N-terminal Module Research Articles

A dominant-negative Arabidopsis cation exchanger 1 (CAX1): N-terminal autoinhibition and membrane topology.

Calcium (Ca2+) is essential for plant growth and cellular homeostasis, with cation exchangers (CAXs) regulating Ca2+ transport into plant vacuoles. In Arabidopsis, multiple CAXs feature a common structural arrangement, comprising an N-terminal autoinhibitory domain followed by two pseudosymmetrical modules. Mutations in CAX1 enhance stress tolerance, notably tolerance to anoxia (a condition marked by oxygen depletion), crucial for flood resilience. Here we engineered a dominant-negative CAX1 variant, named ½N-CAX1, incorporating the autoinhibitory domain and the N-terminal pseudosymmetrical module, which, when expressed in wild-type Arabidopsis plants, phenocopied the anoxia tolerance of cax1. Physiological evaluations, yeast assays, and calcium imaging demonstrated that wild-type plants expressing ½N-CAX1 have phenotypes consistent with inhibition of CAX1, which is likely through direct interaction of ½N-CAX1 with CAX1. Eliminating segments within the N-terminal pseudosymmetrical module, as well as incorporating modules from other plant CAXs and expressing these variants into wild-type plants, failed to produce anoxia tolerance. This underscores the requirement for both the CAX1 autoinhibitory domain and the intact pseudosymmetrical module to produce the dominant-negative phenotype. Our study elucidates the interaction of this ½N-CAX1 variant with CAX1 and its impact on anoxia tolerance, offering insights into further approaches for engineering plant stress tolerance.

Anti-angiogenic activity of a novel angiostatin-like plasminogen fragment produced by a bacterial metalloproteinase

Tumor growth depends on angiogenesis, a process by which new blood vessel are formed from pre-existing normal blood vessels. Proteolytic fragments of plasminogen, containing varying numbers of plasminogen kringle domains, collectively known as angiostatin, are a naturally occurring inhibitor of angiogenesis and inhibit tumor growth. We have developed an “affinity-capture reactor” that enables a single-step method for the production/purification of an angiostatin-like plasminogen fragment from human plasma using an immobilized bacterial metalloproteinase. The resulting fragment, named BL-angiostatin, contains one or two glycosyl chains and the N-terminal PAN module, which are not present in canonical angiostatins tested for cancer treatment. BL-angiostatin inhibited angiogenesis in vitro at 20 nM and the growth of both allograft and human xenograft tumors as well as lung metastasis of primary tumors mice at 0.3–10 mg kg−1. Derivatives of BL angiostatin lacking the PAN module or the terminal sialic acids in the glycosyl chains showed reduced anti-angiogenic activity in vivo, suggesting a role for these functions in activity, possibly via conferring a pharmacokinetic advantage to BL angiostatin compared to recombinant angiostatin lacking both features. These results highlight the potential of BL-angiostatin for therapeutic applications.

Crystal structure of the photosensory module from a PAS-less cyanobacterial phytochrome as Pr shows a mix of dark-adapted and photoactivated features

Phytochromes (Phys) are a diverse collection of photoreceptors that regulate numerous physiological and developmental processes in microorganisms and plants through photointerconversion between red-light absorbing Pr and far-red light-absorbing Pfr states. Light is detected by an N-terminal photosensing module (PSM) sequentially comprised of Period/ARNT/Sim (PAS), cGMP-phosphodiesterase/adenylyl cyclase/FhlA (GAF), and Phy-specific (PHY) domains, with the bilin chromophore covalently-bound within the GAF domain. Phys sense light via the Pr/Pfr ratio measured by light-induced rotation of the bilin D-pyrrole ring that triggers conformational changes within the PSM, which for microbial Phys reaches into an output region. A key step is a β-stranded to α-helical reconfiguration of a hairpin loop extending from the PHY domain to contact the GAF domain. Besides canonical Phys, cyanobacteria express several variants, including a PAS-less subfamily that harbors just the GAF and PHY domains for light detection. Prior 2D-NMR studies of a model PAS-less Phy from Synechococcus_sp._JA-2-3B'a(2-13) (SyB-Cph1) proposed a unique photoconversion mechanism involving an A-pyrrole ring rotation, while magic-angle-spinning NMR probing the chromophore proposed the prototypic D-ring flip. To help solve this conundrum, we determined the crystallographic structure of the GAF-PHY region from SyB-Cph1 as Pr. Surprisingly, this structure differs from canonical Phys by having a Pr ZZZsyn,syn,anti bilin configuration but shifted to the activated position in the binding pocket with consequent folding of the hairpin loop to α-helical, an architecture common for Pfr. Collectively, the PSM of SyB-Cph1 as Pr displayed a mix of dark-adapted and photoactivated features whose co-planar A-C pyrrole rings supports a D-ring flip mechanism.

