An atlas of protein homo-oligomerization across domains of life.

Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes.

Higher-order SPOP assembly reveals a basis for cancer mutant dysregulation.

The speckle-type POZ protein (SPOP) functions in the Cullin3-RING ubiquitin ligase (CRL3) as a receptor for the recognition of substrates involved in cell growth, survival, and signaling. SPOP mutations have been attributed to the development of many types of cancers, including prostate and endometrial cancers. Prostate cancer mutations localize in the substrate-binding site of the substrate recognition (MATH) domain and reduce or prevent binding. However, most endometrial cancer mutations are dispersed in seemingly inconspicuous solvent-exposed regions of SPOP, offering no clear basis for their cancer-causing and peculiar gain-of-function properties. Herein, we present the first structure of SPOP in its oligomeric form, uncovering several new interfaces important for SPOP self-assembly and normal function. Given that many previously unaccounted-for cancer mutations are localized in these newly identified interfaces, we uncover molecular mechanisms underlying dysregulation of SPOP function, with effects ranging from gross structural changes to enhanced self-association, and heightened stability and activity.

Structure, dynamics, assembly, and evolution of protein complexes.

The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.

Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization.

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.

NLRP3 is its own gatekeeper: a group hug of NLRP3 monomers controls inflammation.

A recent study by Hochheiser et al. describes the cryo-electron microscopy (cryoEM) structure of an autoinhibited nucleotide-binding domain-, leucine-rich repeat (LRR)- and pyrin domain-containing protein 3 (NLRP3) decamer that assembles via LRR interactions and is further stabilized by the small-molecule NLRP3-specific inhibitor CRID3 binding into a cleft within the NACHT domain. The study provides a springboard for the development of novel NLRP3-based therapies.

Is a cure for Parkinson's disease hiding inside us?

α-Synuclein (a-syn) oligomers and fibrils are behind neurodegeneration in Parkinson's disease (PD), but therapeutically targeting them is challenging. Amphipathic and cationic helical peptides inhibit amyloid formation and suppress neurotoxicity by selectively binding the solvent-accessible regions in these toxic species. Can endogenous peptides, like LL-37, constitute a new therapeutic paradigm in PD?

The disordered protein SERF promotes α-Synuclein aggregation through liquid-liquid phase separation.

The aggregation of α-Synuclein (α-Syn) into amyloid fibrils is the hallmark of Parkinson's disease. Under stress or other pathological conditions, the accumulation of α-Syn oligomers is the main contributor to the cytotoxicity. A potential approach for treating Parkinson's disease involves preventing the accumulation of these α-Syn oligomers. In this study, we present a novel mechanism involving a conserved group of disorderly proteins known as small EDRK-rich factor (SERF), which promotes the aggregation of α-Syn through a cophase separation process. Using diverse methods like confocal microscopy, fluorescence recovery after photobleaching assays, solution-state NMR spectroscopy, and Western blot, we determined that the N-terminal domain of SERF1a plays a role in the interactions that occur during cophase separation. Within these droplets, α-Syn undergoes a gradual transformation from solid condensates to amyloid fibrils, while SERF1a is excluded from the condensates and dissolves into the solution. Notably, in vivo experiments show that SERF1a cophase separation with α-Syn significantly reduces the deposition of α-Syn oligomers and decreases its cellular toxicity under stress. These findings suggest that SERF1a accelerates the conversion of α-Syn from highly toxic oligomers to less toxic fibrils through cophase separation, thereby mitigating the biological damage of α-Syn aggregation.

Large-scale column-free purification of bovine F-ATP synthase.

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.

In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Elevated concentrations of amyloid-ß oligomers and their proapoptotic effects on age-related cataract.

Recently, amyloid-ß oligomers (AßOs) have been studied as the primary pathogenic substances in Alzheimer's disease (AD). Our previous study revealed that the Aß expression level is closely related to ARC progression. Here, we demonstrated that the accumulation of AßOs in the lens epithelium of age-related cataract (ARC) patients increased during ARC progression and that this alteration was consistent with the changes in mitochondrial function, oxidative stress, and cellular apoptosis. In vitro, human lens epithelial cells (HLECs) treated with AßOs exhibited Ca2+ dyshomeostasis, impaired mitochondrial function, elevated oxidative stress levels, and increased apoptosis. Moreover, the proapoptotic effect of AßOs was alleviated after the uptake of mitochondrial Ca2+ was inhibited. These results establish that AßOs may promote HLEC apoptosis by inducing mitochondrial Ca2+ overload, thus preliminarily revealing the possible association between the accumulation of AßOs and other pathological processes in ARC.

