Interswitch Region Research Articles

Molecular insight into the potential functional role of pseudoenzyme GFOD1 via interaction with NKIRAS2.

The glucose-fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family includes a variety of oxidoreductases with a wide range of substrates that utilize NAD or NADP as redox cofactor. Human contains two members of this family, namely glucose-fructose oxidoreductase domain-containing protein 1 and 2 (GFOD1 and GFOD2). While GFOD1 exhibits low tissue specificity, it is notably expressed in the brain, potentially linked to psychiatric disorders and severe diseases. Nevertheless, the specific function, cofactor preference, and enzymatic activity of GFOD1 remain largely unknown. In this work, we find that GFOD1 does not bind to either NAD or NADP. Crystal structure analysis unveils that GFOD1 exists as a typical homodimer resembling other family members, but lacks essential residues required for cofactor binding, suggesting that it may function as a pseudoenzyme. Exploration of GFOD1-interacting partners in proteomic database identifies NF-κB inhibitor-interacting Ras-like 2 (NKIRAS2) as one potential candidate. Co-immunoprecipitation (co-IP) analysis indicates that GFOD1 interacts with both GTP- and GDP-bound forms of NKIRAS2. The predicted structural model of the GFOD1-NKIRAS2 complex is validated in cells using point mutants and shows that GFOD1 selectively recognizes the interswitch region of NKIRAS2. These findings reveal the distinct structural properties of GFOD1 and shed light on its potential functional role in cellular processes.

Abstract IA05: Uncovering new structural insights into RAS interactions with effectors and regulators

Abstract RAS proteins, which act as binary molecular switches, are implicated in approximately 20% of all cancer cases and are known to play a critical role in the initiation and progression of some of the deadliest cancers, such as lung, colon, and pancreatic cancers. When RAS proteins are in the GTP-bound state, they interact with various effector proteins, such as RAF Kinase, PI 3-Kinase, and RalGDS, leading to the activation of multiple signaling pathways in the cell. Our recent structural studies, including investigations into the KRAS-SIN1 (RBD-PH domain), KRAS-RAF1 (RBD-CRD), and SHOC2-MRAS-PP1C complex, have provided new insights into the vital role played by the switch-II and interswitch regions in the interaction between RAS and downstream effectors and regulators. These findings extend beyond the earlier observations that the switch-I region of RAS proteins alone plays a more significant role in RAS-effector interactions. Furthermore, research into the KRAS4a-SIN1 complex and the reported interaction between KRAS4a and hexokinase have given us a glimpse into the isoform-specific RAS-effector interaction. Finally, the investigation of the SHOC2-MRAS-PP1C complex has also revealed the crucial role played by other members of the RAS subfamily, such as MRAS, in RAF activation by canonical RAS isoforms. Collectively, these studies have shed new light on the mechanisms by which RAS interacts with downstream effectors and regulators. Citation Format: Dhirendra K. Simanshu. Uncovering new structural insights into RAS interactions with effectors and regulators [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA05.

Pressure-based mapping of Arf1 switch transition.

Biomolecular functions are often associated with complex conformational reorganization. These concerted, nontrivial rearrangements among various regions of proteins are essential for their biological functions. Although crystal structures of proteins can provide snapshots of end states essential for their function, a thorough understanding of the structural mechanics of conformational ensemble associated with protein function remains elusive for most proteins. A combination of experimental and computational techniques can assist in understanding these processes. ADP-ribosylation factor 1 (Arf1) is a small GTPase that is implicated in cellular trafficking from the endoplasmic reticulum to the Golgi complex. The transition of Arf1 from its GDP-bound inactive state to the GTP-bound, active state involves a series of conformational transitions resulting in GDP dissociation, a shift in the register of the β-strands in the inter-switch region, and reorganization of the N-terminal helix, and the switch 1 and switch 2 regions. Here we use a combination of molecular modelling, extensive molecular dynamic simulations, pressure perturbation NMR-constrained simulations and pressure perturbation SAXS guided ensemble optimization methods to obtain a comprehensive picture of the functional dynamics of Arf1. Our approach illuminates the events associated with the allosteric communication required for the activation of Arf1.

Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11-WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44-Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events.

Structural basis of human PDZD8\u2013Rab7 interaction for the ER-late endosome tethering

The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation.

Genetic and structural studies of RABL3 reveal an essential role in lymphoid development and function

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a β-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3xm.

