A useful epitope tag derived from maltose binding protein

Maltose binding protein (MBP) is used in recombinant protein expression as an affinity and solubility tag. The monoclonal antibody B48 binds MBP tightly and has no cross‐reactivity to other proteins in an Escherichia coli lysate. This high level of specificity suggested that MBP contains an epitope that could prove useful as a purification and visualization tag for proteins expressed in E. coli. To discover the MBP epitope, a co‐crystal structure was determined for MBP bound to its antibody and four amino acids of MBP were identified as critical for the binding interaction. Fusions of various fragments of MBP to the glutathione S‐transferase protein were engineered in order to identify the smallest fragment still recognized by the α‐MBP antibody. Stabilization of the epitope via mutational engineering resulted in a minimized 14 amino‐acid tag.     

Molecular crowding and RNA synergize to promote phase separation,microtubule interaction,and seeding of Tau condensates

Biomolecular condensation of the neuronal microtubule‐associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate‐mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate‐like density for nuclear‐envelope Tau. These findings suggest that a complex interplay between interaction partners, post‐translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding‐competent Tau and lead to distinct cellular Tau accumulations.     

Alpha‐fetoprotein accelerates the progression of hepatocellular carcinoma by promoting Bcl‐2 gene expression through an RA‐RAR signalling pathway

Previous studies have found that alpha‐fetoprotein (AFP) can promote the proliferation of hepatoma cells and accelerate the progression of hepatocellular carcinoma (HCC). However, the exact mechanism of action remains unclear. Recent bioinformatics studies have predicted the possible interaction between AFP and retinoic acid receptors (RARs). Thus, the purpose of this study was to investigate the molecular mechanism through which AFP promotes tumour cell proliferation by interfering with the RA‐RAR signal pathway. Our data indicated that AFP could significantly promote the proliferation and weaken ATRA‐induced apoptosis of hepatoma cells. Besides, cytoplasmic AFP interacts with RAR, disrupting its entrance into the nucleus, which in turn affects the expression of the Bcl‐2 gene. In addition, knockdown of AFP in HepG2 cells was synchronously associated with an incremental increase of RAR binding to DNA, as well as down‐regulation of Bcl‐2; the opposite effect was observed in AFP gene‐transfected HLE cells. Moreover, a similar effect of AFP was detected in tumour tissues with high serum AFP, but not in adjacent non‐cancerous liver tissues, or HCC tissues with low serum AFP levels. These results indicate that AFP acts as signalling molecule and prevents RAR from entering into the nucleus by interacting with RAR, thereby promoting the expression of Bcl‐2. Our data reveal a novel mechanism through which AFP regulates Bcl‐2 expression and further suggest that AFP may be used as a novel target for treating HCC.     

Engineering defensin α‐helix to produce high‐affinity SARS‐CoV‐2 spike protein binding ligands

The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K _d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.     

NKG2D discriminates diverse ligands through selectively mechano‐regulated ligand conformational changes

Stimulatory immune receptor NKG2D binds diverse ligands to elicit differential anti‐tumor and anti‐virus immune responses. Two conflicting degeneracy recognition models based on static crystal structures and in‐solution binding affinities have been considered for almost two decades. Whether and how NKG2D recognizes and discriminates diverse ligands still remain unclear. Using live‐cell‐based single‐molecule biomechanical assay, we characterized the in situ binding kinetics of NKG2D interacting with different ligands in the absence or presence of mechanical force. We found that mechanical force application selectively prolonged NKG2D interaction lifetimes with the ligands MICA and MICB, but not with ULBPs, and that force‐strengthened binding is much more pronounced for MICA than for other ligands. We also integrated steered molecular dynamics simulations and mutagenesis to reveal force‐induced rotational conformational changes of MICA, involving formation of additional hydrogen bonds on its binding interface with NKG2D, impeding MICA dissociation under force. We further provided a kinetic triggering model to reveal that force‐dependent affinity determines NKG2D ligand discrimination and its downstream NK cell activation. Together, our results demonstrate that NKG2D has a discrimination power to recognize different ligands, which depends on selective mechanical force‐induced ligand conformational changes.     