Structural characterization of regulation by a dynamic N-terminal module in the pentameric ligand-gated ion channel DeCLIC

Structural characterization of regulation by a dynamic N-terminal module in the pentameric ligand-gated ion channel DeCLIC

The amino acid region from 448-517 of CAMTA3 transcription factor containing a part of the TIG domain represses the N-terminal repression module function.

The online version contains supplementary material available at 10.1007/s12298-023-01401-w.

Functional and structural analyses reveal that a dual domain sialidase protects bacteria from complement killing through desialylation of complement factors.

The complement system is the first line of innate immune defense against microbial infections. To survive in humans and cause infections, bacterial pathogens have developed sophisticated mechanisms to subvert the complement-mediated bactericidal activity. There are reports that sialidases, also known as neuraminidases, are implicated in bacterial complement resistance; however, its underlying molecular mechanism remains elusive. Several complement proteins (e.g., C1q, C4, and C5) and regulators (e.g., factor H and C4bp) are modified by various sialoglycans (glycans with terminal sialic acids), which are essential for their functions. This report provides both functional and structural evidence that bacterial sialidases can disarm the complement system via desialylating key complement proteins and regulators. The oral bacterium Porphyromonas gingivalis, a "keystone" pathogen of periodontitis, produces a dual domain sialidase (PG0352). Biochemical analyses reveal that PG0352 can desialylate human serum and complement factors and thus protect bacteria from serum killing. Structural analyses show that PG0352 contains a N-terminal carbohydrate-binding module (CBM) and a C-terminal sialidase domain that exhibits a canonical six-bladed β-propeller sialidase fold with each blade composed of 3-4 antiparallel β-strands. Follow-up functional studies show that PG0352 forms monomers and is active in a broad range of pH. While PG0352 can remove both N-acetylneuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc), it has a higher affinity to Neu5Ac, the most abundant sialic acid in humans. Structural and functional analyses further demonstrate that the CBM binds to carbohydrates and serum glycoproteins. The results shown in this report provide new insights into understanding the role of sialidases in bacterial virulence and open a new avenue to investigate the molecular mechanisms of bacterial complement resistance.

Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

Bacterial NLR-related proteins protect against phage

Bacteria use a wide range of immune pathways to counter phage infection. A subset of these genes shares homology with components of eukaryotic immune systems, suggesting that eukaryotes horizontally acquired certain innate immune genes from bacteria. Here, we show that proteins containing a NACHT module, the central feature of the animal nucleotide-binding domain and leucine-rich repeat containing gene family (NLRs), are found in bacteria and defend against phages. NACHT proteins are widespread in bacteria, provide immunity against both DNA and RNA phages, and display the characteristic C-terminal sensor, central NACHT, and N-terminal effector modules. Some bacterial NACHT proteins have domain architectures similar to the human NLRs that are critical components of inflammasomes. Human disease-associated NLR mutations that cause stimulus-independent activation of the inflammasome also activate bacterial NACHT proteins, supporting a shared signaling mechanism. This work establishes that NACHT module-containing proteins are ancient mediators of innate immunity across the tree of life.

Relaxin family peptide receptors in GtoPdb v.2023.1

Relaxin family peptide receptors (RXFP, nomenclature as agreed by the NC-IUPHAR Subcommittee on Relaxin family peptide receptors [23, 119]) may be divided into two pairs, RXFP1/2 and RXFP3/4. Endogenous agonists at these receptors are heterodimeric peptide hormones structurally related to insulin: relaxin-1, relaxin, relaxin-3 (also known as INSL7), insulin-like peptide 3 (INSL3) and INSL5. Species homologues of relaxin have distinct pharmacology and relaxin interacts with RXFP1, RXFP2 and RXFP3, whereas mouse and rat relaxin selectively bind to and activate RXFP1 [260]. relaxin-3 is the ligand for RXFP3 but it also binds to RXFP1 and RXFP4 and has differential affinity for RXFP2 between species [259]. INSL5 is the ligand for RXFP4 but is a weak antagonist of RXFP3. relaxin and INSL3 have multiple complex binding interactions with RXFP1 [267] and RXFP2 [132] which direct the N-terminal LDLa modules of the receptors together with a linker domain to act as a tethered ligand to direct receptor signaling [262]. INSL5 and relaxin-3 interact with their receptors using distinct residues in their B-chains for binding, and activation, respectively [321, 152].

