Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

Homo-trimeric structure of the ribonuclease for rRNA processing, FAU-1, from Pyrococcus furiosus.

Crystal structure of a ribonuclease for rRNA processing, FAU-1, from Pyrococcus furiosus was determined with the resolution of 2.57 Å in a homo-trimeric form. The monomer structure consists of two domains, N-terminal and C-terminal domains. C-terminal domain forms trimer and each N-terminal domain locates outside of the trimer core. In the obtained crystal, a dinucleotide, pApUp, was bound to the N-terminal domain, indicating that N-terminal domain has the RNA-binding ability. The affinities to RNA of FAU-1 and a fragment corresponding to the N-terminal domain, FAU-ΔC, were confirmed by PAGE and NMR. Interestingly, well dispersed NMR signals were observed at 318 K, indicating that the FAU-ΔC-F18 complex form an ordered structure at higher temperature. As predicted in our previous works, FAU-1 and RNase E show a structural similarity in their RNA binding regions. However, structural similarity between RNase E and FAU-1 could be found in the limited regions of the N-terminal domain. On the other hand, structural similarity between C-terminal domain and some proteins including a phosphatase was found. Thus, it is possible that the catalytic site is located in C-terminal domain.

Synthesis of P-Modified DNA from Boranophosphate DNA as a Precursor via Acyl Phosphite Intermediates.

In this study, we successfully synthesized several kinds of P-modified nucleic acids from boranophosphate DNAs via an acyl phosphite intermediate in solution and on a solid support. In the solution-phase synthesis, phosphorothioate diester, phosphotriester, and phosphoramidate diester were synthesized in a one-pot reaction from boranophosphodiester via the conversion of an acyl phosphite as a key intermediate. In addition, doubly P-modified nucleic acid derivatives which were difficult to synthesize by the phosphoramidite and H-phosphonate methods were also obtained by the conversion reaction. In the solid-phase synthesis, a boranophosphate derivative was synthesized on a solid support using the H-boranophosphonate method. Then, an acyl phosphite intermediate was formed by treatment with pivaloyl chloride in pyridine, followed by appropriate transformations to obtain the P-modified derivatives such as phosphotriester and phosphorothioate diester. Notably, it was suggested that the conversion reaction of a boranophosphate to a phosphorothioate diester proceeded with retention of the stereochemistry of the phosphorous center. In addition, a phosphorothioate/phosphate chimeric dodecamer was successfully synthesized from a boranophosphate/phosphate chimeric dodecamer using the same strategy. Therefore, boranophosphate derivatives are versatile precursors for the synthesis of P-modified DNA, including chimeric derivatives.

Selection of aptamers using ß-1,3-glucan recognition protein-tagged proteins and curdlan beads.

RNA aptamersare nucleic acids that are obtained using the systematic evolution of ligands by exponential enrichment (SELEX) method. When using conventional selection methods to immobilize target proteins on matrix beads using protein tags, sequences are obtained that bind not only to the target proteins but also to the protein tags and matrix beads. In this study, we performed SELEX using ß-1,3-glucan recognition protein (GRP)-tags and curdlan beads to immobilize the acute myeloid leukaemia 1 (AML1) Runt domain (RD) and analysed the enrichment of aptamers using high-throughput sequencing. Comparison of aptamer enrichment using the GRP-tag and His-tag suggested that aptamers were enriched using the GRP-tag as well as using the His-tag. Furthermore, surface plasmon resonance analysis revealed that the aptamer did not bind to the GRP-tag and that the conjugation of the GRP-tag to RD weakened the interaction between the aptamer and RD. The GRP-tag could have acted as a competitor to reduce weakly bound RNAs. Therefore, the affinity system of the GRP-tagged proteins and curdlan beads is suitable for obtaining specific aptamers using SELEX.

Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1.

SURP domains are exclusively found in splicing-related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1-α2-310 -α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α-helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16-residue α-helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio-layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 µM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.

The N93D mutation of the human T-cell leukemia virus type 1 envelope glycoprotein found in symptomatic patients enhances neuropilin-1 b1 domain binding.

