Oligomeric Assemblies Research Articles

SLFN5-mediated chromatin dynamics sculpt higher-order DNA repair topology

Repair of DNA double-strand breaks (DSBs) elicits three-dimensional (3D) chromatin topological changes. A recent finding reveals that 53BP1 assembles into a 3D chromatin topology pattern around DSBs. How this formation of a higher-order structure is configured and regulated remains enigmatic. Here, we report that SLFN5 is a critical factor for 53BP1 topological arrangement at DSBs. Using super-resolution imaging, we find that SLFN5 binds to 53BP1 chromatin domains to assemble a higher-order microdomain architecture by driving damaged chromatin dynamics at both DSBs and deprotected telomeres. Mechanistically, we proposethat 53BP1 topology is shaped by two processes: (1) chromatin mobility driven by the SLFN5-LINC-microtubule axis and (2) the assembly of 53BP1 oligomers mediated by SLFN5. In mammals, SLFN5 deficiency disrupts the DSB repair topology and impairs non-homologous end joining, telomere fusions, class switch recombination, and sensitivity to poly (ADP-ribose) polymerase inhibitor. We establish a molecular mechanism that shapes higher-order chromatin topologies to safeguard genomic stability.

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.

Structural differences between hypoxanthine phosphoribosyltransferase family members highlight opportunities for antiparasitic drug design in neglected diseases

Approaches to identify novel druggable targets for treating neglected diseases include computational studies that predict possible interactions of drugs and their molecular targets. Hypoxanthine phosphoribosyltransferase (HPRT) plays a central role in the purine salvage pathway. This enzyme is essential for the survival of the protozoan parasite T. cruzi, the causal agent of Chagas disease, and other parasites related to neglected diseases. Here we found dissimilar functional behaviours between TcHPRT and the human homologue, HsHPRT, in the presence of substrate analogues that can lie in differences in their oligomeric assemblies and structural features. To shed light on this issue, we carried out a comparative structural analysis between both enzymes. Our results show that HsHPRT is considerably more resistant to controlled proteolysis than TcHPRT. Moreover, we observed a variation in the length of two key loops depending on the structural arrangement of each protein (groups D1T1 and D1T1′). Such variations might be involved in inter-subunit communication or influencing the oligomeric state. Besides, to understand the molecular basis that govern D1T1 and D1T1′ folding groups, we explored the distribution of charges on the interaction surfaces of TcHPRT and HsHPRT, respectively. To know whether the rigidity degree bears effect on the active site, we studied the flexibility of both proteins. The analysis performed here illuminates the underlying reasons and significance behind each protein's preference for one or the other quaternary arrangement that can be exploited for therapeutic approaches.

Oxidative Damages on the Alzheimer’s Related-Aβ Peptide Alters Its Ability to Assemble

Oxidative stress that can lead to oxidation of the amyloid-β (Aβ) peptide is considered a key feature in Alzheimer's disease (AD), influencing the ability of Aβ to assemble into β-sheet rich fibrils that are commonly found in senile plaques of AD patients. The present study aims at investigating the fallouts of Aβ oxidation on the assembly properties of the Aβ peptide. To accomplish this, we performed kinetics and analysis on an oxidized Aβ (oxAβ) peptide, resulting from the attack of reactive oxygen species (ROS) that are formed by the biologically relevant Cu/Aβ/dioxygen/ascorbate system. oxAβ was still able to assemble but displayed ill-defined and small oligomeric assemblies compared to the long and thick β-sheet rich fibrils from the non-oxidized counterpart. In addition, oxAβ does affect the assembly of the parent Aβ peptide. In a mixture of the two peptides, oxAβ has a mainly kinetic effect on the assembly of the Aβ peptide and was able to slow down the formation of Aβ fibril in a wide pH range [6.0-7.4]. However, oxAβ does not change the quantity and morphology of the Aβ fibrils formed to a significant extent. In the presence of copper or zinc di-cations, oxAβ assembled into weakly-structured aggregates rather than short, untangled Cu-Aβ fibrils and long untangled Zn-Aβ fibrils. The delaying effect of oxAβ on metal altered Aβ assembly was also observed. Hence, our results obtained here bring new insights regarding the tight interconnection between (i) ROS production leading to Aβ oxidation and (ii) Aβ assembly, in particular via the modulation of the Aβ assembly by oxAβ. It is the first time that co-assembly of oxAβ and Aβ under various environmental conditions (pH, metal ions …) are reported.

