Structural mechanism of DNA recognition by the p204 HIN domain.

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.

X-ray crystal structure of putative transcription regulator lmo2088 from Listeria monocytogenes.

Listeria monocytogenes is a gram-positive food borne pathogen. The lmo2088 belongs to the TetR family of transcriptional regulators from L. monocytogenes. These transcriptional factors regulate multidrug resistance transporters in L. monocytogenes. Here, we report native protein crystal structure of lmo2088 at a resolution of 1.7 Å. Lmo2088 comprises of an N-terminal DNA binding domain and a variable C-terminal effector binding domain. Furthermore, we identified specific consensus sequences selected by systematic evolution of ligands by exponential enrichment in vitro. The specific binding of lmo2088 with DNA consensus sequence was validated by electrophoretic mobility shift assay, fluorescence polarization and isothermal titration calorimetry. We speculate that the structure of lmo2088 might provide an insight into the regulatory function of lmo2088 of L. monocytogenes.

Structures of RIG-I-Like Receptors and Insights into Viral RNA Sensing.

RIG-I-like receptors (RLRs) are an important family of pattern recognition receptors. They activate the immunological responses against viral infections by sensing RNAs in the cytoplasm. Recent studies provide significant insights into the activation and transduction mechanisms of RLRs family (members including RIG-I, MDA5, and LGP2). Here we review our current understanding of the structures of RLRs. Structural characterizations of RLRs family have revealed the mechanism of their actions at molecular level. The activation mechanisms of RIG-I and MDA5 are different, despite both of them have similar domain organizations and bind to dsRNA ligands. RIG-I, but not MDA5, adopts an auto-suppression conformation in the absence of RNA. In addition to ligand triggered receptor oligomerization, the activities of these receptors are also regulated by several posttranslational modifications, especially ubiquitination. Overall, these structural studies play critical roles in promoting the understanding of viral RNA recognition mechanisms by the host innate immune system, which also contribute to the designing of drugs for treatment of viral infection. However, much work remains to be done in studying the signaling pathway of MDA5 and LGP2, particularly by structural biology.

Identification of novel enriched recurrent chimeric COL7A1-UCN2 in human laryngeal cancer samples using deep sequencing.

BACKGROUND: As hybrid RNAs, transcription-induced chimeras (TICs) may have tumor-promoting properties, and some specific chimeras have become important diagnostic markers and therapeutic targets for cancer. METHODS: We examined 23 paired laryngeal cancer (LC) tissues and adjacent normal mucous membrane tissue samples (ANMMTs). Three of these pairs were used for comparative transcriptomic analysis using high-throughput sequencing. Furthermore, we used real-time polymerase chain reaction (RT-PCR) for further validation in 20 samples. The Kaplan-Meier method and Cox regression model were used for the survival analysis. RESULTS: We identified 87 tumor-related TICs and found that COL7A1-UCN2 had the highest frequency in LC tissues (13/23; 56.5%), whereas none of the ANMMTs were positive (0/23; p < 0.0001). COL7A1-UCN2, generated via alternative splicing in LC tissue cancer cells, had disrupted coding regions, but it down-regulated the mRNA expression of COL7A1 and UCN2. Both COL7A1 and UCN2 were down-expressed in LC tissues as compared to their paired ANMMTs. The COL7A1:ß-actin ratio in COL7A1-UCN2-positive LC samples was significantly lower than that in COL7A1-UCN2-negative samples (p = 0.019). Likewise, the UCN2:ß-actin ratio was also decreased (p = 0.21). Furthermore, COL7A1-UCN2 positivity was significantly associated with the overall survival of LC patients (p = 0.032; HR, 13.2 [95%CI, 1.2-149.5]). CONCLUSION: LC cells were enriched in the recurrent chimera COL7A1-UCN2, which potentially affected cancer stem cell transition, promoted epithelial-mesenchymal transition in LC, and resulted in poorer prognoses.

Structural and biochemical studies reveal UbiG/Coq3 as a class of novel membrane-binding proteins.

