A converged ubiquitin-proteasome pathway for the degradation of TOC and TOM tail-anchored receptors.

In plants, thousands of nucleus-encoded proteins translated in the cytosol are sorted to chloroplasts and mitochondria by binding to specific receptors of the TOC (translocon on the outer chloroplast membrane) and the TOM (translocon on the outer mitochondrial membrane) complexes for import into those organelles. The degradation pathways for these receptors are unclear. Here, we discovered a converged ubiquitin-proteasome pathway for the degradation of Arabidopsis thaliana TOC and TOM tail-anchored receptors. The receptors are ubiquitinated by E3 ligase(s) and pulled from the outer membranes by the AAA+ adenosine triphosphatase CDC48, after which a previously uncharacterized cytosolic protein, transmembrane domain (TMD)-binding protein for tail-anchored outer membrane proteins (TTOP), binds to the exposed TMDs at the C termini of the receptors and CDC48, and delivers these complexes to the 26S proteasome.

Recent advances in plant endomembrane research and new microscopical techniques.

The endomembrane system consists of various membrane-bound organelles including the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), endosomes, and the lysosome/vacuole. Membrane trafficking between distinct compartments is mainly achieved by vesicular transport. As the endomembrane compartments and the machineries regulating the membrane trafficking are largely conserved across all eukaryotes, our current knowledge on organelle biogenesis and endomembrane trafficking in plants has mainly been shaped by corresponding studies in mammals and yeast. However, unique perspectives have emerged from plant cell biology research through the characterization of plant-specific regulators as well as the development and application of the state-of-the-art microscopical techniques. In this review, we summarize our current knowledge on the plant endomembrane system, with a focus on several distinct pathways: ER-to-Golgi transport, protein sorting at the TGN, endosomal sorting on multivesicular bodies, vacuolar trafficking/vacuole biogenesis, and the autophagy pathway. We also give an update on advanced imaging techniques for the plant cell biology research.

Delivering a toxic metal to the active site of urease.

Urease is a nickel (Ni) enzyme that is essential for the colonization of Helicobacter pylori in the human stomach. To solve the problem of delivering the toxic Ni ion to the active site without diffusing into the cytoplasm, cells have evolved metal carrier proteins, or metallochaperones, to deliver the toxic ions to specific protein complexes. Ni delivery requires urease to form an activation complex with the urease accessory proteins UreFD and UreG. Here, we determined the cryo-electron microscopy structures of H. pylori UreFD/urease and Klebsiella pneumoniae UreD/urease complexes at 2.3- and 2.7-angstrom resolutions, respectively. Combining structural, mutagenesis, and biochemical studies, we show that the formation of the activation complex opens a 100-angstrom-long tunnel, where the Ni ion is delivered through UreFD to the active site of urease.

The RRM-mediated RNA binding activity in T. brucei RAP1 is essential for VSG monoallelic expression.

Trypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. Its major surface antigen VSG is expressed from subtelomeric loci in a strictly monoallelic manner. We previously showed that the telomere protein TbRAP1 binds dsDNA through its 737RKRRR741 patch to silence VSGs globally. How TbRAP1 permits expression of the single active VSG is unknown. Through NMR structural analysis, we unexpectedly identify an RNA Recognition Motif (RRM) in TbRAP1, which is unprecedented for RAP1 homologs. Assisted by the 737RKRRR741 patch, TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Mutating conserved RRM residues abolishes the RNA binding activity, significantly decreases the active VSG RNA level, and derepresses silent VSGs. The competition between TbRAP1's RNA and dsDNA binding activities suggests a VSG monoallelic expression mechanism in which the active VSG's abundant RNA antagonizes TbRAP1's silencing effect, thereby sustaining its full-level expression.

Arabidopsis DXO1 activates RNMT1 to methylate the mRNA guanosine cap.

Eukaryotic messenger RNA (mRNA) typically contains a methylated guanosine (m7G) cap, which mediates major steps of mRNA metabolism. Recently, some RNAs in both prokaryotic and eukaryotic organisms have been found to carry a non-canonical cap such as the NAD cap. Here we report that Arabidopsis DXO family protein AtDXO1, which was previously known to be a decapping enzyme for NAD-capped RNAs (NAD-RNA), is an essential component for m7G capping. AtDXO1 associates with and activates RNA guanosine-7 methyltransferase (AtRNMT1) to catalyze conversion of the guanosine cap to the m7G cap. AtRNMT1 is an essential gene. Partial loss-of-function mutations of AtRNMT1 and knockout mutation of AtDXO1 reduce m7G-capped mRNA but increase G-capped mRNAs, leading to similar pleiotropic phenotypes, whereas overexpression of AtRNMT1 partially restores the atdxo1 phenotypes. This work reveals an important mechanism in m7G capping in plants by which the NAD-RNA decapping enzyme AtDXO1 is required for efficient guanosine cap methylation.