All DACs in a Row: Domain Architectures of Bacterial and Archaeal Diadenylate Cyclases.

Cyclic dimeric AMP (c-di-AMP) is a widespread second messenger that controls such key functions as osmotic homeostasis, peptidoglycan biosynthesis, and response to various stresses. C-di-AMP is synthesized by diadenylate cyclases that contain the DAC (DisA_N) domain, which was originally characterized as the N-terminal domain in the DNA integrity scanning protein DisA. In other experimentally studied diadenylate cyclases, DAC domain is typically located at the protein C termini and its enzymatic activity is controlled by one or more N-terminal domains. As in other bacterial signal transduction proteins, these N-terminal modules appear to sense environmental or intracellular signals through ligand binding and/or protein-protein interactions. Studies of bacterial and archaeal diadenylate cyclases also revealed numerous sequences with uncharacterized N-terminal regions. This work provides a comprehensive review of the N-terminal domains of bacterial and archaeal diadenylate cyclases, including the description of five previously undefined domains and three PK_C-related domains of the DacZ_N superfamily. These data are used to classify diadenylate cyclases into 22 families, based on their conserved domain architectures and the phylogeny of their DAC domains. Although the nature of the regulatory signals remains obscure, the association of certain dac genes with anti-phage defense CBASS systems and other phage-resistance genes suggests that c-di-AMP might also be involved in the signaling of phage infection.

Mechanism and linkage specificities of the dual retaining β-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-β-d-manno-oct-2-ulosonic acid (β-Kdo) from cytidine-5'-monophospho-β-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with β-(2→4) and β-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining β-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the β-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/β domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

Structural characterization of pH- and calcium-modulated open and closed states of the pentameric ligand-gated ion channel DeCLIC.

Pentameric ligand-gated ion channels are critical mediators of electrochemical signal transduction in a variety of excitable cells. Biophysical and pharmacological development in this family relies heavily on high-quality structural data in multiple distinct functional states. Bacterial pLGICs can provide valuable insight into fundamental mechanisms of gating, as they often desensitize to a lesser extent than eukaryotic channels, while retaining sensitivity to modulatory conditions such as pH and environmental calcium. Recently, a new prokaryotic family member (DeCLIC) from a Desulfofustis deltaproteobacterium was identified, including a previously uncharacterized N-terminal domain. The channel was found to be modulated by calcium ions, and with Ca2+ present it crystallized with a dramatically expanded pore. Here, we used cryo-electron microscopy to identify multiple conformations of the channel under acidic conditions. When Ca2+ was present, two classes of particles of roughly equal proportion were identified, one class was similar to the crystallographic closed state, while the second class represented an apparent open state notably divergent from the open X-ray structure. When Ca2+ was absent, the predominant class had a similar open pore, while a second class had a closed pore and evidence for a highly dynamic N-terminal domain. These data provide new insight into pH modulation and the under-characterized open state of pentameric channels, as well as a novel mechanism of dynamic ion-channel regulation via an N-terminal module.

Molecular Characterization and Function of the Nogo-66 Receptor (NgR1) Gene in the Chinese Tree Shrew

Nogo-66 receptor (NgR1) is a glycosylphosphatidylinositol-linked cell surface receptor with high affinity for Nogo-66. The binding of Nogo-66 to NgR1 plays a key role in inhibiting neurite growth, limiting synaptic plasticity and mediating Mammalian Reovirus (MRV) infection. The Chinese tree shrew (Tupaia belangeri chinensis) is, a new and valuable experimental animal that is widely used in biomedical research. Although susceptible to MRV, little is known about tree shrew NgR1 and its role in MRV infection. In this study, we cloned NgR1 form the Chinese tree shrew by RACE technology and analyzed its characteristics, spatial structure and its tissue expression. We also examined the expression pattern of NgR1 in the response of tree shrew primary nerve cells (tNC) to MRV1/TS/2011 infection. Tree shrew NgR1 was found to have a closer relationship to human NgR1 (90.34%) than to mouse NgR1. Similar to the protein structure of human NgR1, the tree shrew NgR1 has the same leucine-rich repeat (LRR) domain structure that is capped by C-terminal and N-terminal cysteine-rich modules. The tree shrew NgR1 mRNAs were predominantly detected in the central nervous system (CNS), and tree shrew NgR1 can mediate infection by MRV1/TS/2011. Taken together, these results help to elucidate the function of NgR1 and provide a basis for using the tree shrew as an animal model for studies of the nervous system and infectious diseases.