SOBA: Development and testing of a soluble oligomer binding assay for detection of amyloidogenic toxic oligomers.

The formation of toxic Amyloid ß-peptide (Aß) oligomers is one of the earliest events in the molecular pathology of Alzheimer's Disease (AD). These oligomers lead to a variety of downstream effects, including impaired neuronal signaling, neuroinflammation, tau phosphorylation, and neurodegeneration, and it is estimated that these events begin 10 to 20 y before the presentation of symptoms. Toxic Aß oligomers contain a nonstandard protein structure, termed α-sheet, and designed α-sheet peptides target this main-chain structure in toxic oligomers independent of sequence. Here we show that a designed α-sheet peptide inhibits the deleterious effects on neuronal signaling and also serves as a capture agent in our soluble oligomer binding assay (SOBA). Pre-incubated synthetic α-sheet-containing Aß oligomers produce strong SOBA signals, while monomeric and ß-sheet protofibrillar Aß do not. α-sheet containing oligomers were also present in cerebrospinal fluid (CSF) from an AD patient versus a noncognitively impaired control. For the detection of toxic oligomers in plasma, we developed a plate coating to increase the density of the capture peptide. The proof of concept was achieved by testing 379 banked human plasma samples. SOBA detected Aß oligomers in patients on the AD continuum, including controls who later progressed to mild cognitive impairment. In addition, SOBA discriminated AD from other forms of dementia, yielding sensitivity and specificity of 99% relative to clinical and neuropathological diagnoses. To explore the broader potential of SOBA, we adapted the assay for a-synuclein oligomers and confirmed their presence in CSF from patients with Parkinson's disease and Lewy body dementia.

Structure of the prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum reveals an open hexamer conformation.

Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans.

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin- or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20 µM with a Vmax of 0.079 nmol/(µg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

Molecular, structural, and functional characterization of delta subunit of T-complex protein-1 from Leishmania donovani.

Chaperonins/Heat shock protein 60 are ubiquitous multimeric protein complexes that assist in the folding of partially and/or misfolded proteins using metabolic energy into their native stage. The eukaryotic group II chaperonin, also referred as T-complex protein-1 ring complex (TRiC)/T-complex protein-1 (TCP1)/chaperonin containing T-complex protein (CCT), contains 8-9 paralogous subunits, arranged in each of the two rings of hetero-oligomeric complex. In Leishmania, till date, only one subunit, LdTCP1γ, has been well studied. Here, we report the molecular, structural, and functional characterization of TCP1δ subunit of Leishmania donovani (LdTCP1δ), the causative agent of Indian kala-azar. LdTCP1δ gene exhibited only 27.9% identity with LdTCP1γ and clustered in a separate branch in the phylogenic tree of LdTCP1 subunits. The purified recombinant protein formed a high molecular weight complex (0.75 MDa), arranged into 16-mer assembly, and performed in vitro chaperonin activity as assayed by ATP-dependent luciferase folding. LdTCP1δ exhibits 1.8-fold upregulated expression in metabolically active, rapidly dividing log phase promastigotes. Over-expression of LdTCP1δ in promastigotes results in increased infectivity and rate of multiplication of intracellular amastigotes. The study thus establishes the existence of an individual functionally active homo-oligomeric complex of LdTCP1δ chaperonin with its role in parasite infectivity and multiplication.

Activated human Orai1 channel in lipid biolayer may exist as a pentamer.

The human Orai1 (hOrai1) channel plays a crucial role in extracellular Ca2+ influx and has emerged as an attractive drug target for various diseases. However, the activated structure of the hOrai1 channel assembly within a lipid bilayer remains unknown. In this study, we expressed and purified the hOrai1 channel covalently linked to two SOAR tandems (HOSS). Patch-clamp experiments in whole-cell configuration showed that HOSS is constitutively active. Biochemical characterization confirmed that the purified HOSS channels were successfully incorporated into MSP1E3D1 nanodiscs. Negative staining revealed that the HOSS channels resemble a mushroom, with the body representing the hOrai1 channel and the leg representing the SOAR domain. Surprisingly, 2D analysis of cryo-EM data demonstrated a pentameric assembly of HOSS in a lipid bilayer. Our findings suggest that the hOrai1 channel may assemble into different oligomeric states in response to varying membrane environments.