Structures of N-terminally processed KRAS provide insight into the role of N-acetylation

Although post-translational modification of the C-terminus of RAS has been studied extensively, little is known about N-terminal processing. Mass spectrometric characterization of KRAS expressed in mammalian cells showed cleavage of the initiator methionine (iMet) and N-acetylation of the nascent N-terminus. Interestingly, structural studies on GDP- and GMPPNP-bound KRAS lacking the iMet and N-acetylation resulted in Mg2+-free structures of KRAS with flexible N-termini. In the Mg2+-free KRAS-GDP structure, the flexible N-terminus causes conformational changes in the interswitch region resulting in a fully open conformation of switch I. In the Mg2+-free KRAS-GMPPNP structure, the flexible N-terminus causes conformational changes around residue A59 resulting in the loss of Mg2+ and switch I in the inactive state 1 conformation. Structural studies on N-acetylated KRAS-GDP lacking the iMet revealed the presence of Mg2+ and a conformation of switch regions also observed in the structure of GDP-bound unprocessed KRAS with the iMet. In the absence of the iMet, the N-acetyl group interacts with the central beta-sheet and stabilizes the N-terminus and the switch regions. These results suggest there is crosstalk between the N-terminus and the Mg2+ binding site, and that N-acetylation plays an important role by stabilizing the N-terminus of RAS upon excision of the iMet.

Molecular Characterization of Differentially Expressed ADP Ribosylation Factor from WSSV Infected Black Tiger Shrimp Penaeus monodon

Suppression subtractive hybridization (SSH) cDNA library was constructed using hepatopancreas of the WSSV infected black tiger shrimp, Penaeus monodon. The differentially expressed ESTs of SSH cDNA library represented different functional categories belonging to energy and metabolism, osmoregulation, cytoskeletal and structural proteins, membrane traffic and organelle structure, immune genes, protein folding, transport and synthesis, ovarian and oocyte development, nucleic acid processing, cell cycle and heat shock protein. The ADP ribosylation factor (Arf) was one of the differentially expressed gene which was identified from the SSH cDNA library. The full length cDNA of P. monodon Arf (pmArf) was cloned by 3′- and 5′-rapid amplification of cDNA ends (3′/5′ RACE). The complete nucleotide sequence of pmArf encoded protein sequence of 180 amino acids revealed the characteristic conserved features of the Arf family proteins represented by the P-loop, the switch regions and interswitch region. The sequence homology indicated that the pmArf belonged to class II of Arf family. The gene expression analysis of pmArf and other immune related genes (penaeidin, anti-lipopolysaccharide factor and QM) in the gills, hepatopancreas and gut tissues of the WSSV infected shrimp was carried out by Real-Time PCR. The pmArf gene was up-regulated with highest gene expression levels at 24 h (1.33-fold) in the gill tissues and at 48 h in the gut tissues (1.44-fold) and hepatopancreas (1.46-fold) of WSSV infected shrimp, indicating its potential role in immune response against WSSV infection.

Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure

C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD(+) to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3.

The Function of Embryonic Stem Cell-expressed RAS (E-RAS), a Unique RAS Family Member, Correlates with Its Additional Motifs and Its Structural Properties

E-RAS is a member of the RAS family specifically expressed in embryonic stem cells, gastric tumors, and hepatic stellate cells. Unlike classical RAS isoforms (H-, N-, and K-RAS4B), E-RAS has, in addition to striking and remarkable sequence deviations, an extended 38-amino acid-long unique N-terminal region with still unknown functions. We investigated the molecular mechanism of E-RAS regulation and function with respect to its sequence and structural features. We found that N-terminal extension of E-RAS is important for E-RAS signaling activity. E-RAS protein most remarkably revealed a different mode of effector interaction as compared with H-RAS, which correlates with deviations in the effector-binding site of E-RAS. Of all these residues, tryptophan 79 (arginine 41 in H-RAS), in the interswitch region, modulates the effector selectivity of RAS proteins from H-RAS to E-RAS features.

Crystal Structure of the Rab9A-RUTBC2 RBD Complex Reveals the Molecular Basis for the Binding Specificity of Rab9A with RUTBC2

Rab9 plays a vital role in regulating the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network through interactions with various effectors. Here, we report the crystal structure of GTP-bound Rab9A in complex with the Rab-binding domain (RBD) of the effector RUTBC2. RUTBC2 RBD assumes a pleckstrin homology domain fold that uses a binding site consisting of mainly β1 and the η1 insertion to interact with the switch and interswitch regions of Rab9A. The C-terminal hypervariable region of Rab9A is disordered and thus not required for RUTBC2 binding. The conformational plasticity of the switch and interswitch regions of Rab9A primarily determines the specificity for RUTBC2. Our biochemical and biological data confirm these findings and further show that Rab9B can bind to RUTBC2 probably in a similar manner as Rab9A. These results together reveal the molecular basis for the binding specificity of Rab9A with RUTBC2.