UbiG and Coq3 (orthologue in eukaryotes) are SAM-MTases (S-adenosylmethionine-dependent methyltransferases) that catalyse both O-methylation steps in CoQ biosynthesis from prokaryotes to eukaryotes. However, the detailed molecular mechanism by which they function remains elusive. In the present paper, we report that UbiG/Coq3 defines a novel class of membrane-binding proteins. Escherichia coli UbiG binds specifically to liposomes containing PG (phosphatidylglycerol) or CL (cardiolipin, or diphosphatidylglycerol), two major lipid components of the E. coli plasma membrane, whereas human and yeast Coq3 display a strong preference for liposomes enriched with CL, a signature lipid of the mitochondrial membrane. The crystal structure of UbiG from E. coli was determined at 2.1 Å (1 Å = 0.1 nm) resolution. The structure exhibits a typical Class I SAM-MTase fold with several variations, including a unique insertion between strand ß5 and helix α10. This insertion is highly conserved and is required for membrane binding. Mutation of the key residues renders UbiG unable to efficiently bind liposome in vitro and the mutant fails to rescue the phenotype of ΔubiG strain in vivo. Taken together, our results shed light on a novel biochemical function of the UbiG/Coq3 protein.

Structure of the DNA-binding domain of the response regulator SaeR from Staphylococcus aureus.

The SaeR/S two-component regulatory system is essential for controlling the expression of many virulence factors in Staphylococcus aureus. SaeR, a member of the OmpR/PhoB family, is a response regulator with an N-terminal regulatory domain and a C-terminal DNA-binding domain. In order to elucidate how SaeR binds to the promoter regions of target genes, the crystal structure of the DNA-binding domain of SaeR (SaeR(DBD)) was solved at 2.5 Å resolution. The structure reveals that SaeR(DBD) exists as a monomer and has the canonical winged helix-turn-helix module. EMSA experiments suggested that full-length SaeR can bind to the P1 promoter and that the binding affinity is higher than that of its C-terminal DNA-binding domain. Five key residues on the winged helix-turn-helix module were verified to be important for binding to the P1 promoter in vitro and for the physiological function of SaeR in vivo.

Structure of Rot, a global regulator of virulence genes in Staphylococcus aureus.

Staphylococcus aureus is a highly versatile pathogen that can infect human tissue by producing a large arsenal of virulence factors that are tightly regulated by a complex regulatory network. Rot, which shares sequence similarity with SarA homologues, is a global regulator that regulates numerous virulence genes. However, the recognition model of Rot for the promoter region of target genes and the putative regulation mechanism remain elusive. In this study, the 1.77 Šresolution X-ray crystal structure of Rot is reported. The structure reveals that two Rot molecules form a compact homodimer, each of which contains a typical helix-turn-helix module and a ß-hairpin motif connected by a flexible loop. Fluorescence polarization results indicate that Rot preferentially recognizes AT-rich dsDNA with ~30-base-pair nucleotides and that the conserved positively charged residues on the winged-helix motif are vital for binding to the AT-rich dsDNA. It is proposed that the DNA-recognition model of Rot may be similar to that of SarA, SarR and SarS, in which the helix-turn-helix motifs of each monomer interact with the major grooves of target dsDNA and the winged motifs contact the minor grooves. Interestingly, the structure shows that Rot adopts a novel dimerization model that differs from that of other SarA homologues. As expected, perturbation of the dimer interface abolishes the dsDNA-binding ability of Rot, suggesting that Rot functions as a dimer. In addition, the results have been further confirmed in vivo by measuring the transcriptional regulation of α-toxin, a major virulence factor produced by most S. aureus strains.

TGFB1 induces fetal reprogramming and enhances intestinal regeneration.

The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.

Alpinetin promotes hair regeneration via activating hair follicle stem cells.