Characteristic Evaluation of Recombinant MiSp/Poly(lactic-co-glycolic) Acid (PLGA) Nanofiber Scaffolds as Potential Scaffolds for Bone Tissue Engineering.

Biomaterial-based nanofibrous scaffolds are the most effective alternative to bone transplantation therapy. Here, two recombinant minor ampullate spidroins (spider silk proteins), R1SR2 and NR1SR2C, were blended with Poly(lactic-co-glycolic) Acid (PLGA), respectively, to generate nanofiber scaffolds by electrospinning. The N-terminal (N), C-terminal (C), repeating (R1 and R2) and spacer (S) modules were all derived from the minor ampullate spidroins (MiSp). The physical properties and structures of the blended scaffolds were measured by scanning electron microscopy (SEM), water contact angle measurement, Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and Tensile mechanical testing. The results showed that blending of MiSp (R1SR2 and NR1SR2C) reduced the diameter of nanofibers, increased the porosity and glass transition temperatures of nanofibrous scaffolds, and effectively improved the hydrophilicity and ultimate strain of scaffolds. It is worth noting that the above changes were more significant in the presence of the N- and C-termini of MiSp. In cell culture assays, human bone mesenchymal stem cells (HBMSCs) grown on NR1SR2C/PLGA (20/80) scaffolds displayed markedly enhanced proliferative and adhesive abilities compared with counterparts grown on pure PLGA scaffolds. Jointly, these findings indicated recombinant MiSp/PLGA, particularly NR1SR2C/PLGA (20/80) blend nanofibrous scaffolds, is promising for bone tissue engineering.

The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions

The evolutionary conserved CCR4-NOT complex functions in the cytoplasm as the main mRNA deadenylase in both constitutive mRNA turnover and regulated mRNA decay pathways. The versatility of this complex is underpinned by its modular multi-subunit organization, with distinct structural modules actuating different functions. The structure and function of all modules are known, except for that of the N-terminal module. Using different structural approaches, we obtained high-resolution data revealing the architecture of the human N-terminal module composed of CNOT1, CNOT10, and CNOT11. The structure shows how two helical domains of CNOT1 sandwich CNOT10 and CNOT11, leaving the most conserved domain of CNOT11 protruding into solvent as an antenna. We discovered that GGNBP2, a protein identified as a tumor suppressor and spermatogenic factor, is a conserved interacting partner of the CNOT11 antenna domain. Structural and biochemical analyses thus pinpoint the N-terminal CNOT1-CNOT10-CNOT11 module as a conserved protein-protein interaction platform.

Discovery and Characterization of the Metallopterin-Dependent Ergothioneine Synthase from Caldithrix abyssi.

Ergothioneine is a histidine derivative with a 2-mercaptoimidazole side chain and a trimethylated α-amino group. Although the physiological function of this natural product is not yet understood, the facts that many bacteria, some archaea, and most fungi produce ergothioneine and that plants and animals have specific mechanisms to absorb and distribute ergothioneine in specific tissues suggest a fundamental role in cellular life. The observation that ergothioneine biosynthesis has emerged multiple times in molecular evolution points to the same conclusion. Aerobic bacteria and fungi attach sulfur to the imidazole ring of trimethylhistidine via an O2-dependent reaction that is catalyzed by a mononuclear non-heme iron enzyme. Green sulfur bacteria and archaea use a rhodanese-like sulfur transferase to attach sulfur via oxidative polar substitution. In this report, we describe a third unrelated class of enzymes that catalyze sulfur transfer in ergothioneine production. The metallopterin-dependent ergothioneine synthase from Caldithrix abyssi contains an N-terminal module that is related to the tungsten-dependent acetylene hydratase and a C-terminal domain that is a functional cysteine desulfurase. The two modules cooperate to transfer sulfur from cysteine onto trimethylhistidine. Inactivation of the C-terminal desulfurase blocks ergothioneine production but maintains the ability of the metallopterin to exchange sulfur between ergothioneine and trimethylhistidine. Homologous bifunctional enzymes are encoded exclusively in anaerobic bacterial and archaeal species.

Modular Adapters Utilizing Binders of Different Molecular Types Expand Cell-Targeting Options for Adenovirus Gene Delivery.