The in vitro structure and functions of the disordered late embryogenesis abundant three proteins

Late embryogenesis abundant (LEA) proteins are produced during seed embryogenesis and in vegetative tissue in response to various abiotic stressors. A correlation has been established between LEA expression and stress tolerance, yet their precise biochemical mechanism remains elusive. LEA proteins are very rich in hydrophilic amino acids, and they have been found to be intrinsically disordered proteins (IDPs) in vitro. Here, we perform biochemical and structural analyses of the four LEA3 proteins from Arabidopsis thaliana (AtLEA3). We show that the LEA3 proteins are disordered in solution but have regions with propensity for order. All LEA3 proteins were effective cryoprotectants of LDH in the freeze/thaw assays, while only one member, AtLEA3‐4, was shown to bind Cu²⁺ and Fe³⁺ ions with micromolar affinity. As well, only AtLEA3‐4 showed binding and a gain in α‐helicity in the presence of the membrane mimic dodecylphosphocholine (DPC). We explored this interaction in greater detail using ¹⁵N‐heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance, and demonstrate that two sets of conserved motifs present in AtLEA3‐4 are involved in the interaction with the DPC micelles, which themselves gain α‐helical structure.     

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.     

An ultra‐high affinity protein–protein interface displaying sequence‐robustness

Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra‐high affinity interactions (K _d <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra‐high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra‐high affinity complex (K _d of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra‐high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra‐high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra‐high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild‐type, could occur without loss of affinity. This demonstrates that ultra‐high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.     

Multi‐modal adaptor‐clathrin contacts drive coated vesicle assembly

Clathrin‐coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated‐pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo‐EM structure of clathrin cages assembled in the presence of β2 hinge‐appendage (β2HA). We find that the β2‐appendage binds in at least two positions in the cage, demonstrating that multi‐modal binding is a fundamental property of clathrin‐AP2 interactions. In one position, β2‐appendage cross‐links two adjacent terminal domains from different triskelia. Functional analysis of β2HA‐clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin‐box motif on the hinge and the “sandwich site” on the appendage. We propose that β2‐appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.     

Structural insights into the tropomodulin assembly at the pointed ends of actin filaments

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1‐Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three‐dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin‐binding sites of tropomodulin and generated a three‐dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self‐consistent three‐dimensional model of tropomodulin assembly at the pointed end. The model of the pointed‐end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.     

Potential enzyme toxicity of perfluorooctanoic acid

Using equilibrium dialysis, isothermal titration calorimetry (ITC) and circular dichroism (CD), the interactions of perfluorooctanoic acid (PFOA) and lysozyme were investigated under normal human physiological conditions, i.e., at pH 4.40, 6.00 and 7.40 at 37°C in 0.15 M electrolyte. A simple and rapid spectrophotometric method was developed for determining PFOA concentrations. Interactions between PFOA and lysozyme were found to result from non-specific non-covalent bonds—F/N and F/O affinity, ion-pair attraction, hydrogen bond, hydrophobic interaction and van der Waals force—and were affected by chemical adsorption to monolayers. The results indicated that binding of PFOA altered the secondary structure and activity of lysozyme. This work provides a useful experimental strategy for research into the enzyme toxicity of organic chemicals, e.g., food additives and organic contaminants, and it may help to elucidate the molecular toxicology of human health risks.     

Cryo‐EM structure of native human uromodulin,a zona pellucida module polymer

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization, and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) “domain”. Despite the conservation of this element from hydra to humans, no detailed information is available on the filamentous conformation of any ZP module protein. Here, we report a cryo‐electron microscopy study of uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant protein in human urine and an archetypal ZP module‐containing molecule, in its mature homopolymeric state. UMOD forms a one‐start helix with an unprecedented 180‐degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD‐based models of heteromeric vertebrate egg coat filaments identify a common sperm‐binding region at the interface between subunits.     