A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1

The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X-linked disease caused by mutations of the inositol-5-phosphatase OCRL1. OCRL1 is a membrane-bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab-binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α-helical structure elements, the Rab-binding interface of OCRL1 consists mainly of the IgG-like β-strand structure of the ASPM-SPD-2-Hydin domain as well as one α-helix. Our results give a deeper structural understanding of disease-causing mutations of OCRL1 affecting Rab binding.

Insight into the Role of Dynamics in the Conformational Switch of the Small GTP-binding Protein Arf1

Activation of the small GTP-binding protein Arf1, a major regulator of cellular traffic, follows an ordered sequence of structural events, which have been pictured by crystallographic snapshots. Combined with biochemical analysis, these data lead to a model of Arf1 activation, in which opening of its N-terminal helix first translocates Arf1-GDP to membranes, where it is then secured by a register shift of the interswitch β-strands, before GDP is eventually exchanged for GTP. However, how Arf1 rearranges its central β-sheet, an event that involves the loss and re-formation of H-bonds deep within the protein core, is not explained by available structural data. Here, we used Δ17Arf1, in which the N-terminal helix has been deleted, to address this issue by NMR structural and dynamics analysis. We first completed the assignment of Δ17Arf1 bound to GDP, GTP, and GTPγS and established that NMR data are fully consistent with the crystal structures of Arf1-GDP and Δ17Arf1-GTP. Our assignments allowed us to analyze the kinetics of both protein conformational transitions and nucleotide exchange by real-time NMR. Analysis of the dynamics over a very large range of timescale by (15)N relaxation, CPMG relaxation dispersion and H/D exchange reveals that while Δ17Arf1-GTP and full-length Arf1-GDP dynamics is restricted to localized fast motions, Δ17Arf1-GDP features unique intermediate and slow motions in the interswitch region. Altogether, the NMR data bring insight into how that membrane-bound Arf1-GDP, which is mimicked by the truncation of the N-terminal helix, acquires internal motions that enable the toggle of the interswitch.

GDP-bound and Nucleotide-free Intermediates of the Guanine Nucleotide Exchange in the Rab5·Vps9 System

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

Structural basis for Rab GTPase recognition and endosome tethering by the C 2 H 2 zinc finger of Early Endosomal Autoantigen 1 (EEA1)

Regulation of endosomal trafficking by Rab GTPases depends on selective interactions with multivalent effectors, including EEA1 and Rabenosyn-5, which facilitate endosome tethering, sorting, and fusion. Both EEA1 and Rabenosyn-5 contain a distinctive N-terminal C(2)H(2) zinc finger that binds Rab5. How these C(2)H(2) zinc fingers recognize Rab GTPases remains unknown. Here, we report the crystal structure of Rab5A in complex with the EEA1 C(2)H(2) zinc finger. The binding interface involves all elements of the zinc finger as well as a short N-terminal extension but is restricted to the switch and interswitch regions of Rab5. High selectivity for Rab5 and, to a lesser extent Rab22, is observed in quantitative profiles of binding to Rab family GTPases. Although critical determinants are identified in both switch regions, Rab4-to-Rab5 conversion-of-specificity mutants reveal an essential requirement for additional substitutions in the proximal protein core that are predicted to indirectly influence recognition through affects on the structure and conformational stability of the switch regions.

Molecular cloning and characterization of a class II ADP ribosylation factor from the shrimp Marsupenaeus japonicus

In shrimp, small GTPases in the Ras superfamily can regulate hemolytic phagocytosis against WSSV infection. However, the ADP ribosylation factors (Arfs), also belonging to the regulatory GTP-binding proteins and playing a central role in membrane trafficking, have not been reported yet in shrimp and their relationship with WSSV infection is completely unknown to date. Here, a novel class II Arf (designated as MjArf4) was cloned and characterized from the shrimp Marsupenaeus japonicus. Like other Arfs, MjArf4 contains an N-terminal myristoylated site, a p loop, switch regions, as well as an interswitch region. In High Five cells, when MjArf4 was in its GDP-bound form, it dispersed into the whole cell, whereas in the GTP-bound form it promoted formation of a punctuate Golgi-like structure, indicating that the MjArf4 distribution was dependent on its GDP/GTP binding. After challenge with WSSV, the mRNA level of MjArf4 was up-regulated significantly as WSSV propagated. Thus, a member of the Arf family was characterized for the first time in shrimp and found to be involved in WSSV infection.

