Biophysical Characterization of p51 and p66 Monomers of HIV-1 Reverse Transcriptase with Their Inhibitors.

Human immunodeficiency virus (HIV)-1 reverse transcriptase (HIV-1 RT) is responsible for the transcription of viral RNA genomes into DNA genomes and has become an important target for the treatment of acquired immune deficiency syndrome (AIDS). This study used biophysical techniques to characterize the HIV-1 RT structure, monomer forms, and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) bound forms. Inactive p66W401A and p51W401A were selected as models to study the HIV-1 RT monomer structures. Nuclear magnetic resonance (NMR) spectroscopy revealed that the unliganded forms of p66W401A protein and p51W401A protein had similar conformation to each other in solution. The complexes of p66W401A or p51W401A with inhibitors showed similar conformations to p66 in the RT heterodimer bound to the NNRTIs. Furthermore, the results of paramagnetic relaxation enhancement (PRE)-assisted NMR revealed that the unliganded forms of the p66W401A and p51W401A conformations were different from the unliganded heterodimer, characterized by a greater distance between the fingers and thumb subdomains. Small-angle X-ray scattering (SAXS) experiments confirmed that p66W401A and p51W401A can bind with inhibitors, similar to the p66/p51 heterodimer. The findings of this study increase the structural knowledge base of HIV-1 RT monomers, which may be helpful in the future design of potent viral inhibitors.

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status.

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

The Double-Layered Structure of Amyloid-ß Assemblage on GM1-Containing Membranes Catalytically Promotes Fibrillization.

Alzheimer's disease (AD) is associated with progressive accumulation of amyloid-ß (Aß) cross-ß fibrils in the brain. Aß species tightly associated with GM1 ganglioside, a glycosphingolipid abundant in neuronal membranes, promote amyloid fibril formation; therefore, they could be attractive clinical targets. However, the active conformational state of Aß in GM1-containing lipid membranes is still unknown. The present solid-state nuclear magnetic resonance study revealed a nonfibrillar Aß assemblage characterized by a double-layered antiparallel ß-structure specifically formed on GM1 ganglioside clusters. Our data show that this unique assemblage was not transformed into fibrils on GM1-containing membranes but could promote conversion of monomeric Aß into fibrils, suggesting that a solvent-exposed hydrophobic layer provides a catalytic surface evoking Aß fibril formation. Our findings offer structural clues for designing drugs targeting catalytically active Aß conformational species for the development of anti-AD therapeutics.

Mutational and Environmental Effects on the Dynamic Conformational Distributions of Lys48-Linked Ubiquitin Chains.

In multidomain proteins, individual domains connected by flexible linkers are dynamically rearranged upon ligand binding and sensing changes in environmental factors, such as pH and temperature. Here, we characterize dynamic domain rearrangements of Lys48-linked ubiquitin (Ub) chains as models of multidomain proteins in which molecular surfaces mediating intermolecular interactions are involved in intramolecular domain-domain interactions. Using NMR and other biophysical techniques, we characterized dynamic conformational interconversions of diUb between open and closed states regarding solvent exposure of the hydrophobic surfaces of each Ub unit, which serve as binding sites for various Ub-interacting proteins. We found that the hydrophobic Ub-Ub interaction in diUb was reinforced by cysteine substitution of Lys48 of the distal Ub unit because of interaction between the cysteinyl thiol group and the C-terminal segment of the proximal Ub unit. In contrast, the replacement of the isopeptide linker with an artificial ethylenamine linker minimally affected the conformational distributions. Furthermore, we demonstrated that the mutational modification allosterically impacted the exposure of the most distal Ub unit in triUb. Thus, the conformational interconversion of Ub chains offers a unique design framework in Ub-based protein engineering not only for developing biosensing probes but also for allowing new opportunities for the allosteric regulation of multidomain proteins.

Hysteresis behavior in the unfolding/refolding processes of a protein trapped in metallo-cages.

Confinement of molecules in a synthetic host can physically isolate even their unstable temporary structures, which has potential for application to protein transient structure analysis. Here we report the NMR snapshot observation of protein unfolding and refolding processes by confining a target protein in a self-assembled coordination cage. With increasing acetonitrile content in CD3CN/H2O media (50 to 90 vol%), the folding structure of a protein sharply denatured at 83 vol%, clearly revealing the regions of initial unfolding. Unfavorable aggregation of the protein leading to irreversible precipitation is completely prevented because of the spatial isolation of the single protein molecule in the cage. When the acetonitrile content reversed (84 to 70 vol%), the once-denatured protein started to regain its original folded structure at 80 vol%, showing that the protein folding/unfolding process can be referred to as a phase transition with hysteresis behavior.