Incredible affinity of Kattosh with PPAR‐γ receptors attenuates STZ‐induced pancreas and kidney lesions evidenced in chemicobiological interactions

Since ancient times, plants have been used as green bioresources to ensure a healthier life by recovering from different diseases. Kattosh (Lasia spinosa L. Thwaites) is a local plant with various traditional uses, especially for arthritis, constipation and coughs. This research investigated the effect of Kattosh stem extract (LSES) on streptozotocin‐induced damage to the pancreas, kidney, and liver using in vitro, in vivo and in silico methods. In vitro phytochemical, antioxidative and anti‐inflammatory effects of LSES were accomplished by established methods followed by antidiabetic actions in in vivo randomized controlled intervention in STZ‐induced animal models for four weeks. In an in silico study, LSES phytocompounds interacted with antidiabetic receptors of peroxisome proliferator‐activated receptor‐gamma (PPAR, PDB ID: 3G9E), AMP‐activated protein kinase (AMPK, PDB ID: 4CFH) and α‐amylase enzyme (PDB ID: 1PPI) to verify the in vivo results. In addition, LSES showed promising in vitro antioxidative and anti‐inflammatory effects. In contrast, it showed a decrease in weekly blood glucose level, normalized lipid profile, ameliorated liver and cardiac markers, managed serum AST and ALT levels, and increased glucose tolerance ability in the animal model study. Restoration of pancreatic and kidney damage was reflected by improving histopathological images. In ligand–receptor interaction, ethyl α‐d‐glucopyranoside of Kattosh showed the highest affinity for the α‐amylase enzyme, PPAR, and AMPK receptors. Results demonstrate that the affinity of Kattosh phytocompounds potentially attenuates pancreatic and kidney lesions and could be approached as an alternative antidiabetic source with further clarification.     

Molecular Structure-Affinity Relationship of Bufadienolides and Human Serum Albumin In Vitro and Molecular Docking Analysis

The development of bufadienolides as anti-tumor agents is limited due to poor pharmacokinetic properties regarding drug half-lives and toxicity in vivo. These serious factors might be improved by increasing the drug/albumin-binding ratio. This study therefore investigated the relationship between the structural properties of nine bufadienolides and their affinities for human serum albumin (HSA) by a fluorescence spectroscopy-based analysis and molecular docking. Fluorescence quenching data showed that the interaction of each bufadienolide with HSA formed a non-fluorescent complex, while thermodynamic parameters revealed negative ΔS and ΔH values, corresponding to changes in enthalpy and entropy, respectively. The structural differences between the various bufadienolides markedly influenced their binding affinity for HSA. With the exception of a C = O bond at the C12 position that decreased the binding affinity for HSA, other polar groups tended to increase the affinity, especially a hydroxyl (OH) group at assorted bufadienolide sites. The rank order of binding affinities for drugs with tri-hydroxyl groups was as follows: 11-OH > 5-OH > 16-OH; in addition, 16-acetoxy (OAc), 10-aldehyde and 14-epoxy constituents notably enhanced the binding affinity. Among these groups, 11-OH and 16-acetyl were especially important for a seamless interaction between the bufadienolides and HSA. Furthermore, molecular docking analysis revealed that either an 11-OH or a 16-OAc group spatially close to a five-membered lactone ring significantly facilitated the anchoring of these compounds within site I of the HSA pocket via hydrogen bonding (H-bonding) with Tyr150 or Lys199, respectively. In summary, bufadienolide structure strongly affects binding with HSA, and 11-OH or 16-OAc groups improve the drug association with key amino acid residues. This information is valuable for the prospective development of bufadienolides with improved pharmacological profiles as novel anti-tumor drugs.     

CTD of SARS‐CoV‐2 N protein is a cryptic domain for binding ATP and nucleic acid that interplay in modulating phase separation