Amyloid oligomers as on-pathway precursors or off-pathway competitors of fibrils.

Amyloid Diseases involve the growth of disease specific proteins into amyloid fibrils and their deposition in protein plaques. Amyloid fibril formation is typically preceded by oligomeric intermediates. Despite significant efforts, the specific role fibrils or oligomers play in the etiology of any given amyloid disease remains controversial. In neurodegenerative disease, though, amyloid oligomers are widely considered critical contributors to disease symptoms. Aside from oligomers as inevitable on-pathway precursors of fibril formation, there is significant evidence for off-pathway oligomer formation competing with fibril growth. The distinct mechanisms and pathways of oligomer formation directly affect our understanding under which conditions oligomers emerge in vivo, and whether their formation is directly coupled to, or distinct from, amyloid fibril formation. In this review, we will discuss the basic energy landscapes underlying the formation of on-pathway vs. off-pathway oligomers, their relation to the related amyloid aggregation kinetics, and their resulting implications for disease etiology. We will review evidence on how differences in the local environment of amyloid assembly can dramatically shift the relative preponderance of oligomers vs. fibrils. Finally, we will comment on gaps in our knowledge of oligomer assembly, of their structure, and on how to assess their relevance to disease etiology.

Template-free prediction of a new monotopic membrane protein fold and oligomeric assembly by AlphaFold2.

AlphaFold2 (AF2) has revolutionized the field of protein structural prediction. Here, we test its ability to predict the tertiary and quaternary structure of a previously undescribed scaffold with new folds and unusual architecture, the monotopic membrane protein caveolin-1 (CAV1). We recently found that CAV1 can assemble a disc-shaped structure composed of 11 symmetrically arranged CAV1 protomers, each of which assumes an identical new fold, that also contains the largest parallel β-barrel known to exist in nature. As this novel fold and assembly was not part of the PDB when AF2 was trained, it presents a blind and challenging test case for the algorithm. AF2 predicts the fold of the protomer despite CAV1's lack of structural similarity to other proteins in the PDB. It also assembles multiple copies of CAV1 into disc-shaped complexes similar in overall organization to those observed experimentally, and predicts several interfaces between protomers accurately at atomic-detail including helix-helix interactions and interactions between conserved residues in loops that stabilize the outer rim of the complex. Interestingly, AF2 predicts additional closed disc structures containing between 7 and 15 CAV1 protomers. However, some of these multimers are energetically strained compared to the experimental structure, especially the parallel β-barrel located in the center of the CAV1 complex. These findings highlight the ability of AF2 to correctly predict new protein folds and assemblies at a granular level, identify important protein interaction interfaces, and predict the overall organization of homo-oligomeric assemblies, while missing some elements of higher order complexes. This work posits a new direction for the continued development of AF2 and other deep learning protein structure prediction approaches.

Client-induced elongation, expansion, and co-aggregation of the lens alpha-crystallins.