The B domain of protein A retains residual structures in 6 M guanidinium chloride as revealed by hydrogen/deuterium-exchange NMR spectroscopy.

The characterization of residual structures persistent in unfolded proteins is an important issue in studies of protein folding, because the residual structures present, if any, may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the residual structures of the isolated B domain (BDPA) of staphylococcal protein A in 6 M guanidinium chloride. BDPA is a small three-helix-bundle protein, and until recently its folding/unfolding reaction has been treated as a simple two-state process between the native and the fully unfolded states. We employed a dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange 2D NMR techniques with the use of spin desalting columns, which allowed us to investigate the H/D-exchange behavior of individually identified peptide amide (NH) protons. We obtained H/D-exchange protection factors of the 21 NH protons that form an α-helical hydrogen bond in the native structure, and the majority of these NH protons were significantly protected with a protection factor of 2.0-5.2 in 6 M guanidinium chloride, strongly suggesting that these weakly protected NH protons form much stronger hydrogen bonds under native folding conditions. The results can be used to deduce the structure of an early folding intermediate, when such an intermediate is shown by other methods. Among three native helical regions, the third helix in the C-terminal side was highly protected and stabilized by side-chain salt bridges, probably acting as the folding initiation site of BDPA. The present results are discussed in relation to previous experimental and computational findings on the folding mechanisms of BDPA.

Key Residue for Aggregation of Amyloid-ß Peptides.

It is known that oligomers of amyloid-ß (Aß) peptide are associated with Alzheimer's disease. Aß has two isoforms: Aß40 and Aß42. Although the difference between Aß40 and Aß42 is only two additional C-terminal residues, Aß42 aggregates much faster than Aß40. It is unknown what role the C-terminal two residues play in accelerating aggregation. Since Aß42 is more toxic than Aß40, its oligomerization process needs to be clarified. Moreover, clarifying the differences between the oligomerization processes of Aß40 and Aß42 is essential to elucidate the key factors of oligomerization. Therefore, to investigate the dimerization process, which is the early oligomerization process, Hamiltonian replica-permutation molecular dynamics simulations were performed for Aß40 and Aß42. We identified a key residue, Arg5, for the Aß42 dimerization. The two additional residues in Aß42 allow the C-terminus to form contact with Arg5 because of the electrostatic attraction between them, and this contact stabilizes the ß-hairpin. This ß-hairpin promotes dimer formation through the intermolecular ß-bridges. Thus, we examined the effects of amino acid substitutions of Arg5, thereby confirming that the mutations remarkably suppressed the aggregation of Aß42. Moreover, the mutations of Arg5 suppressed the Aß40 aggregation. It was found by analyzing the simulations that Arg5 is important for Aß40 to form intermolecular contacts. Thus, it was clarified that the role of Arg5 in the oligomerization process varies due to the two additional C-terminal residues.

Conformational Variability of Amyloid-ß and the Morphological Diversity of Its Aggregates.

Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many minima for different competing low-energy structures and, therefore, is much more enigmatic than that of multiple folding pathways. Thus, to understand the entire energy landscape of protein aggregation, it is important to elucidate the full picture of conformational changes and polymorphisms of amyloidogenic proteins. This review provides an overview of the conformational diversity of amyloid-ß (Aß) characterized from experimental and theoretical approaches. Aß exhibits a high degree of conformational variability upon transiently interacting with various binding molecules in an unstructured conformation in a solution, forming an α-helical intermediate conformation on the membrane and undergoing a structural transition to the ß-conformation of amyloid fibrils. This review also outlines the structural polymorphism of Aß amyloid fibrils depending on environmental factors. A comprehensive understanding of the energy landscape of amyloid formation considering various environmental factors will promote drug discovery and therapeutic strategies by controlling the fibril formation pathway and targeting the consequent morphology of aggregated structures.

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science.

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride.

Overall structure of fully assembled cyanobacterial KaiABC circadian clock complex by an integrated experimental-computational approach.