BACKGROUND: Alopecia affects millions of individuals globally, with hair loss becoming more common among young people.  Various traditional Chinese medicines (TCM) have been used clinically for treating alopecia, however, the effective compounds and underlying mechanism are less known. We sought to investigate the effect of Alpinetin (AP), a compound extracted from Fabaceae and Zingiberaceae herbs, in hair regeneration. METHODS: Animal model for hair regeneration was mimicked by depilation in C57BL/6J mice. The mice were then topically treated with 3 mg/ml AP, minoxidil as positive control (PC), or solvent ethanol as vehicle control (VC) on the dorsal skin. Skin color changes which reflected the hair growth stages were monitored and pictured, along with H&E staining and hair shaft length measurement. RNA-seq analysis combined with immunofluorescence staining and qPCR analysis were used for mechanism study. Meanwhile, Gli1CreERT2; R26RtdTomato and Lgr5EGFP-CreERT2; R26RtdTomato transgenic mice were used to monitor the activation and proliferation of Gli1+ and Lgr5+ HFSCs after treatment. Furthermore, the toxicity of AP was tested in keratinocytes and fibroblasts from both human and mouse skin to assess the safety. RESULTS: When compared to minoxidil-treated and vehicle-treated control mice, topical application of AP promoted anagen initiation and delayed catagen entry, resulting in a longer anagen phase and hair shaft length. Mechanistically, RNA-seq analysis combined with immunofluorescence staining of Lef1 suggested that Lgr5+ HFSCs in lower bulge were activated by AP via Wnt signaling. Other HFSCs, including K15+, Lef1+, and Gli1+ cells, were also promoted into proliferating upon AP treatment. In addition, AP inhibited cleaved caspase 3-dependent apoptosis at the late anagen stage to postpone regression of hair follicles. More importantly, AP showed no cytotoxicity in keratinocytes and fibroblasts from both human and mouse skin. CONCLUSION: This study clarified the effect of AP in promoting hair regeneration by activating HFSCs via Wnt signaling. Our findings may contribute to the development of a new generation of pilatory that is more efficient and less cytotoxic for treating alopecia.

Phase Change Behavior of NaCl-H2O Binary Solution Under the Control of AC Electric Field.

Cryopreservation, which refers to preservation of cells or tissues at subzero temperatures, inevitably involves the problem of cryoinjury caused by ice crystals. The application of an external electric field during the freezing process has been shown to be a promising approach to produce miniature ice grains and decrease the fraction of ice crystallization at a slow cooling rate. Thus, the dielectric and thermodynamic properties of NaCl-H2O binary solutions at subzero temperatures were tremendously important for understanding the mechanism of ice formation under the manipulation of an AC electric field in biopreservation. However, there was still a lack of relevant information in the literature. The first objective of this study was to systematically measure the dielectric spectrum of 0.9% NaCl-H2O binary solutions at temperatures ranging from -100°C to 0°C with a cooling/heating rate of 2°C/min. We further measured the thermodynamic properties of a 0.9% NaCl-H2O binary solution while applying a series of electric fields near its dielectric relaxation frequency. The effect of the electric field on the crystal morphology was studied last. Pure water was selected as the control group. The results showed that an AC electric field can alter the thermodynamic process and thus the phase transition and ice crystal structure could be manipulated. It was concluded that the AC electric-assistant preservation method will be a promising technology in cryopreservation.

Activation and Immune Regulation Mechanisms of PYHIN Family During Microbial Infection.

The innate immune system defenses against pathogen infections via patten-recognition receptors (PRRs). PRRs initiate immune responses by recognizing pathogen-associated molecular patterns (PAMPs), including peptidoglycan, lipopolysaccharide, and nucleic acids. Several nucleic acid sensors or families have been identified, such as RIG-I-like receptors (RLRs), Toll-like receptors (TLRs), cyclic GMP-AMP synthase (cGAS), and PYHIN family receptors. In recent years, the PYHIN family cytosolic DNA receptors have increased attention because of their important roles in initiating innate immune responses. The family members in humans include Absent in melanoma 2 (AIM2), IFN-γ inducible protein 16 (IFI16), interferon-inducible protein X (IFIX), and myeloid cell nuclear differentiation antigen (MNDA). The PYHIN family members are also identified in mice, including AIM2, p202, p203, p204, and p205. Herein, we summarize recent advances in understanding the activation and immune regulation mechanisms of the PYHIN family during microbial infection. Furthermore, structural characterizations of AIM2, IFI16, p202, and p204 provide more accurate insights into the signaling mechanisms of PYHIN family receptors. Overall, the molecular details will facilitate the development of reagents to defense against viral infections.