SARS‐CoV‐2 nucleocapsid (N) protein plays essential roles in many steps of the viral life cycle, thus representing a key drug target. N protein contains the folded N‐/C‐terminal domains (NTD/CTD) and three intrinsically disordered regions, while its functions including liquid–liquid phase separation (LLPS) depend on the capacity in binding various viral/host‐cell RNA/DNA of diverse sequences. Previously NTD was established to bind various RNA/DNA while CTD to dimerize/oligomerize for forming high‐order structures. By NMR, here for the first time we decrypt that CTD is not only capable of binding S2m, a specific probe derived from SARS‐CoV‐2 gRNA but with the affinity even higher than that of NTD. Very unexpectedly, ATP, the universal energy currency for all living cells with high cellular concentrations (2–16 mM), specifically binds CTD with Kd of 1.49 ± 0.28 mM. Strikingly, the ATP‐binding residues of NTD/CTD are identical in the SARS‐CoV‐2 variants while ATP and S2m interplay in binding NTD/CTD, as well as in modulating LLPS critical for the viral life cycle. Results together not only define CTD as a novel binding domain for ATP and nucleic acid, but enforce our previous proposal that ATP has been evolutionarily exploited by SARS‐CoV‐2 to complete its life cycle in the host cell. Most importantly, the unique ATP‐binding pockets on NTD/CTD may offer promising targets for design of specific anti‐SARS‐CoV‐2 molecules to fight the pandemic. Fundamentally, ATP emerges to act at mM as a cellular factor to control the interface between the host cell and virus lacking the ability to generate ATP.     

Liquid–liquid phase separation of Tau by self and complex coacervation

The liquid–liquid phase separation (LLPS) of Tau has been postulated to play a role in modulating the aggregation property of Tau, a process known to be critically associated with the pathology of a broad range of neurodegenerative diseases including Alzheimer''s Disease. Tau can undergo LLPS by homotypic interaction through self‐coacervation (SC) or by heterotypic association through complex‐coacervation (CC) between Tau and binding partners such as RNA. What is unclear is in what way the formation mechanisms for self and complex coacervation of Tau are similar or different, and the addition of a binding partner to Tau alters the properties of LLPS and Tau. A combination of in vitro experimental and computational study reveals that the primary driving force for both Tau CC and SC is electrostatic interactions between Tau‐RNA or Tau‐Tau macromolecules. The liquid condensates formed by the complex coacervation of Tau and RNA have distinctly higher micro‐viscosity and greater thermal stability than that formed by the SC of Tau. Our study shows that subtle changes in solution conditions, including molecular crowding and the presence of binding partners, can lead to the formation of different types of Tau condensates with distinct micro‐viscosity that can coexist as persistent and immiscible entities in solution. We speculate that the formation, rheological properties and stability of Tau droplets can be readily tuned by cellular factors, and that liquid condensation of Tau can alter the conformational equilibrium of Tau.     

Identification of high affinity fatty acid binding sites on human serum albumin by MM-PBSA method

Human serum albumin (HSA) has seven common fatty acid (FA) binding sites. In this study, we used the molecular mechanics Poisson-Boltzmann surface area method to identify high affinity FA binding sites on HSA in terms of binding free energy. Using multiple HSA-FA (myristate, palmitate) complex models constructed by molecular dynamics simulations, two methods were performed in molecular mechanics Poisson-Boltzmann surface area, the “three-trajectory method” and the “single-trajectory method”. The former, which is less precise than the latter but may be more accurate as it includes the effects of conformational change upon binding, was used to classify high and low affinity sites. As a result, Sites 2, 4, and 5 were identified as high affinity sites for both FAs. The latter method, which is precise because energies are calculated from snapshots of the same trajectory for HSA-FAcomplex, was performed to compare the magnitude of binding free energy for these sites. The order of magnitude was 5 > 4 > 2, identical to that of a previous publication by others. In this way, a combination of the two methods was effectively used to identify high affinity sites. This study therefore provides an insight into the quantitative identification of high affinity FA binding sites on HSA.     

Solution structures of the Shewanella woodyi H‐NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP‐051

Heme‐nitric oxide/oxygen binding (H‐NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H‐NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H‐NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H‐NOX and coiled‐coil domains. Here, we report the full NMR structure for CO‐ligated Sw H‐NOX in the presence and absence of stimulator compound IWP‐051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi‐empirical quantum potential energy approach. Although IWP‐051 binding is weak, a single binding conformation was found at the interface of the two H‐NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix‐connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure‐based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H‐NOX proteins. The largest dynamical loop lies at the interface between Sw H‐NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.