Moving nickel along the hydrogenase-urease maturation pathway.

Hydrogenases and ureases play vital metabolic functions in all three domains of life. However, nickel ions are cytotoxic because they can inactivate enzymes that require less competitive ions (e.g. Mg2+) in the Irving-Williams series to function. Life has evolved elegant mechanisms to solve the problem of delivering the toxic metal to the active site of nickel-containing enzymes inside the cells. Here, we review our current understanding of nickel trafficking along the hydrogenase and urease maturation pathways. Metallochaperones and accessory proteins (SlyD, HypA, HypB, UreD, UreE, UreF, and UreG) form specific protein complexes to allow the transfer of nickel from one protein to another without releasing the toxic metal into the cytoplasm. The role of SlyD is not fully understood, but it can interact with and transfer its nickel to HypB. In the hydrogenase maturation pathway, nickel is transferred from HypB to HypA, which can then deliver its nickel to the hydrogenase large subunit precursor. In Helicobacter pylori, the urease maturation pathway receives its nickel from HypA of the hydrogenase maturation pathway via the formation of a HypA/UreE2 complex. Guanosine triphosphate (GTP) binding promotes the formation of a UreE2G2 complex, where UreG receives a nickel from UreE. In the final step of the urease maturation, nickel/GTP-bound UreG forms an activation complex with UreF, UreD, and apo-urease. Upon GTP hydrolysis, nickel is released from UreG to the urease. Finally, some common themes learned from the hydrogenase-urease maturation pathway are discussed.

The flexible N-terminal motif of uL11 unique to eukaryotic ribosomes interacts with P-complex and facilitates protein translation.

Eukaryotic uL11 contains a conserved MPPKFDP motif at the N-terminus that is not found in archaeal and bacterial homologs. Here, we determined the solution structure of human uL11 by NMR spectroscopy and characterized its backbone dynamics by 15N-1H relaxation experiments. We showed that these N-terminal residues are unstructured and flexible. Structural comparison with ribosome-bound uL11 suggests that the linker region between the N-terminal domain and C-terminal domain of human uL11 is intrinsically disordered and only becomes structured when bound to the ribosomes. Mutagenesis studies show that the N-terminal conserved MPPKFDP motif is involved in interacting with the P-complex and its extended protuberant domain of uL10 in vitro. Truncation of the MPPKFDP motif also reduced the poly-phenylalanine synthesis in both hybrid ribosome and yeast mutagenesis studies. In addition, G→A/P substitutions to the conserved GPLG motif of helix-1 reduced poly-phenylalanine synthesis to 9-32% in yeast ribosomes. We propose that the flexible N-terminal residues of uL11, which could extend up to ∼25 Šfrom the N-terminal domain of uL11, can form transient interactions with the uL10 that help to fetch and fix it into a position ready for recruiting the incoming translation factors and facilitate protein synthesis.

A Sixty-Year Research and Development of Trichosanthin, a Ribosome-Inactivating Protein.

Tian Hua Fen, a herbal powder extract that contains trichosanthin (TCS), was used as an abortifacient in traditional Chinese medicine. In 1972, TCS was purified to alleviate the side effects. Because of its clinical applications, TCS became one of the most active research areas in the 1960s to the 1980s in China. These include obtaining the sequence information in the 1980s and the crystal structure in 1995. The replication block of TCS on human immunodeficiency virus in lymphocytes and macrophages was found in 1989 and started a new chapter of its development. Clinical studies were subsequently conducted. TCS was also found to have the potential for gastric and colorectal cancer treatment. Studies on its mechanism showed TCS acts as an rRNA N-glycosylase (EC 3.2.2.22) by hydrolyzing and depurinating A-4324 in α-sarcin/ricin loop on 28S rRNA of rat ribosome. Its interaction with acidic ribosomal stalk proteins was revealed in 2007, and its trafficking in mammalian cells was elucidated in the 2000s. The adverse drug reactions, such as inducing immune responses, short plasma half-life, and non-specificity, somehow became the obstacles to its usage. Immunotoxins, sequence modification, or coupling with polyethylene glycerol and dextran were developed to improve the pharmacological properties. TCS has nicely shown the scientific basis of traditional Chinese medicine and how its research and development have expanded the knowledge and applications of ribosome-inactivating proteins.

Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins.

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein-protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel ß-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor-cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge-charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

Mechanistic insights into an atypical interaction between ATG8 and SH3P2 in Arabidopsis thaliana.