Human T-cell leukemia virus type 1 (HTLV-1) infection of host cells is mainly mediated by interactions with the viral envelope glycoprotein surface unit (SU) and three host receptors: heparan sulfate proteoglycan, neuropilin-1 (Nrp1), and glucose transporter type 1. Residues 90-94 of SU are considered as a Nrp1 binding site, and our previous results show that an SU peptide consisting of residues 85-94 can bind directly to the Nrp1 b1 domain with a binding affinity of 7.4 µM. Therefore, the SU peptide is expected to be a good model to investigate the SU-Nrp1 interaction. Recently, the N93D mutation in the Nrp1 b1 binding region of the SU was identified in symptomatic patients with HTLV-1 infections in the Brazilian Amazon. However, it remains unclear how the SU-N93D mutation affects Nrp1 b1 binding. To elucidate the impact of the substituted Asp93 of SU on Nrp1 b1 binding, we analyzed the interaction between the SU-N93D peptide and Nrp1 b1 using isothermal titration calorimetry and nuclear magnetic resonance. The SU-N93D peptide binds directly to Nrp1 b1 with a binding affinity of 3.5 µM, which is approximately two-fold stronger than wild-type. This stronger binding is likely a result of the interaction between the substituted residue Asp93 of the N93D peptide and the four residues Trp301, Lys347, Glu348, and Thr349 of Nrp1 b1. Our results suggest that the interaction of SU Asp93 with the four residues of Nrp1 b1 renders the high affinity of the N93D mutant for Nrp1 b1 binding during HTLV-1 entry.

Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE.

Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome , initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE® technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome . This study provides a proof of concept for the application of this method on large complexes.

Specific inhibition of FGF5-induced cell proliferation by RNA aptamers.

Fibroblast growth factor 5 (FGF5) is a crucial regulator of hair growth and an oncogenic factor in several human cancers. To generate FGF5 inhibitors, we performed Systematic Evolution of Ligands by EXponential enrichment and obtained novel RNA aptamers that have high affinity to human FGF5. These aptamers inhibited FGF5-induced cell proliferation, but did not inhibit FGF2-induced cell proliferation. Surface plasmon resonance demonstrated that one of the aptamers, F5f1, binds to FGF5 tightly (Kd = 0.7 ± 0.2 nM), but did not fully to FGF1, FGF2, FGF4, FGF6, or FGFR1. Based on sequence and secondary structure similarities of the aptamers, we generated the truncated aptamer, F5f1_56, which has higher affinity (Kd = 0.118 ± 0.003 nM) than the original F5f1. Since the aptamers have high affinity and specificity to FGF5 and inhibit FGF5-induced cell proliferation, they may be candidates for therapeutic use with FGF5-related diseases or hair disorders.

Amelioration of Alzheimer's Disease by Gut-Pancreas-Liver-Brain Interaction in an App Knock-In Mouse Model.

In this study, we observed disease progression, changes in the gut microbiota, and interactions among the brain, liver, pancreas, and intestine in a mouse model of Alzheimer's disease (AD), in addition to attempting to inhibit disease progression through the dietary supplementation of L-arginine and limonoids. Wild-type mice (WC) and AD mice were fed a normal diet (AC), a diet supplemented with L-arginine and limonoids (ALA), or a diet containing only limonoids (AL) for 12-64 weeks. The normal diet-fed WC and AC mice showed a decrease in the diversity of the gut microbiota, with an increase in the Firmicutes/Bacteroidetes ratio, and bacterial translocation. Considerable bacterial translocation to the pancreas and intense inflammation of the pancreas, liver, brain, and intestinal tissues were observed in the AC mice from alterations in the gut microbiota. The ALA diet or AL diet-fed mice showed increased diversity of the bacterial flora and suppressed oxidative stress and inflammatory responses in hepatocytes and pancreatic cells, bacterial translocation, and neurodegeneration of the brain. These findings suggest that L-arginine and limonoids help in maintaining the homeostasis of the gut microbiota, pancreas, liver, brain, and gut in AD mice.

A G-quadruplex-forming RNA aptamer binds to the MTG8 TAFH domain and dissociates the leukemic AML1-MTG8 fusion protein from DNA.

MTG8 (RUNX1T1) is a fusion partner of AML1 (RUNX1) in the leukemic chromosome translocation t(8;21). The AML1-MTG8 fusion gene encodes a chimeric transcription factor. One of the highly conserved domains of MTG8 is TAFH which possesses homology with human TAF4 [TATA-box binding protein-associated factor]. To obtain specific inhibitors of the AML1-MTG8 fusion protein, we isolated RNA aptamers against the MTG8 TAFH domain using systematic evolution of ligands by exponential enrichment. All TAF aptamers contained guanine-rich sequences. Analyses of a TAF aptamer by NMR, CD, and mutagenesis revealed that it forms a parallel G-quadruplex structure in the presence of K+ . Furthermore, the aptamer could bind to the AML1-MTG8 fusion protein and dissociate the AML1-MTG8/DNA complex, suggesting that it can inhibit the dominant negative effects of AML1-MTG8 against normal AML1 function and serve as a potential therapeutic agent for leukemia.