In the cyanobacterial circadian clock system, KaiA, KaiB and KaiC periodically assemble into a large complex. Here we determined the overall structure of their fully assembled complex by integrating experimental and computational approaches. Small-angle X-ray and inverse contrast matching small-angle neutron scatterings coupled with size-exclusion chromatography provided constraints to highlight the spatial arrangements of the N-terminal domains of KaiA, which were not resolved in the previous structural analyses. Computationally built 20 million structural models of the complex were screened out utilizing the constrains and then subjected to molecular dynamics simulations to examine their stabilities. The final model suggests that, despite large fluctuation of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions. Thus, our integrative approach provides a useful tool to resolve large complex structures harboring dynamically fluctuating domains.

Experimental and computational characterization of dynamic biomolecular interaction systems involving glycolipid glycans.

On cell surfaces, carbohydrate chains that modify proteins and lipids mediate various biological functions, which are exerted not only through carbohydrate-protein interactions but also through carbohydrate-carbohydrate interactions. These glycans exhibit considerable degrees of conformational variability and often form clusters providing multiple binding sites. The integration of nuclear magnetic resonance spectroscopy and molecular dynamics simulation has made it possible to delineate the dynamical structures of carbohydrate chains. This approach has facilitated the remodeling of oligosaccharide conformational space in the prebound state to improve protein-binding affinity and has been applied to visualize dynamic carbohydrate-carbohydrate interactions that control glycoprotein-glycoprotein complex formation. Functional glycoclusters have been characterized by experimental and computational approaches applied to various model membranes and artificial self-assembling systems. This line of investigation has provided dynamic views of molecular assembling on glycoclusters, giving mechanistic insights into physiological and pathological molecular events on cell surfaces as well as clues for the design and creation of molecular systems exerting improved glycofunctions. Further development and accumulation of such studies will allow detailed understanding and artificial control of the "glycosynapse" foreseen by Dr. Sen-itiroh Hakomori.

Biophysical Characterization of Novel DNA Aptamers against K103N/Y181C Double Mutant HIV-1 Reverse Transcriptase.

The human immunodeficiency virus type-1 Reverse Transcriptase (HIV-1 RT) plays a pivotal role in essential viral replication and is the main target for antiviral therapy. The anti-HIV-1 RT drugs address resistance-associated mutations. This research focused on isolating the potential specific DNA aptamers against K103N/Y181C double mutant HIV-1 RT. Five DNA aptamers showed low IC50 values against both the KY-mutant HIV-1 RT and wildtype (WT) HIV-1 RT. The kinetic binding affinity forms surface plasmon resonance of both KY-mutant and WT HIV-1 RTs in the range of 0.06-2 µM and 0.15-2 µM, respectively. Among these aptamers, the KY44 aptamer was chosen to study the interaction of HIV-1 RTs-DNA aptamer complex by NMR experiments. The NMR results indicate that the aptamer could interact with both WT and KY-mutant HIV-1 RT at the NNRTI drug binding pocket by inducing a chemical shift at methionine residues. Furthermore, KY44 could inhibit pseudo-HIV particle infection in HEK293 cells with nearly 80% inhibition and showed low cytotoxicity on HEK293 cells. These together indicated that the KY44 aptamer could be a potential inhibitor of both WT and KY-mutant HIV-RT.

Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade.

Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada.

BACKGROUND: Many limno-terrestrial tardigrades can enter an ametabolic state, known as anhydrobiosis, upon desiccation, in which the animals can withstand extreme environments. Through genomics studies, molecular components of anhydrobiosis are beginning to be elucidated, such as the expansion of oxidative stress response genes, loss of stress signaling pathways, and gain of tardigrade-specific heat-soluble protein families designated CAHS and SAHS. However, to date, studies have predominantly investigated the class Eutardigrada, and molecular mechanisms in the remaining class, Heterotardigrada, still remains elusive. To address this gap in the research, we report a multiomics study of the heterotardigrade Echiniscus testudo, one of the most desiccation-tolerant species which is not yet culturable in laboratory conditions. RESULTS: In order to elucidate the molecular basis of anhydrobiosis in E. testudo, we employed a multi-omics strategy encompassing genome sequencing, differential transcriptomics, and proteomics. Using ultra-low input library sequencing protocol from a single specimen, we sequenced and assembled the 153.7 Mbp genome annotated using RNA-Seq data. None of the previously identified tardigrade-specific abundant heat-soluble genes was conserved, while the loss and expansion of existing pathways were partly shared. Furthermore, we identified two families novel abundant heat-soluble proteins, which we named E. testudo Abundant Heat Soluble (EtAHS), that are predicted to contain large stretches of disordered regions. Likewise the AHS families in eutardigrada, EtAHS shows structural changes from random coil to alphahelix as the water content was decreased in vitro. These characteristics of EtAHS proteins are analogous to those of CAHS in eutardigrades, while there is no conservation at the sequence level. CONCLUSIONS: Our results suggest that Heterotardigrada have partly shared but distinct anhydrobiosis machinery compared with Eutardigrada, possibly due to convergent evolution within Tardigrada. (276/350).

Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible ß-Barrel Structure in Solution and Keeps This Structure in Dehydration.

Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the ßD and ßE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the ß-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.

Deuteration Aiming for Neutron Scattering.

The distinguished feature of neutron as a scattering probe is an isotope effect, especially the large difference in neutron scattering length between hydrogen and deuterium. The difference renders the different visibility between hydrogenated and deuterated proteins. Therefore, the combination of deuterated protein and neutron scattering enables the selective visualization of a target domain in the complex or a target protein in the multi-component system. Despite of this fascinating character, there exist several problems for the general use of this method: difficulty and high cost for protein deuteration, and control and determination of deuteration ratio of the sample. To resolve them, the protocol of protein deuteration techniques is presented in this report. It is strongly expected that this protocol will offer more opportunity for conducting the neutron scattering studies with deuterated proteins.

Cold Atmospheric Plasma Modification of Amyloid ß.

Cold atmospheric plasma (CAP) has attracted much attention in the fields of biotechnology and medicine owing to its potential utility in clinical applications. Recently accumulating evidence has demonstrated that CAP influences protein structures. However, there remain open questions regarding the molecular mechanisms behind the CAP-induced structural perturbations of biomacromolecules. Here, we investigated the potential effects of CAP irradiation of amyloid ß (Aß), an amyloidogenic protein associated with Alzheimer's disease. Using nuclear magnetic resonance spectroscopy, we observed gradual spectral changes in Aß after a 10 s CAP pretreatment, which also suppressed its fibril formation, as revealed by thioflavin T assay. As per mass spectrometric analyses, these effects were attributed to selective oxidation of the methionine residue (Met) at position 35. Interestingly, this modification occurred when Aß was dissolved into a pre-irradiated buffer, indicating that some reactive species oxidize the Met residue. Our results strongly suggest that the H2O2 generated in the solution by CAP irradiation is responsible for Met oxidation, which inhibits Aß amyloid formation. The findings of the present study provide fundamental insights into plasma biology, giving clues for developing novel applications of CAP.

Characterization of New DNA Aptamers for Anti-HIV-1 Reverse Transcriptase.

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC50 value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.

Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR.

The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution. Based on the backbone resonance assignment of ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 60 identified peptide amide groups in the ubiquitin sequence. Although a majority of these amide groups were not protected, certain amide groups involved in a middle helix (residues 23-34) and an N-terminal ß-hairpin (residues 2-16) were significantly protected with a protection factor of 2.1-4.2, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were more than 52% hydrogen bonded in the residual structures. We show that the hydrogen-bonded residual structures in the α-helix and the ß-hairpin are formed even in 6 M guanidinium chloride, suggesting that these residual structures may function as a folding initiation site to guide the subsequent folding reactions of ubiquitin.

NMR Characterization of Conformational Interconversions of Lys48-Linked Ubiquitin Chains.

Ubiquitin (Ub) molecules can be enzymatically connected through a specific isopeptide linkage, thereby mediating various cellular processes by binding to Ub-interacting proteins through their hydrophobic surfaces. The Lys48-linked Ub chains, which serve as tags for proteasomal degradation, undergo conformational interconversions between open and closed states, in which the hydrophobic surfaces are exposed and shielded, respectively. Here, we provide a quantitative view of such dynamic processes of Lys48-linked triUb and tetraUb in solution. The native and cyclic forms of Ub chains are prepared with isotope labeling by in vitro enzymatic reactions. Our comparative NMR analyses using monomeric Ub and cyclic diUb as reference molecules enabled the quantification of populations of the open and closed states for each Ub unit of the native Ub chains. The data indicate that the most distal Ub unit in the Ub chains is the most apt to expose its hydrophobic surface, suggesting its preferential involvement in interactions with the Ub-recognizing proteins. We also demonstrate that a mutational modification of the distal end of the Ub chain can remotely affect the solvent exposure of the hydrophobic surfaces of the other Ub units, suggesting that Ub chains could be unique design frameworks for the creation of allosterically controllable multidomain proteins.