Efficient and cell-specific delivery of DNA is essential for the effective and safe use of gene delivery technologies. Consequently, a large variety of technologies have been developed and applied in a wide range of ex vivo and in vivo applications, including multiple approaches based on viral vectors. However, widespread success of a technology is largely determined by the versatility of the method and the ease of use. The rationally designed adapter technology previously developed redirects widely used human adenovirus serotype 5 (HAdV-C5) to a defined cell population, by binding and blocking the adenoviral knob tropism while simultaneously allowing fusions of an N-terminal retargeting module. Here we expand modularity, and thus applicability of this adapter technology, by extending the nature of the cell-binding portion. We report successful receptor-specific transduction mediated by a retargeting module consisting of either a DARPin, a single-chain variable fragment (scFv) of an antibody, a peptide, or a small molecule ligand. Furthermore, we show that an adapter can be engineered to carry more than one specificity, allowing dual targeting. Specific HAdV-C5 retargeting was thus demonstrated to human epidermal growth factor receptor 2 (HER2), human folate receptorα, and neurotensin receptor 1, effective at vector concentrations as low as a multiplicity of infection of 2.5. Therefore, we report a modular design which allows plug-and-play combinations of different binding modules, leading to efficient and specific mono- or dual-targeting while circumventing tedious optimization procedures. This extends the technology to combinational applications of cell-specific binding, supporting research in gene therapy, synthetic biology, and biotechnology.

English

Bacteriophytochromes (BphPs) are light-regulated biliprotein photoreceptors widely occurring in bacteria. BphPs exists in two spectral distinct states: the red-light absorbing state (Pr) and the far-red light-absorbing state (Pfr). BphPs are composed of an N-terminal photosensory core module and a light-controlled C-terminal histidine kinase module. The detailed mechanism on how photoconversion, initiated by a light-triggered isomerization of the biliverdin chromophore, is conducted from the N-terminal to the C-terminal domain and how the histidine kinase is modulated is still not clear. In this work Agp1 from Agrobacterium tumefaciens is used as a model phytochrome. The method of labeling full-length Agp1 mutants with single accessible Cys residue using fluorescence probes is described. Different absorption spectra showed that neither mutagenesis nor fluorescence labeling exerts significant effects on the photoconversion behavior of various Agp1 mutants. The histidine kinase activity assay revealed that the labeled holoproteins exhibit wild-types like autophosphorylation activity that depends on the Pr and the Pfr state. Fluorescence spectroscopic data suggested that fluorescence resonance energy transfer occurs from the donor Atto495 to the acceptor Atto565 in double-labeled Agp1-A362C, K514C, H554C, R603C, I653C, and V674C mutants. Red light irradiation gave rise to small changes on fluorescence emission intensity in some mutants but in some no remarkable inter-subunit distance changes occurred during the photo-conversion from Pr to Pfr in full-length Agp1. In contrast to double-labeled samples, significant changes in fluorescence intensity were observed in single-labeled samples.   Key words: Bacteriophytochromes (BphPs), C-terminal domain, photosensory, phytochrome. 

Noncanonical function of Capicua as a growth termination signal in Drosophila oogenesis

Capicua (Cic) proteins are conserved HMG-box transcriptional repressors that control receptor tyrosine kinase (RTK) signaling responses and are implicated in human neurological syndromes and cancer. While Cic is known to exist as short (Cic-S) and long (Cic-L) isoforms with identical HMG-box and associated core regions but distinct N termini, most previous studies have focused on Cic-S, leaving the function of Cic-L unexplored. Here we show that Cic-L acts in two capacities during Drosophila oogenesis: 1) as a canonical sensor of RTK signaling in somatic follicle cells, and 2) as a regulator of postmitotic growth in germline nurse cells. In these latter cells, Cic-L behaves as a temporal signal that terminates endoreplicative growth before they dump their contents into the oocyte. We show that Cic-L is necessary and sufficient for nurse cell endoreplication arrest and induces both stabilization of CycE and down-regulation of Myc. Surprisingly, this function depends mainly on the Cic-L-specific N-terminal module, which is capable of acting independently of the Cic HMG-box-containing core. Mirroring these observations, basal metazoans possess truncated Cic-like proteins composed only of Cic-L N-terminal sequences, suggesting that this module plays unique, ancient roles unrelated to the canonical function of Cic.

Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism

P-Rex (PI(3,4,5)P3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gβγ and PI(3,4,5)P3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gβγ and PI(3,4,5)P3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.