular platform that induces the posttranslational modification of microtubules around the inclusion. These cages are then used to reposition the Golgi complex during infection and promote the development of the inclusion. This study provides the first evidence that ARF1 and ARF4 play critical roles in controlling posttranslationally modified microtubules around the inclusion and that Chlamydia trachomatis hijacks this novel function of ARF to reposition the Golgi complex.

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface.

Arf GTPases assemble protein complexes on membranes to carry out major functions in cellular traffic. An essential step is their activation by guanine nucleotide exchange factors (GEFs), whose Sec7 domain stimulates GDP/GTP exchange. ArfGEFs form 2 major families: ArfGEFs with DCB, HUS and HDS domains (GBF1 and BIG1/BIG2 in humans), which act at the Golgi; and ArfGEFs with a C-terminal PH domain (cytohesin, EFA6 and BRAG), which function at the plasma membrane and endosomes. In addition, pathogenic bacteria encode an ArfGEF with a unique membrane-binding domain. Here we review the allosteric regulation of Arf GTPases and their GEFs at the membrane interface. Membranes contribute several regulatory layers: at the GTPase level, where activation by GTP is coupled to membrane recruitment by a built-in structural device; at the Sec7 domain, which manipulates this device to ensure that Arf-GTP is attached to membranes; and at the level of non-catalytic ArfGEF domains, which form direct or GTPase-mediated interactions with membranes that enable a spectacular diversity of regulatory regimes. Notably, we show here that membranes increase the efficiency of a large ArfGEF (human BIG1) by 32-fold by interacting directly with its N-terminal DCB and HUS domains. The diversity of allosteric regulatory regimes suggests that ArfGEFs can function in cascades and circuits to modulate the shape, amplitude and duration of Arf signals in cells. Because Arf-like GTPases feature autoinhibitory elements similar to those of Arf GTPases, we propose that their activation also requires allosteric interactions of these elements with membranes or other proteins.

RET recognition of GDNF-GFRα1 ligand by a composite binding site promotes membrane-proximal self-association.

The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.

Biochemical and structural insights into microtubule perturbation by CopN from Chlamydia pneumoniae.

Although the actin network is commonly hijacked by pathogens, there are few reports of parasites targeting microtubules. The proposed member of the LcrE protein family from some Chlamydia species (e.g. pCopN from C. pneumoniae) binds tubulin and inhibits microtubule assembly in vitro. From the pCopN structure and its similarity with that of MxiC from Shigella, we definitively confirm CopN as the Chlamydia homolog of the LcrE family of bacterial proteins involved in the regulation of type III secretion. We have also investigated the molecular basis for the pCopN effect on microtubules. We show that pCopN delays microtubule nucleation and acts as a pure tubulin-sequestering protein at steady state. It targets the ß subunit interface involved in the tubulin longitudinal self-association in a way that inhibits nucleotide exchange. pCopN contains three repetitions of a helical motif flanked by disordered N- and C-terminal extensions. We have identified the pCopN minimal tubulin-binding region within the second and third repeats. Together with the intriguing observation that C. trachomatis CopN does not bind tubulin, our data support the notion that, in addition to the shared function of type III secretion regulation, these proteins have evolved different functions in the host cytosol. Our results provide a mechanistic framework for understanding the C. pneumoniae CopN-specific inhibition of microtubule assembly.

The determinants that govern microtubule assembly from the atomic structure of GTP-tubulin.

Tubulin alternates between a soluble curved structure and a microtubule straight conformation. GTP binding to αß-tubulin is required for microtubule assembly, but whether this triggers conversion into a straighter structure is still debated. This is due, at least in part, to the lack of structural data for GTP-tubulin before assembly. Here, we report atomic-resolution crystal structures of soluble tubulin in the GDP and GTP nucleotide states in a complex with a stathmin-like domain. The structures differ locally in the neighborhood of the nucleotide. A loop movement in GTP-bound tubulin favors its recruitment to the ends of growing microtubules and facilitates its curved-to-straight transition, but this conversion has not proceeded yet. The data therefore argue for the conformational change toward the straight structure occurring as microtubule-specific contacts are established. They also suggest a model for the way the tubulin structure is modified in relation to microtubule assembly.