In selective macroautophagy/autophagy, cargo receptors are recruited to the forming autophagosome by interacting with Atg8 (autophagy-related 8)-family proteins and facilitate the selective sequestration of specific cargoes for autophagic degradation. In addition, Atg8 interacts with a number of adaptors essential for autophagosome biogenesis, including ATG and non-ATG proteins. The majority of these adaptors and receptors are characterized by an Atg8-family interacting motif (AIM) for binding to Atg8. However, the molecular basis for the interaction mode between ATG8 and regulators or cargo receptors in plants remains largely unclear. In this study, we unveiled an atypical interaction mode for Arabidopsis ATG8f with a plant unique adaptor protein, SH3P2 (SH3 domain-containing protein 2), but not with the other two SH3 proteins. By structure analysis of the unbound form of ATG8f, we identified the unique conformational changes in ATG8f upon binding to the AIM sequence of a plant known autophagic receptor, NBR1. To compare the binding affinity of SH3P2-ATG8f with that of ATG8f-NBR1, we performed a gel filtration assay to show that ubiquitin-associated domain of NBR1 outcompetes the SH3 domain of SH3P2 for ATG8f interaction. Biochemical and cellular analysis revealed that distinct interfaces were employed by ATG8f to interact with NBR1 and SH3P2. Further subcellular analysis showed that the AIM-like motif of SH3P2 is essential for its recruitment to the phagophore membrane but is dispensable for its trafficking in endocytosis. Taken together, our study provides an insightful structural basis for the ATG8 binding specificity toward a plant-specific autophagic adaptor and a conserved autophagic receptor.Abbreviations: ATG, autophagy-related; AIM, Atg8-family interacting motif; BAR, Bin-Amphiphysin-Rvs; BFA, brefeldin A; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; CCV, clathrin-coated-vesicle; CLC2, clathrin light chain 2; Conc A, concanamycin A; ER, endoplasmic reticulum; LDS, LIR docking site; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; PE, phosphatidylethanolamine; SH3P2, SH3 domain containing protein 2; SH3, Src-Homology-3; UBA, ubiquitin-associated; UIM, ubiquitin-interacting motif.

Arabidopsis PUB2 and PUB4 connect signaling components of pattern-triggered immunity.

Plants use pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity (PTI). Precise regulation of information from PRRs to downstream signaling components is vital to mounting an appropriate immune response and requires dynamic interactions of these PTI components. We used transcriptome profiling, phenotypic analysis, molecular genetics, and protein-protein interaction analysis to understand the roles of the Arabidopsis plant U-box (PUB) proteins PUB2 and PUB4 in disease resistance and PTI signaling. Loss of function of both PUB2 and PUB4 diminishes the PAMP-triggered oxidative bursts and dampens mitogen-activated protein kinase signaling, resulting in a severe compromise in resistance to not only pathogenic but also nonpathogenic strains of Pseudomonas syringae. Within PUB4, the E3 ligase activity is dispensable, but the armadillo repeat region is essential and sufficient for its function in immunity. PUB2 and PUB4 interact with PTI signaling components, including FLS2, BIK1, PBL27, and RbohD, and enhance FLS2-BIK1 and BIK1-RbohD interactions. Our study reveals that PUB2 and PUB4 are critical components of plant immunity and connect PTI components to positively regulate defense responses.

Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection.

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.

Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV.

A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana.

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Structural and Mutagenesis Studies Evince the Role of the Extended Protuberant Domain of Ribosomal Protein uL10 in Protein Translation.

The lateral stalk of ribosomes constitutes the GTPase-associated center and is responsible for recruiting translation factors to the ribosomes. The eukaryotic stalk contains a P-complex, in which one molecule of uL10 (formerly known as P0) protein binds two copies of P1/P2 heterodimers. Unlike bacterial uL10, eukaryotic uL10 has an extended protuberant (uL10ext) domain inserted into the N-terminal RNA-binding domain. Here, we determined the solution structure of the extended protuberant domain of Bombyx mori uL10 by nuclear magnetic resonance spectroscopy. Comparison of the structures of the B. mori uL10ext domain with eRF1-bound and eEF2-bound ribosomes revealed significant structural rearrangement in a "hinge" region surrounding Phe183, a residue conserved in eukaryotic but not in archaeal uL10. 15N relaxation analyses showed that residues in the hinge region have significantly large values of transverse relaxation rates. To test the role of the conserved phenylalanine residue, we created a yeast mutant strain expressing an F181A variant of uL10. An in vitro translation assay showed that the alanine substitution increased the level of polyphenylalanine synthesis by ∼33%. Taken together, our results suggest that the hinge motion of the uL10ext domain facilitates the binding of different translation factors to the GTPase-associated center during protein synthesis.

Structural and Functional Investigation and Pharmacological Mechanism of Trichosanthin, a Type 1 Ribosome-Inactivating Protein.