Kinetic and Thermodynamic Analyses of RNA-Protein Interactions.

Recently created biophysical methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), have been widely used to quantitatively study biomolecule interactions. The dissociation constant of the interaction with kinetic parameters, such as association rate constant and dissociation rate constant, can be obtained using SPR analysis. With thermodynamic parameters, such as enthalpy change and entropy change, the dissociation constant can be obtained by ITC analysis. Both methods differ not only in the type of information obtained but also in throughput and sample concentration. Analyzing the biophysical parameters of RNA-protein interactions will help us understand their functions in biological processes. In this chapter, we describe step-by-step SPR and ITC protocols suitable to study the kinetics and thermodynamics of RNA-protein interactions.

Altered-specificity mutants of the HIV Rev arginine-rich motif-RRE IIB interaction.

Arginine-rich motifs (ARMs) bind RNA structures with high affinity and specificity, and the human immunodeficiency virus (HIV) exploits ARM-RNA interactions to regulate its lifecycle. The expression of HIV structural genes relies on recognition between the ARM of its Rev protein and its primary binding site, an internal loop in the viral RNA, the Rev-response element region IIB (IIB). Many functional variants of the Rev ARM-IIB interaction have been discovered, yet how easily it can evolve new specificities is poorly explored. A double mutant of Rev ARM, R35G-N40 V, uses an unknown strategy to recognize IIB. Here, isothermal titration calorimetry and gel shift assays show that the R35G-N40V-IIB interaction has high affinity and specificity in vitro and a larger unfavorable entropy change upon binding than that of wild-type Rev ARM-IIB. In stark contrast with the critical dependence of wild-type Rev on Arg35, Arg39, Asn40, and Arg44, mutational profiling shows R35G-N40V is highly mutable at positions 40 and 44 and dependent on Gly35, Arg38, Arg39, Arg42, and Arg43. Affinity measurements in vitro and reporter assay measurements in vivo are consistent with the wild-type Rev ARM and R35G-N40V maintaining their recognition strategies when binding IIB mutants specific to wild-type Rev ARM and R35G-N40V, respectively. Some single amino acid mutants of wild-type Rev ARM and R35G-N40V have enhanced specificity, recognizing mutant IIBs yet not wild-type IIB. These results provide another example of viral ARM-RNA interactions evolving new specificities with few mutations, consistent with neutral theories of evolution.

ITC Measurement for High-Affinity Aptamers Binding to Their Target Proteins.

Aptamers are nucleic acid molecules that bind to a target molecule with high affinity and specificity, which are generated by a process known as systematic evolution of ligands by exponential enrichment (SELEX). Because of their high affinity and specificity, aptamers were developed as therapeutic agents. Although aptamers are investigated as promising therapeutic agents, the mechanism of their high affinity and specificity is not clear. Therefore, structural and biophysical studies are important to know that. To date, ITC is increasingly being used to study the thermodynamic basis of aptamer-target protein interactions. Understanding the mechanism of aptamer binding would contribute to their development for therapeutic applications. In this chapter, we describe the protocol to study the thermodynamics of aptamer-protein interactions.

Crystal structure and calcium-induced conformational changes of diacylglycerol kinase α EF-hand domains.

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that phosphorylate diacylglycerol into phosphatidic acid, modulating the levels of these key signaling lipids. Recently, increasing attention has been paid to DGKα isozyme as a potential target for cancer immunotherapy. We have previously shown that DGKα is positively regulated by Ca2+ binding to its N-terminal EF-hand domains (DGKα-EF). However, little progress has been made for the structural biology of mammalian DGKs and the molecular mechanism underlying the Ca2+ -triggered activation remains unclear. Here we report the first crystal structure of Ca2+ -bound DGKα-EF and analyze the structural changes upon binding to Ca2+ . DGKα-EF adopts a canonical EF-hand fold, but unexpectedly, has an additional α-helix (often called a ligand mimic [LM] helix), which is packed into the hydrophobic core. Biophysical and biochemical analyses reveal that DGKα-EF adopts a protease-susceptible "open" conformation without Ca2+ that tends to form a dimer. Cooperative binding of two Ca2+ ions dissociates the dimer into a well-folded monomer, which resists to proteolysis. Taken together, our results provide experimental evidence that Ca2+ binding induces substantial conformational changes in DGKα-EF, which likely regulates intra-molecular interactions responsible for the activation of DGKα and suggest a possible role of the LM helix for the Ca2+ -induced conformational changes. SIGNIFICANCE STATEMENT: Diacylglycerol kinases (DGKs), which modulates the levels of two lipid second messengers, diacylglycerol and phosphatidic acid, is still structurally enigmatic enzymes since its first identification in 1959. We here present the first crystal structure of EF-hand domains of diacylglycerol kinase α in its Ca2+ bound form and characterize Ca2+ -induced conformational changes, which likely regulates intra-molecular interactions. Our study paves the way for future studies to understand the structural basis of DGK isozymes.