Structural aspects of Rab6–effector complexes

The small GTPase Rab6 regulates vesicle trafficking at the level of Golgi. Recently, the crystal structures of Rab6 in complexes with two unrelated effectors have been determined. The structure of Rab6a-GTP in complex with a 378-residue internal fragment of the effector Rab6IP1 (Rab6-interacting protein 1) has been solved. In addition, the structure of Rab6 with the golgin, GCC185, has also been determined. In both complexes, two alpha-helices from the effector mediate binding to switch I, switch II and the interswitch region of Rab6. Comparisons of the complexes reveal significant conformational changes in the conserved hydrophobic triad of Rab6. Thus conformational flexibility in the triad mediates recognition of compositionally distinct alpha-helical coiled coils, providing a rationale for the promiscuity of Rab6 in effector recruitment.

Structural insights into host GTPase isoform selection by a family of bacterial GEF mimics

The Escherichia coli type III effector Map belongs to a large family of bacterial virulence factors that activate host Rho GTPase signaling pathways through an unknown molecular mechanism. Here we report direct evidence that Map functions as a potent and selective guanine-nucleotide exchange factor (GEF) for Cdc42. The 2.3-A structure of the Map-Cdc42 complex revealed that Map mimics the GEF strategy of the mammalian Dbl family but has a three-dimensional architecture that is nearly identical to the bacterial GEF Salmonella spp. SopE. A comparative analysis between human and bacterial GEFs revealed a previously uncharacterized pairing mechanism between Map and the variable beta2-3 interswitch region of Cdc42. We propose a GTPase selection model that is experimentally validated by the preferential activation Rac1 and RhoA by the Shigella spp. effectors IpgB1 and IpgB2, respectively. These results significantly expand the repertoire of bacterial GEF mimics and unify a GEF selection mechanism for host GTPase substrates.

Structural Basis for Recruitment of Rab6-Interacting Protein 1 to Golgi via a RUN Domain

Small GTPase Rab6 regulates vesicle trafficking at the level of Golgi via recruitment of numerous and unrelated effectors. The crystal structure of Rab6a(GTP) in complex with a 378-residue internal fragment of the effector Rab6IP1 was solved at 3.2 angstroms resolution. This Rab6IP1 region encompasses an all alpha-helical RUN domain followed in tandem by a PLAT domain that adopts a beta sandwich fold. The structure reveals that the first and last alpha helices of the RUN domain mediate binding to switch I, switch II, and the interswitch region of Rab6. It represents the largest Rab-effector complex determined to date. Comparisons with the recent structure of Rab6 in complex with an unrelated effector, human golgin GCC185, reveals significant conformational changes in the conserved hydrophobic triad of Rab6. Flexibility in the switch and interswitch regions of Rab6 mediates recognition of compositionally distinct alpha-helical coiled coils, thereby contributing to Rab6 promiscuity in effector recruitment.

Crystal structure of the Sec4p:Sec2p complex in the nucleotide exchanging intermediate state

Vesicular transport during exocytosis is regulated by Rab GTPase (Sec4p in yeast), which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. Here, we report the crystal structure of the Sec2p GEF domain in a complex with the nucleotide-free Sec4p at 2.7 Å resolution. Upon complex formation, the Sec2p helices approach each other, flipping the side chain of Phe-109 toward Leu-104 and Leu-108 of Sec2p. These three residues provide a hydrophobic platform to attract the side chains of Phe-49, Ile-53, and Ile-55 in the switch I region as well as Phe-57 and Trp-74 in the interswitch region of Sec4p. Consequently, the switch I and II regions are largely deformed, to create a flat hydrophobic interface that snugly fits the surface of the Sec2p coiled coil. These drastic conformational changes disrupt the interactions between switch I and the bound guanine nucleotide, which facilitates the GDP release. Unlike the recently reported 3.3 Å structure of the Sec4p·Sec2p complex, our structure contains a phosphate ion bound to the P-loop, which may represent an intermediate state of the nucleotide exchange reaction.