Gestational Weight Gain and Its Effects on Maternal and Neonatal Outcome in Women With Twin Pregnancies: A Systematic Review and Meta-Analysis.

Background: The incidence of twin pregnancies has risen recently. Such pregnancies are associated with an increased risk for poor maternal and infant outcomes. Gestational weight gain, particularly in singleton pregnancies, has been well-linked with maternal and infant outcomes. The aim of the current meta-analysis was to evaluate the effects of gestational weight gain on maternal and fetal outcomes in women with twin pregnancies. Methods: A systematic search was conducted using the PubMed, Scopus, and Google Scholar databases. Studies, either retrospective or prospective in design, evaluating the effects of gestational weight gain (defined using Institute of Medicine (IOM) guidelines) maternal and/or fetal/neonatal outcomes in women with twin pregnancies were included. Statistical analysis was performed using STATA software. Results: Eleven studies were included in the meta-analysis. Mothers with inadequate weight gain had increased risk for gestational diabetes mellitus (OR 1.19; 95% CI: 1.01, 1.40) and decreased risk for gestational hypertension (OR 0.58; 95% CI: 0.49, 0.68) and cesarean section (OR 0.94; 95% CI: 0.93, 0.96). Neonates born to mothers with inadequate weight gain were susceptible to increased risk for preterm delivery (OR 1.17; 95% CI: 1.03, 1.34), very preterm delivery (gestational age <32 weeks) (OR 1.84; 95% CI: 1.36, 2.48), small for gestational age status (OR 1.41; 95% CI: 1.15, 1.72), low birth weight status (<2,500 g) (OR 1.27; 95% CI: 1.17, 1.38), and neonatal intensive care unit (NICU) admission (OR 1.16; 95% CI: 1.08, 1.24). The pooled findings indicate an increased risk for gestational hypertension (OR 1.82; 95% CI: 1.60, 2.06) and cesarean section (OR 1.07; 95% CI: 1.05, 1.08) among mothers with excessive weight gain. Neonates born to mothers with excessive weight gain were susceptible to increased risk for preterm delivery and very preterm delivery, but were associated with a decreased risk for low birth weight status and small for gestational age status. Conclusions: Gestational weight gain in twin pregnancy, either lower or higher than IOM recommended guidelines, is associated with poor maternal and neonatal outcomes. Our findings call for incorporating counseling on optimal weight gain during pregnancy as part of routine antenatal visits.

Reciprocal signals between nerve and epithelium: how do neurons talk with epithelial cells?

Most epithelium tissues continuously undergo self-renewal through proliferation and differentiation of epithelial stem cells (known as homeostasis), within a specialized stem cell niche. In highly innervated epithelium, peripheral nerves compose perineural niche and support stem cell homeostasis by releasing a variety of neurotransmitters, hormones, and growth factors and supplying trophic factors to the stem cells. Emerging evidence has shown that both sensory and motor nerves can regulate the fate of epithelial stem cells, thus influencing epithelium homeostasis. Understanding the mechanism of crosstalk between epithelial stem cells and neurons will reveal the important role of the perineural niche in physiological and pathological conditions. Herein, we review recent discoveries of the perineural niche in epithelium mainly in tissue homeostasis, with a limited touch in wound repair and pathogenesis.

Identification of a Novel Major Allergen in Buckwheat Seeds: Fag t 6.

