pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Molecular Mechanism of Adenovirus Late Protein L4-100K Chaperones the Trimerization of Hexon.

Assembly of the adenovirus capsid protein hexon depends on the assistance of the molecular chaperone L4-100K. However, the chaperone mechanisms remain unclear. In this study, we found that L4-100K was involved in the hexon translation process and could prevent hexon degradation by the proteasome in cotransfected human cells. Two nonadjacent domains, 84-133 and 656-697, at the N-terminal and C-terminal regions of human adenovirus type 5 L4-100K, respectively, were found to be crucial and cooperatively responsible for hexon trimer expression and assembly. These two chaperone-related domains were conserved in the sequence of L4-100K and in the function of hexon assembly across different adenovirus serotypes. Different degrees of cross-activity of hexon trimerization with different serotypes were detected in subgroups B, C, and D, which were proven to be controlled by the interaction between the C-terminal chaperone-related domain of L4-100K and hypervariable regions (HVR) of hexon. Additionally, HVR-chimeric hexon mutants were successfully assembled with the assistance of the 1-697 mutant. Structural analysis of 656-697 by nuclear magnetic resonance and structural prediction of L4-100K using Robetta showed that the two conserved domains are mainly composed of α-helices and are located on the surface of the highly folded core region. Our research provides a more complete understanding of hexon assembly and guidance for the development of hexon-chimeric adenovirus vectors that will be safer, smarter, and more efficient. IMPORTANCE Adenovirus vectors have been widely used in clinical trials of vaccines and gene therapy, although some deficiencies remain. Chimeric modification of the hexon was expected to improve the potency of preexisting immune evasion and targeting, but in many cases, viral packaging is prevented by the inability of the chimeric hexon to assemble correctly. So far, few studies have examined the mechanisms of hexon trimer assembly. Here, we show how the chaperone protein L4-100K contributes to the assembly of the adenovirus capsid protein hexon, and these data will provide a guide for novel adenovirus vector design and development, as we desired.

Mechanochemical Reactions from Individual Impacts to Global Transformation Kinetics.

Typically induced by the mechanical processing of powders in ball mills, mechanochemical transformations are considered to result from the application of mechanical force to solid reactants. However, the undeniable deep connection between the dynamic compaction of powders during impacts and the overall transformation degree has yet to be disclosed. In the present work, we show that the square planar bis(dibenzoylmethanato)NiII coordination compound undergoes trimerization when its powder experiences even a single ball impact. Based on systematic experiments with individual ball impacts and analysis by Raman spectroscopy, we provide here quantitative mapping of the transformation in the powder compact and deduce bulk reaction kinetics from multiple individual impacts.

A missense mutation sheds light on a novel structure-function relationship of RANKL.

The tumor necrosis factor (TNF)-like core domain of receptor activator of nuclear factor-κB ligand (RANKL) is a functional domain critical for osteoclast differentiation. One of the missense mutations identified in patients with osteoclast-poor autosomal recessive osteopetrosis (ARO) is located in residue methionine 199 that is replaced with lysine (M199K) amid the TNF-like core domain. However, the structure-function relationship of this mutation is not clear. Sequence-based alignment revealed that the fragment containing human M199 is highly conserved and equivalent to M200 in rat. Using site-directed mutagenesis, we generated three recombinant RANKL mutants M200K/A/E (M200s) by replacing the methionine 200 with lysine (M200K), alanine (M200A), and glutamic acid (M200E), representative of distinct physical properties. TRAcP staining and bone pit assay showed that M200s failed to support osteoclast formation and bone resorption, accompanied by impaired osteoclast-related signal transduction. However, no antagonistic effect was found in M200s against wild-type rat RANKL. Analysis of the crystal structure of RANKL predicted that this methionine residue is located within the hydrophobic core of the protein, thus, likely to be crucial for protein folding and stability. Consistently, differential scanning fluorimetry analysis suggested that M200s were less stable. Western blot analysis analyses further revealed impaired RANKL trimerization by M200s. Furthermore, receptor-ligand binding assay displayed interrupted interaction of M200s to its intrinsic receptors. Collectively, our studies revealed the molecular basis of human M199-induced ARO and elucidated the indispensable role of rodent residue M200 (equivalent to human M199) for the RANKL function.