The long-lived nature of the eye lens presents unique challenges to the maintenance of protein stability and function. Age-related accumulation of chemical modifications to proteins in the lens promote the formation of light-scattering aggregates that disrupt vision, leading to cataract: a leading cause of blindness worldwide. To counteract these effects, ∼40% of the lens cytosol is composed of α-crystallins (αAc and αBc isoforms): ATP-independent chaperone “holdases” that work to prevent formation of protein aggregates capable of scattering light. Like other members of the small heat shock protein (sHSP) family of chaperones, the α-crystallins form large and polydisperse oligomeric assemblies that recognize and sequester destabilized proteins, through the formation of soluble chaperone/client complexes. Over time, saturating conditions of unfolding clientele overwhelm lens chaperone capacity, leading to light-scattering co-aggregates. A mechanistic basis for the chaperone/client co-aggregation pathway is not well understood. Here, we applied single-particle electron microscopy and other biophysical techniques to define the morphological transitions associated with chaperone/client co-aggregation, using the model client lysozyme. We observe a mechanism where αAc and αBc chaperone/client sequestration progresses through an “initiation complex” (∼15–20 nm diameter, akin to apo-state α-crystallins), and proceeds through an intermediate elongation/expansion stage where co-aggregates reach dimensions of 50–200 nm. Ultimately, under saturating client conditions, the elongation/expansion complexes appear to cluster (or collapse) to form large light-scattering aggregates (microns in diameter). Ensemble and single-particle analysis techniques show αAc and αBc adopt a similar overall mechanism of expansion/elongation, while some unique isoform-specific features may be attributed to characteristic differences in activity toward lysozyme client. Overall, this work provides a mechanistic basis for understanding how α-crystallins (and perhaps other sHSP's) accommodate destabilized clients and depicts a potential client-induced co-aggregation pathway leading to lens opacification and age-related vision loss.

Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase.

Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.

Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts.

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F1F0-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c15-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.

Structural Characterization of Murine Phosphodiesterase 5 Isoforms and Involvement of Cysteine Residues in Supramolecular Assembly.

Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms' oligomeric assembly and the loss of enzymatic activity when modified.

RanBP9 controls the oligomeric state of CTLH complex assemblies

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8β subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8β and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8β-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.

Machine-learning reveals the virtual screening strategies of solid hydrogen-bonded oligomeric assemblies for thermo-responsive applications

The assembly of hydrogen-bonding (H-bonding) motifs enables the phase transition of oligomeric materials, and facilitates thermo-responsive properties such as self-healing and melt-castable capabilities. However, the discovery of solid H-bonded oligomers remains in the laboratory because of the absence of practical virtual screening (VS) strategies. Herein, a machine-learning (ML) approach is proposed to realize the vS for solid H-bonded oligomers with high accuracy (>93 %). A synthetic library comprising 770 oligo-dimethylsiloxane (oDMS) with structurally diverse H-bonding motifs was constructed, and the solid/fluid-state labels of oDMS were obtained via rheological measurements. Tailored descriptors for H-bonding motifs, which were derived from quantum chemistry calculation and analysis, were adopted for ML algorithms, including interaction energy, AlogP98, molecular flexibility, van der Waals area and Connolly surface occupied volume values. The eXtreme Gradient Boosting (XGBoost) model presents the best prediction accuracy among the investigated ML algorithms and the interpretation of XGBoost model provides the feasible vS routines for the discovery of solid H-bonded oligomers.

Microtubule Severing Enzymes Oligomerization and Allostery: A Tale of Two Domains.

Severing proteins are nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily whose function is to remodel the largest cellular filaments, microtubules. The standard AAA+ machines adopt hexameric ring structures for functional reasons, while being primarily monomeric in the absence of the nucleotide. Both major severing proteins, katanin and spastin, are believed to follow this trend. However, studies proposed that they populate lower-order oligomers in the presence of cofactors, which are functionally relevant. Our simulations show that the preferred oligomeric assembly is dependent on the binding partners and on the type of severing protein. Essential dynamics analysis predicts that the stability of an oligomer is dependent on the strength of the interface between the helical bundle domain (HBD) of a monomer and the convex face of the nucleotide binding domain (NBD) of a neighboring monomer. Hot spots analysis found that the region consisting of the HBD tip and the C-terminal (CT) helix is the only common element between the allosteric networks responding to nucleotide, substrate, and intermonomer binding. Clustering analysis indicates the existence of multiple pathways for the transition between the secondary structure of the HBD tip in monomers and the structure(s) it adopts in oligomers.