Buckwheat is one of the five main allergenic foods (eggs, milk, wheat, buckwheat, and peanuts). Oleosin is an important type of allergen in some allergic foods. However, although most diagnostic nut and seed extracts are defatted, some patients with food allergies may have false negative diagnostic results of oleosin in vitro. Recently, we found that the serum of buckwheat allergic patients responded strongly to an 18 kDa protein. Mass spectrometry analysis showed it is the oleosin protein family. We further purified and evaluated the allergenicity of this buckwheat oleosin-type allergen, which is involved in the formation of buckwheat oil bodies. The tartary buckwheat oleosin allergen was named Fag t 6, according to the WHO/IUIS Allergen Nomenclature Subcommittee criteria. The DNA sequence of tartary buckwheat oleosin was cloned. Dot blot and enzyme-linked immunosorbent assay (ELISA) showed half of the 20 buckwheat allergic patients' serum had strong reactivity with purified buckwheat Fag t 6. Circular dichroism experiment analysis of its thermal stability showed a Tm of 64.65 ± 0.65 °C. A buckwheat allergy showed possible cross-reaction with a wheat allergy. In summary, this study not only increases our understanding of buckwheat allergies and oil-soluble allergens in general, it may also be used to improve diagnostic tests for buckwheat allergies in the future.

Atomic-resolution structures of type I ribosome inactivating protein alpha-momorcharin with different substrate analogs.

Alpha-momorcharin (Alpha-MMC) from the seed of bitter melon is a type I ribosome inactivating protein (RIP) that removes a specific adenine from 28S rRNA and inhibits protein biosynthesis. Here, we report seven crystal complex structures of alpha-MMC with different substrate analogs (adenine, AMP, cAMP, dAMP, ADP, GMP, and xanthosine) at 1.08 Å to 1.52 Å resolution. These structures reveal that not only adenine, but also guanine and their analogs can effectively bind to alpha-MMC. The side chain of Tyr93 adopts two conformations, serving as a switch to open and close the substrate binding pocket of alpha-MMC. Although adenine, AMP, GMP, and guanine are located in a similar active site in different RIPs, residues involved in the interaction between RIPs and substrate analogs are slightly different. Complex structures of alpha-MMC with different substrate analogs solved in this study provide useful information on its enzymatic mechanisms and may enable the development of new inhibitors to treat the poisoning of alpha-MMC.

Structure determination of CAMP factor of Mobiluncus curtisii and insights into structural dynamics.

Bacterial vaginosis (BV) is a common type of vaginal inflammation caused by a proliferation of pathogenic bacteria, among which Mobiluncus curtisii. In our previous studies on M. curtisii genome, we identified the presence of a genomic fragment encoding a 25 kDa pore-forming toxin, the CAMP factor, which is known to be involved in the synergistic lysis of erythrocytes namely CAMP reaction. However, whether this hypothetical gene product has hemolytic activity is unknown. Moreover, its relative structure and function are not yet solved. Here we found that the M. curtisii CAMP factor is a monomer at pH 4.4 and oligomer at pH > 4.6. Hemolysis assays showed that M. curtisii CAMP factor could lyse sheep red blood cells efficiently in pH 5.4-7.4. Negative staining electron microscope analysis of the CAMP factor revealed ring-like structures at pH above 4.6. Additionally, the crystal structure of M. curtisii CAMP factor, determineded at 1.85 Å resolution, reveals a 5 + 3 helix motif. Further functional analysis suggested that the structural rearrangement of the N-terminal domain might be required for protein function. In conclusion, this structure-function relationship study of CAMP factor provides a new perspective of the M. curtisii role in BV development.

Structure determination of the CAMP factor of Streptococcus agalactiae with the aid of an MBP tag and insights into membrane-surface attachment.