The transmembrane domain and luminal C-terminal region independently support invariant chain trimerization and assembly with MHCII into nonamers.

BACKGROUND: Invariant chain (CD74, Ii) is a multifunctional protein expressed in antigen presenting cells. It assists the ER exit of various cargos and serves as a receptor for the macrophage migration inhibitory factor. The newly translated Ii chains trimerize, a structural feature that is not readily understood in the context of its MHCII chaperoning function. Two segments of Ii, the luminal C-terminal region (TRIM) and the transmembrane domain (TM), have been shown to participate in the trimerization process but their relative importance and impact on the assembly with MHCII molecules remains debated. Here, we addressed the requirement of these domains in the trimerization of human Ii as well as in the oligomerization with MHCII molecules. We used site-directed mutagenesis to generate series of Ii and DR mutants. These were transiently transfected in HEK293T cells to test their cell surface expression and analyse their interactions by co-immunoprecipitations. RESULTS: Our results showed that the TRIM domain is not essential for Ii trimerization nor for intracellular trafficking with MHCII molecules. We also gathered evidence that in the absence of TM, TRIM allows the formation of multi-subunit complexes with HLA-DR. Similarly, in the absence of TRIM, Ii can assemble into high-order structures with MHCII molecules. CONCLUSIONS: Altogether, our data show that trimerization of Ii through either TM or TRIM sustains nonameric complex formation with MHCII molecules.

Interactions Determining the Structural Integrity of the Trimer of Plant Light Harvesting Complex in Lipid Membranes.

The structural basis for the stability of the trimeric form of the light harvesting complex (LHCII), a pigmented protein from green plants pivotal for photosynthesis, remains elusive till date. The protein embedded in a dipalmitoylphosphatidylcholine (DPPC) lipid membrane is investigated using all-atom molecular dynamics simulations to find out the interactions responsible for the structural integrity of the trimer and its relation to antenna function. Central association of chlorophyll a (CLA) molecules near the LHCII chains is attributed to a conserved coordination between the Mg of CLA and the oxygen of a specific residue of the first helix of a chain. The residue forms a salt-bridge with the fourth helix of the same chain of the trimer, not of the monomer. In an earlier experiment, three residues (WYR) at each chain of the trimer have been found indispensable for the trimerization and referred to as trimerization motif. We find that the residues of the trimerization motif are connected to the lipids or pigments by a chain of interactions rather than a direct contact. Synergistic effects of sequentially located hydrogen bonds and salt-bridges within monomers of the trimer keep the trimer conformation stable in association with the pigments or the lipids. These interactions are exclusively present in the pigmented trimer and not present in the monomer or in the unpigmented trimer. Thus, our results provide a molecular basis for the inherent stability of the LHCII trimer in a lipid membrane and explain many pre-existing experimental data.

Comparative Studies on Thermal Decompositions of Dinitropyrazole-Based Energetic Materials.

Dinitropyrazole is an important structure for the design and synthesis of energetic materials. In this work, we reported the first comparative thermal studies of two representative dinitropyrazole-based energetic materials, 4-amino-3,5-dinitropyrazole (LLM-116) and its novel trimer derivative (LLM-226). Both the experimental and theoretical results proved the active aromatic N-H moiety would cause incredible variations in the physicochemical characteristics of the obtained energetic materials. Thermal behaviors and kinetic studies of the two related dinitropyrazole-based energetic structures showed that impressive thermal stabilization could be achieved after the trimerization, but also would result in a less concentrated heat-release process. Detailed analysis of condensed-phase systems and the gaseous products during the thermal decomposition processes, and simulation studies based on ReaxFF force field, indicated that the ring opening of LLM-116 was triggered by hydrogen transfer of the active aromatic N-H moiety. In contrast, the initial decomposition of LLM-226 was caused by the rupture of carbon-nitrogen bonds at the diazo moiety.