Oligomerization of the heteromeric γ-aminobutyric acid receptor GABAB in a eukaryotic cell-free system

Understanding the assembly mechanism and function of membrane proteins is a fundamental problem in biochemical research. Among the membrane proteins, G protein-coupled receptors (GPCRs) represent the largest class in the human body and have long been considered to function as monomers. Nowadays, the oligomeric assembly of GPCRs is widely accepted, although the functional importance and therapeutic intervention remain largely unexplored. This is partly due to difficulties in the heterologous production of membrane proteins. Cell-free protein synthesis (CFPS) with its endogenous endoplasmic reticulum-derived structures has proven as a technique to address this issue. In this study, we investigate for the first time the conceptual CFPS of a heteromeric GPCR, the γ-aminobutyric acid receptor type B (GABAB), from its protomers BR1 and BR2 using a eukaryotic cell-free lysate. Using a fluorescence-based proximity ligation assay, we provide evidence for colocalization and thus suggesting heterodimerization. We prove the heterodimeric assembly by a bioluminescence resonance energy transfer saturation assay providing the manufacturability of a heterodimeric GPCR by CFPS. Additionally, we show the binding of a fluorescent orthosteric antagonist, demonstrating the feasibility of combining the CFPS of GPCRs with pharmacological applications. These results provide a simple and powerful experimental platform for the synthesis of heteromeric GPCRs and open new perspectives for the modelling of protein–protein interactions. Accordingly, the presented technology enables the targeting of protein assemblies as a new interface for pharmacological intervention in disease-relevant dimers.

Slc25a12 regulates Opa1 oligomeric assembly and cristae maintenance which is implicated in cardiac pathophysiology

Slc25a12 regulates Opa1 oligomeric assembly and cristae maintenance which is implicated in cardiac pathophysiology

Structures of the holo CRISPR RNA-guided transposon integration complex

CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism1–3. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA–TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC–TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein–DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.

Ligand-Based Discovery of a Small Molecule as Inhibitor of α-Synuclein Amyloid Formation

α-Synuclein (α-Syn) aggregates are implicated in Parkinson's disease (PD), so inhibitors of α-Syn aggregation have been intensively explored. It has been demonstrated that small molecules might be able to reduce α-Syn aggregation in fibrils, thus exerting neuroprotective effects in models of PD. To expand our knowledge about the structural requirements for blocking the recognition process into the oligomeric assembly of α-Syn aggregates, we performed a ligand-based virtual screening procedure using two well-known α-Syn aggregation inhibitors, SynuClean-D and ZPD-2, as query compounds. A collection of thirty-four compounds bearing distinct chemical functionalities and mutual chemical features were studied in a Th-T fluorescence test, thus identifying 5-(2,6-dinitro-4-(trifluoromethyl)benzyl)-1-methyl-1H-tetrazole (named MeSC-04) as a potent α-Syn amyloid formation inhibitor that demonstrated similar behavior when compared to SynuClean-D in the thioflavin-T-monitored kinetic assays, with both molecules reducing the number and size of amyloid fibrils, as evidenced by electron microscopy. Molecular modeling studies suggested the binding mode of MeSC-04 through the identification of putative druggable pockets on α-syn fibrils and a subsequent consensus docking methodology. Overall, this work could furnish new insights in the development of α-Syn amyloid inhibitors from synthetic sources.

Inhibition of Amyloid Nucleation by Steric Hindrance.