Experimental modification in thermal stability of oligomers by alanine substitution and site saturation mutagenesis of interfacial residues.

For certain industrial applications, the stability of protein oligomers is important. In this study, we demonstrated an efficient method to improve the thermal stability of oligomers using the trimeric protein chloramphenicol acetyltransferase (CAT) as the model. We substituted all interfacial residues of CAT with alanine to detect residues critical for oligomer stability. Mutation of six of the forty-nine interfacial residues enhanced oligomer thermal stability. Site saturation mutagenesis was performed on these six residues to optimize the side chains. About 15% of mutations enhanced thermal stability by more than 0.5 °C and most did not disrupt activity of CAT. Certain combinations of mutations further improved thermal stability and resistance against heat treatment. The quadruple mutant, H17V/N34S/F134A/D157C, retained the same activity as the wild-type after heat treatment at 9 °C higher temperature than the wild-type CAT. Furthermore, combinations with only alanine substitutions also improved thermal stability, suggesting the method we developed can be used for rapid modification of industrially important proteins.

Amyloid-ß oligomer-induced neurotoxicity by exosomal interactions between neuron and microglia.

A hallmark of Alzheimer's disease (AD) is amyloid-ß (Aß) plaque deposition in the brain, causing deficits in cognitive function. Amyloid-beta oligomers (AßOs), the soluble precursor peptides producing Aß plaques, also produce neurotoxicity and microgliosis together with glycolytic reprogramming. Recently, monocarboxylate transporter 1 (MCT1), a key glycolysis regulator, and its ancillary protein, CD147, are found to play an important role in the secretion of exosomes, 30-200 nm vesicles in size, which are considered as toxic molecule carriers in AD. However, the effect of low-concentration AßOs (1 nM) on microglia MCT1 and CD147 expression as well as 1 nM AßOs-treated microglia-derived exosomes on neuronal toxicity remain largely elusive. In this study, 1 nM AßOs induce significant axonopathy and microgliosis. Furthermore, 1 nM AßOs-treated neurons- or microglia-derived exosomes produce axonopathy through their autologous or heterologous uptake by neurons, supporting the role of exosomes as neurotoxicity mediators in AD. Interestingly, MCT1 and CD147 are enhanced in microglia by treatment with 1 nM AßOs or exosomes from 1 nM AßOs-treated- microglia or neurons, suggesting the implication of AßOs-induced enhanced MCT1 and CD147 in microglia with AD neuropathogenesis, which is consistent with the in-silico analysis of the single cell RNA sequencing data from microglia in mouse models of AD and AD patients.

Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis.

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

Early Events in ß2AR Dimer Dynamics Mediated by Activation-Related Microswitches.

G-Protein-Coupled Receptors (GPCRs) make up around 3-4% of the human genome and are the targets of one-third of FDA-approved drugs. GPCRs typically exist as monomers but also aggregate to form higher-order oligomers, including dimers. ß2AR, a pharmacologically relevant GPCR, is known to be targeted for the treatment of asthma and cardiovascular diseases. The activation of ß2AR at the dimer level remains under-explored. In the current study, molecular dynamics (MD) simulations have been performed to understand activation-related structural changes in ß2AR at the dimer level. The transition from inactive to active and vice versa has been studied by starting the simulations in the apo, agonist-bound, and inverse agonist-bound ß2AR dimers for PDB ID: 2RH1 and PDB ID: 3P0G, respectively. A cumulative total of around 21-µs simulations were performed. Residue-based distances, RMSD, and PCA calculations suggested that either of the one monomer attained activation-related features for the apo and agonist-bound ß2AR dimers. The TM5 and TM6 helices within the two monomers were observed to be in significant variation in all the simulations. TM5 bulge and proximity of TM2 and TM7 helices may be contributing to one of the early events in activation. The dimeric interface between TM1 and helix 8 were observed to be well maintained in the apo and agonist-bound simulations. The presence of inverse agonists favored inactive features in both the monomers. These key features of activation known for monomers were observed to have an impact on ß2AR dimers, thereby providing an insight into the oligomerization mechanism of GPCRs.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

IMPORTANCE: Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.