Enhancing the activity of membrane remodeling epsin-peptide by trimerization.

Modulating the structural dynamics of biomembranes by inducing bilayer curvature and lipid packing defects has been highlighted as a practical tool to modify membrane-dependent cellular processes. Previously, we have reported on an amphipathic helical peptide derived from the N-terminal segment (residues 1-18, EpN18) of epsin-1, which can promote membrane remodeling including lipid packing defects in cell membranes. However, a high concentration is required to exhibit a pronounced effect. In this study, we demonstrate a significant increase in the membrane-remodeling effect of EpN18 by constructing a branched EpN18 homotrimer. Both monomer and trimer could enhance cell internalization of octaarginine (R8), a cell-penetrating peptide. The EpN18 trimer, however, promoted the uptake of R8 at an 80-fold lower concentration than the monomer. Analysis of the generalized polarization of a polarity-sensitive dye (di-4-ANEPPDHQ) revealed a higher efficacy of trimeric EpN18 in loosening the lipid packing in the cell membrane. Circular dichroism measurements in the presence of lipid vesicles showed that the EpN18 trimer has a higher α-helix content compared with the monomer. The stronger ability of the EpN18 trimer to impede negative bilayer curvature is also corroborated by solid-state 31P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.

A Rational Insight into the Effect of Dimethyl Sulfoxide on TNF-α Activity.

Direct inhibition of tumor necrosis factor-alpha (TNF-α) action is considered a promising way to prevent or treat TNF-α-associated diseases. The trimeric form of TNF-α binds to its receptor (TNFR) and activates the downstream signaling pathway. The interaction of TNF-α with molecular-grade dimethyl sulfoxide (DMSO) in an equal volumetric ratio renders TNF-α inert, in this state, TNF-α fails to activate TNFR. Here, we aimed to examine the inhibition of TNF-α function by various concentrations of DMSO. Its higher concentration led to stronger attenuation of TNF-α-induced cytokine secretion by fibroblasts, and of their death. We found that this inhibition was mediated by a perturbation in the formation of the functional TNF-α trimer. Molecular dynamics simulations revealed a transient interaction between DMSO molecules and the central hydrophobic cavity of the TNF-α homodimer, indicating that a brief interaction of DMSO with the TNF-α homodimer may disrupt the formation of the functional homotrimer. We also found that the sensitizing effect of actinomycin D on TNF-α-induced cell death depends upon the timing of these treatments and on the cell type. This study will help to select an appropriate concentration of DMSO as a working solvent for the screening of water-insoluble TNF-α inhibitors.

Oxidative Dearomative Cross-Dehydrogenative Coupling of Indoles with Diverse C-H Nucleophiles: Efficient Approach to 2,2-Disubstituted Indolin-3-ones.

The oxidative, dearomative cross-dehydrogenative coupling of indoles with various C-H nucleophiles is developed. This process features a broad substrate scope with respect to both indoles and nucleophiles, affording structurally diverse 2,2-disubstituted indolin-3-ones in high yields (up to 99%). The oxidative dimerization and trimerization of indoles has also been demonstrated under the same conditions.

The Trimerization of Isocyanate-Functionalized Prepolymers: An Effective Method for Synthesizing Well-Defined Polymer Networks.

For the study of polymer networks, having access to polymer networks with a controlled and well-defined microscopic network structure is of great importance. However, typically, such networks are difficult to synthesize. In this work, a simple, effective, and widely applicable method is presented for synthesizing polymer networks with a well-defined network structure. This is done by the functionalization of polymeric diols using a diisocyanate, and their subsequent trimerization. Using hexamethylene diisocyanate and hydroxyl-group-terminated poly(ε-caprolactone) and poly(ethylene glycol), it is shown that both hydrophobic and hydrophilic poly(urethane-isocyanurate) networks with a well-defined network structure can readily be synthesized. By using in situ infrared spectroscopy, it is shown that the trimerization of isocyanate endgroups is clearly the predominant reaction pathway of network formation, supporting the proposed mechanism and network structure. The resulting networks possess excellent mechanical properties in both the dry and in the wet state.

Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation.

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.

Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation.

The p75 neurotrophin receptor (p75(NTR)) is a multifunctional receptor that participates in many critical processes in the nervous system, ranging from apoptosis to synaptic plasticity and morphological events. It is a member of the tumor necrosis factor receptor (TNFR) superfamily, whose members undergo trimeric oligomerization. Interestingly, p75(NTR) interacts with dimeric ligands (i.e., proneurotrophins or mature neurotrophins), but several of the intracellular adaptors that mediate p75(NTR) signaling are trimeric (i.e., TNFR-associated factor 6 or TRAF6). Consequently, the active receptor signaling unit remains uncertain. To identify the functional receptor complex, we evaluated its oligomerization in vitro and in mice brain tissues using a combination of biochemical techniques. We found that the most abundant homotypic arrangement for p75(NTR) is a trimer and that monomers and trimers coexist at the cell surface. Interestingly, trimers are not required for ligand-independent or ligand-dependent p75(NTR) activation in a growth cone retraction functional assay. However, monomers are capable of inducing acute morphological effects in neurons. We propose that p75(NTR) activation is regulated by its oligomerization status and its levels of expression. These results indicate that the oligomeric state of p75(NTR) confers differential responses and offers an explanation for the diverse and contradictory actions of this receptor in the nervous system. SIGNIFICANCE STATEMENT: The p75 neurotrophin receptor (p75(NTR)) regulates a wide range of cellular functions, including apoptosis, neuronal processes remodeling, and synaptic plasticity. The goal of our work was to inquire whether oligomers of the receptor are required for function. Here we report that p75(NTR) predominantly assembles as a trimer, similar to other tumor necrosis factor receptors. Interestingly, monomers and trimers coexist at the cell surface, but trimers are not required for p75(NTR) activation in a functional assay. However, monomers are capable of inducing acute morphological effects in neurons. Identification of the oligomerization state of p75(NTR) begins to provide insights to the mechanisms of signal initiation of this noncatalytic receptor, as well as to develop therapeutic interventions to diminish its activity.

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin: MOLECULAR INSIGHTS INTO A CRITICAL PREREQUISITE OF MEMBRANE-BOUND MONOMERS.

The insecticidal feature of the three-domain Cry δ-endotoxins from Bacillus thuringiensis is generally attributed to their capability to form oligomeric pores, causing lysis of target larval midgut cells. However, the molecular description of their oligomerization process has not been clearly defined. Here a stable prepore of the 65-kDa trypsin-activated Cry4Ba mosquito-specific toxin was established through membrane-mimetic environments by forming an ∼200-kDa octyl-ß-D-glucoside micelle-induced trimer. The SDS-resistant trimer caused cytolysis to Sf9 insect cells expressing Aedes-mALP (a Cry4Ba receptor) and was more effective than a toxin monomer in membrane perturbation of calcein-loaded liposomes. A three-dimensional model of toxin trimer obtained by negative-stain EM in combination with single-particle reconstruction at ∼5 nm resolution showed a propeller-shaped structure with 3-fold symmetry. Fitting the three-dimensional reconstructed EM map with a 100-ns molecular dynamics-simulated Cry4Ba structure interacting with an octyl-ß-D-glucoside micelle showed relative positioning of individual domains in the context of the trimeric complex with a major protrusion from the pore-forming domain. Moreover, high-speed atomic force microscopy imaging at nanometer resolution and a subsecond frame rate demonstrated conformational transitions from a propeller-like to a globularly shaped trimer upon lipid membrane interactions, implying prepore-to-pore conversion. Real-time trimeric arrangement of monomers associated with L-α-dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid bicelle membranes was also envisaged by successive high-speed atomic force microscopy imaging, depicting interactions among three individual subunits toward trimer formation. Together, our data provide the first pivotal insights into the structural requirement of membrane-induced conformational changes of Cry4Ba toxin monomers for the molecular assembly of a prepore trimer capable of inserting into target membranes to generate a lytic pore.