Despite recent experiments and simulations suggesting that small-molecule inhibitors and some post-translational modifications (e.g., glycosylation and ubiquitination) can suppress the pathogenic aggregation of proteins due to steric hindrance, the effect of steric hindrance on amyloid formation has not been systematically studied. Based on Monte Carlo simulations using a coarse-grained model for amyloidogenic proteins and a hard sphere acting as steric hindrance, we investigated how steric hindrance on proteins could affect amyloid formation, particularly two steps of primary nucleation, namely, oligomerization and conformational conversion into a β-sheet-enriched nucleus. We found that steric spheres played an inhibitory role in oligomerization with the effect proportional to the sphere radius RS, which we attributed to the decline in the nonspecific attractions between proteins. During the second step, small steric spheres facilitated the conformational conversion of proteins while large ones suppressed the conversion. The overall steric effect on amyloid nucleation was inhibitory regardless of RS. As RS increased, oligomeric assemblies changed from amorphous into sheet-like, structurally ordered species, reminiscent of the structure of amyloid fibrils. The oligomers with large RS were off-pathway with their ordered structures induced by the competition between steric hindrance and nonspecific attractions of soluble proteins. Interestingly, the equimolar mixture of proteins with and without steric hindrance amplified the sterically inhibitory effect by increasing the energy barrier of protein's conformational conversion. The physical mechanisms and biological implications of the above results are discussed. Our findings improve the current understanding of how nature regulates protein aggregation and amyloid formation by steric hindrance.

Signal peptide stabilizes folding and inhibits misfolding of serum amyloid A.

Signal peptide (SP) plays an important role in membrane targeting for insertion of secretory and membrane proteins during translocation processes in prokaryotes and eukaryotes. Beside the targeting functions, SP has also been found to affect the stability and folding of several proteins. Serum amyloid A (SAA) proteins are apolipoproteins responding to acute-phase inflammation. The fibrillization of SAA results in a protein misfolding disease named amyloid A (AA) amyloidosis. The main disease-associated isoform of human SAA, SAA1.1, is expressed as a precursor protein with an N-terminal signal peptide composed of 18 residues. The cleavage of the SP generates mature SAA1.1. To investigate whether the SP affects properties of SAA1.1, we systematically examined the structure, protein stability, and fibrillization propensity of pre-SAA1.1, which possesses the SP, and Ser-SAA1.1 without the SP but containing with an additional N-terminal serine residue. We found that the presence of the SP did not significantly affect the predominant helical structure but changed the tertiary conformation as evidenced by intrinsic fluorescence and exposed hydrophobic surfaces. Pre-SAA1.1 and Ser-SAA1.1 formed distinct oligomeric assemblies in which pre-SAA1.1 populated as tetramer and octamer, whereas Ser-SAA1.1 existed as a predominant hexamer. Pre-SAA1.1 was found significantly more stable than Ser-SAA1.1 upon thermal and chemical unfolding. Ser-SAA1.1, but not pre-SAA1.1, is capable of forming amyloid fibrils in protein misfolding study, indicating a protective role of the SP. Altogether, our results demonstrated a novel role of the SP in SAA folding and misfolding and provided a novel direction for therapeutic development of AA amyloidosis.

Template-free prediction of a new monotopic membrane protein fold and assembly by AlphaFold2

AlphaFold2 (AF2) has revolutionized the field of protein structural prediction. Here, we test its ability to predict the tertiary and quaternary structure of a previously undescribed scaffold with new folds and unusual architecture, the monotopic membrane protein caveolin-1 (CAV1). CAV1 assembles into a disc-shaped oligomer composed of 11 symmetrically arranged protomers, each assuming an identical new fold, and contains the largest parallel β-barrel known to exist in nature. Remarkably, AF2 predicts both the fold of the protomers and the interfaces between them. It also assembles between seven and 15 copies of CAV1 into disc-shaped complexes. However, the predicted multimers are energetically strained, especially the parallel β-barrel. These findings highlight the ability of AF2 to correctly predict new protein folds and oligomeric assemblies at a granular level while missing some elements of higher-order complexes, thus positing a new direction for the continued development of deep-learning protein structure prediction approaches.