CAMP factor is a unique α-helical bacterial toxin that is known for its co-hemolytic activity in combination with staphylococcal sphingomyelinase. It was first discovered in the human pathogen Streptococcus agalactiae (also known as group B streptococcus), but homologous genes have been found in many other Gram-positive pathogens. In this study, the efforts that led to the determination of the first structure of a CAMP-family toxin are reported. Initially, it was possible to produce crystals of the native protein which diffracted to near 2.45 Šresolution. However, a series of technical obstacles were encountered on the way to structure determination. Over a period of more than five years, many methods, including selenomethionine labeling, mutations, crystallization chaperones and heavy-atom soaking, were attempted, but these attempts resulted in limited progress. The structure was finally solved using a combination of iodine soaking and molecular replacement using the crystallization chaperone maltose-binding protein (MBP) as a search model. Analysis of native and MBP-tagged CAMP-factor structures identified a conserved interaction interface in the C-terminal domain (CTD). The positively charged surface may be critical for binding to acidic ligands. Furthermore, mutations on the interaction interface at the CTD completely abolished its co-hemolytic activities. This study provides novel insights into the mechanism of the membrane-permeabilizing activity of CAMP factor.

Screening of Nanobody Specific for Peanut Major Allergen Ara h 3 by Phage Display.

Peanut allergy is a major health problem worldwide. Detection of food allergens is a critical aspect of food safety. The VHH domain of single chain antibody from camelids, also known as nanobody (Nb), showed its advantages in the development of biosensors because of its high stability, small molecular size, and ease of production. However, no nanobody specific to peanut allergens has been developed. In this study, we constructed a library with random triplets (NNK) in its CDR regions of a camel nanobody backbone. We screened the library with peanut allergy Ara h 3 and obtained several candidate nanobodies. One of the promising nanobodies, Nb16 was further biochemical characterization by gel filtration, isothermal titration calorimetry (ITC), cocrystallization, and Western blot in terms of its interaction with Ara h 3. Nb16 specifically binds to peanut major allergen Ara h 3 with a dissociation constant of 400 nM. Furthermore, we obtained the Ara h 3-Nb16 complex crystals. Structure analysis shows the packing mode is completely different between the Ara h 3-Nb16 complex crystal and the native Ara h 3 crystal. Structural determination of Ara h 3-Nb16 will provide the necessary information to understand the allergenicity of this important peanut allergen. The nanobody Nb16 may have application in the development of biosensors for peanut allergen detection.

High-resolution crystal structure of Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that plays critical roles in bacterial pathogenesis in some pathogenic bacteria. In this study, the crystal structure of group B streptococcus GAPDH was determined at 1.36 Šresolution. The structure contained an asymmetric mixed holo tetramer, with two NAD ligands bound to two protomers. Further structural analysis identified interesting phosphate ion-binding sites, which shed light on its catalytic mechanism.

Structural insights into the interaction of the ribosomal P stalk protein P2 with a type II ribosome-inactivating protein ricin.

Ricin is a type II ribosome-inactivating protein (RIP) that depurinates A4324 at the sarcin-ricin loop of 28 S ribosomal RNA (rRNA), thus inactivating the ribosome by preventing elongation factors from binding to the GTPase activation centre. Recent studies have disclosed that the conserved C-terminal domain (CTD) of eukaryotic ribosomal P stalk proteins is involved in the process that RIPs target ribosome. However, the details of the molecular interaction between ricin and P stalk proteins remain unknown. Here, we report the structure of ricin-A chain (RTA) in a complex with the CTD of the human ribosomal protein P2. The structure shows that the Phe111, Leu113 and Phe114 residues of P2 insert into a hydrophobic pocket formed by the Tyr183, Arg235, Phe240 and Ile251 residues of RTA, while Asp115 of P2 forms hydrogen bonds with Arg235 of RTA. The key residues in RTA and P2 for complex formation were mutated, and their importance was determined by pull-down assays. The results from cell-free translation assays further confirmed that the interaction with P stalk proteins is essential for the inhibition of protein synthesis by RTA. Taken together, our results provide a structural basis that will improve our understanding of the process by which ricin targets the ribosome, which will benefit the development of effective small-molecule inhibitors for use as therapeutic agents.