A DFT Mechanistic Study on Ethylene Tri- and Tetramerization with Cr/PNP Catalysts: Single versus Double Insertion Pathways.

The mechanism of ethylene trimerization and tetramerization with a chromium-diphosphinoamine (Cr-PNP) catalyst system has been studied by theoretical (DFT) methods. Two representative ligands have been explored, namely Ph2 PN(Me)PPh2 and (o-MeC6 H4 )2 PN(Me)P(o-MeC6 H4 )2 . Calculations on the former ligand reveal how a combination of single and double ethylene insertion mechanisms may lead to 1-hexene, 1-octene and the major side products (cyclopentanes and n-alkanes). For the latter ligand, introduction of o-alkyl substitution leads to a more sterically congested active species, which suppresses the available pathways for tetramerization and side product formation. Hence, the high selectivity of o-aryl substituted PNP ligands for trimerization can be rationalized.

Access to Acyclic Z-Enediynes by Alkyne Trimerization: Cooperative Bimetallic Catalysis Using Air as the Oxidant.

Presented herein is a mild, operationally simple, mix-and-go procedure for the synthesis of acyclic trisubstituted Z-enediynes, from readily available terminal alkynes, in good yields. This method stems from a serendipitous discovery, and makes use of cooperative palladium/copper bimetallic catalysis and air as an oxidant to effect an intriguing alkyne trimerization to yield the valuable Z-enediyne moiety.

Crystal structure and molecular imaging of the Nav channel ß3 subunit indicates a trimeric assembly.

The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated ß subunits. Here, we report the crystal structure of the human ß3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the ß3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length ß3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length ß3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that ß3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the ß3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

Synthesis of Branched α-Olefins via Trimerization and Tetramerization of Ethylene.

α-Olefins are very important bulk and fine chemicals and their synthesis from ethylene, an abundantly available and inexpensive feedstock, is highly attractive. Unfortunately, the direct or on-purpose synthesis of olefins from ethylene is limited to three examples, 1-butene, 1-hexene, and 1-octene, all having a linear structure. Herein, the direct synthesis of 3-methylenepentane and 4-ethylhex-1-ene, branched trimerization, and tetramerization products of ethylene, respectively, is reported. Different molecular titanium catalysts, all highly active, with a selectivity toward the formation of the branched ethylene trimer or tetramer, the employment of different activators, and different reaction conditions are the key to selective product formation. The long-time stability of selected catalysts employed permits upscaling as demonstrated for the synthesis of 4-ethylhex-1-ene (52 g isolated, TON(ethylene) 10.7 · 106).

A potent neutralizing nanobody targeting a unique epitope on the receptor-binding domain of SARS-CoV-2 spike protein.

SARS-CoV-2 and its variants continue to threaten public health. Nanobodies that block the attachment of the RBD to host cell angiotensin-converting enzyme 2 (ACE2) represent promising drug candidates. In this study, we reported the identification and structural biological characterization of a nanobody from a RBD-immunized alpaca. The nanobody, termed as 2S-1-19, shows outstanding neutralizing activity against both pseudotyped and authentic SARS-CoV-2 viruses. The crystal structure of 2S-1-19 bound to SARS-CoV-2 RBD reveals an epitope that overlaps with the binding site for ACE2. We also showed that 2S-1-19 reserves promising, though compromised, neutralizing activity against the Delta variant and that the trivalent form of 2S-1-19 remarkably increases its neutralizing capacity. Despite this, neither the monomeric or trimeric 2S-1-19 could neutralize the Omicron BA.1.1 variant, possibility due to the E484A and Q493K mutations found within this virus variant. These data provide insights into immune evasion caused by SARS-CoV-2 variants.