Artificial cationic peptides that increase nuclease resistance of siRNA without disturbing RNAi activity.

Properties of cationic peptides bearing amino or guanidino groups with various side chain lengths that bind to double stranded RNAs (dsRNAs) were investigated. Peptides with shorter side chain lengths effectively bound to dsRNAs (12mers) increasing their thermal stability. NMR measurements suggested that the cationic peptide binds to the inner side of the major groove of dsRNA. These peptides also increased the thermal stability of siRNA and effectively protected from RNase A digestion. On the other hand, both peptides containing amino groups and guanidine groups did not disturb RNAi activity.

Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5'-untraslated region.

Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions -202 to -117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5'-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.

Synthesis of 4'-C-aminoalkyl-2'-O-methyl modified RNA and their biological properties.

In this paper, we describe the synthesis of 4'-C-aminoalkyl-2'-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4'-C-aminoethyl and 4'-C-aminopropyl-2'-O-methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4'-C-aminoalkyl-2'-O-methyluridine adopts an C2'-endo sugar puckering in dsRNA. The 4'-C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4'-C-aminoethyl and 4'-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4'-C-aminoethyl and 4'-C-aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules.

Characterisation of an aptamer against the Runt domain of AML1 (RUNX1) by NMR and mutational analyses.

Since the invention of systematic evolution of ligands by exponential enrichment, many short oligonucleotides (or aptamers) have been reported that can bind to a wide range of target molecules with high affinity and specificity. Previously, we reported an RNA aptamer that shows high affinity to the Runt domain (RD) of the AML1 protein, a transcription factor with roles in haematopoiesis and immune function. From kinetic and thermodynamic studies, it was suggested that the aptamer recognises a large surface area of the RD, using numerous weak interactions. In this study, we identified the secondary structure by nuclear magnetic resonance spectroscopy and performed a mutational study to reveal the residue critical for binding to the RD. It was suggested that the large contact area was formed by a DNA-mimicking motif and a multibranched loop, which confers the high affinity and specificity of binding.

Thermodynamic study of aptamers binding to their target proteins.

Aptamers are nucleic acids that bind to a target molecule with high affinity and specificity, which are selected from systematic evolution of ligands by exponential enrichment (SELEX). Aptamers feature high affinity and specificity to their target molecule and a large structural diversity; biophysical tools, together with structural studies, are essential to reveal the mechanism of aptamers recognition. Furthermore, understanding the mechanism of action would also contribute to their development for therapeutic applications. Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. In a single experiment, it provides all thermodynamic parameters of a molecular interaction, including dissociation constant, Kd; Gibbs free energy change, ΔG; enthalpy change, ΔH; entropy change, ΔS; and stoichiometry, N. The development of modern microcalorimeters significantly contributed to the expansion of the ITC use in biological systems. Therefore, ITC has been applied to the development of small therapeutic agents that bind to target proteins and is increasingly being used to study aptamer-target protein interactions. This review focuses on thermodynamic approaches for understanding the molecular principles of aptamer-target interactions.

Conjugation of two RNA aptamers improves binding affinity to AML1 Runt domain.

To develop a high-affinity aptamer against AML1 Runt domain, two aptamers were conjugated based on their structural information. The newly designed aptamer Apt14 was generated by the conjugation of two RNA aptamers (Apt1 and Apt4) obtained by SELEX against AML1 Runt domain, resulting in improvement in its binding performance. The residues of AML1 Runt domain in contact with Apt14 were predicted in silico and confirmed by mutation and NMR analyses. It was suggested that the conjugated internal loop renders additional contacts and is responsible for the enhancement in the binding affinity. Conjugation of two aptamers that bind to different sites of the target protein is a facile and robust strategy to develop an aptamer with higher performance.