Trichosanthin (TCS) is an RNA N-glycosidase that depurinates adenine-4324 in the conserved α-sarcin/ricin loop (α-SRL) of rat 28 S ribosomal RNA (rRNA). TCS has only one chain, and is classified as type 1 ribosome-inactivating protein (RIP). Our structural studies revealed that TCS consists of two domains, with five conserved catalytic residues Tyr70, Tyr111, Glu160, Arg163 and Phe192 at the active cleft formed between them. We also found that the structural requirements of TCS to interact with the ribosomal stalk protein P2 C-terminal tail. The structural analyses suggest TCS attacks ribosomes by first binding to the C-terminal domain of ribosomal P protein. TCS exhibits a broad spectrum of biological and pharmacological activities including anti-tumor, anti-virus, and immune regulatory activities. This review summarizes an updated knowledge in the structural and functional studies and the mechanism of its multiple pharmacological effects.

FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors.

Plant-type ferredoxins in Arabidopsis transfer electrons from the photosystem I to multiple redox-driven enzymes involved in the assimilation of carbon, nitrogen, and sulfur. Leaf-type ferredoxins also modulate the switch between the linear and cyclic electron routes of the photosystems. Recently, two novel ferredoxin homologs with extra C-termini were identified in the Arabidopsis genome (AtFdC1, AT4G14890; AtFdC2, AT1G32550). FdC1 was considered as an alternative electron acceptor of PSI under extreme ferredoxin-deficient conditions. Here, we showed that FdC1 could interact with some, but not all, electron acceptors of leaf-type Fds, including the ferredoxin-thioredoxin reductase (FTR), sulfite reductase (SiR), and nitrite reductase (NiR). Photoreduction assay on cytochrome c and enzyme assays confirmed its capability to receive electrons from PSI and donate electrons to the Fd-dependent SiR and NiR but not to the ferredoxin-NADP+ oxidoreductase (FNR). Hence, FdC1 and leaf-type Fds may play differential roles by channeling electrons from photosystem I to different downstream electron acceptors in photosynthetic tissues. In addition, the median redox potential of FdC1 may allow it to receive electrons from FNR in non-photosynthetic plastids.

Structural insights into how GTP-dependent conformational changes in a metallochaperone UreG facilitate urease maturation.

The ability of metallochaperones to allosterically regulate the binding/release of metal ions and to switch protein-binding partners along the metal delivery pathway is essential to the metallation of the metalloenzymes. Urease, catalyzing the hydrolysis of urea into ammonia and carbon dioxide, contains two nickel ions bound by a carbamylated lysine in its active site. Delivery of nickel ions for urease maturation is dependent on GTP hydrolysis and is assisted by four urease accessory proteins UreE, UreF, UreG, and UreH(UreD). Here, we determined the crystal structure of the UreG dimer from Klebsiella pneumoniae in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound Helicobacter pylori UreG (HpUreG) in the UreG2F2H2 complex reveals large conformational changes in the G2 region and residues near the 66CPH68 metal-binding motif. Upon GTP binding, the side chains of Cys66 and His68 from each of the UreG protomers rotate toward each other to coordinate a nickel ion in a square-planar geometry. Mutagenesis studies on HpUreG support the conformational changes induced by GTP binding as essential to dimerization of UreG, GTPase activity, in vitro urease activation, and the switching of UreG from the UreG2F2H2 complex to form the UreE2G2 complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreG2F2H2 complex could activate urease in vitro in the presence of GTP. Based on our results, we propose a mechanism of how conformational changes of UreG during the GTP hydrolysis/binding cycle facilitate urease maturation.

Using Homology Modeling to Understand the Structural Basis of Specific Interaction of a Plant-Specific AtSar1a-AtSec23a Pair Involved in Protein ER Export.

Homology modeling allows the prediction of a protein structure based on sequence similarity to a known structure of homologous proteins. In this chapter, we use a plant-specific AtSar1a-Atsec23a pair of proteins as a case study to illustrate how to use homology modeling to understand the specificity of the pairwise interaction between AtSar1a and AtSec23a. The detailed procedures described here are also useful in structure prediction of other protein complexes.

Structures and Ribosomal Interaction of Ribosome-Inactivating Proteins.

Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA N-glycosidases that depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. RIPs are grouped into three types according to the number of subunits and the organization of the precursor sequences. RIPs are two-domain proteins, with the active site located in the cleft between the N- and C-terminal domains. It has been found that the basic surface residues of the RIPs promote rapid and specific targeting to the ribosome and a number of RIPs have been shown to interact with the C-terminal regions of the P proteins of the ribosome. At present, the structural basis for the interaction of trichosanthin and ricin-A chain toward P2 peptide is known. This review surveys the structural features of the representative RIPs and discusses how they approach and interact with the ribosome.