Oligomeric Assemblies Research Articles

Free Cholesterol Accelerates Aβ Self-Assembly on Membranes at Physiological Concentration.

The effects of membranes on the early-stage aggregation of amyloid β (Aβ) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer’s disease. We have shown that direct Aβ-membrane interactions dramatically enhance the Aβ aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aβ monomers and significantly changes the conformational sampling of Aβ monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aβ-lipid interaction is an important factor in the disease prone amyloid assembly process.

Macrocyclic Peptides Derived from Familial Alzheimer's Disease Mutants Show Charge-Dependent Oligomeric Assembly and Toxicity.

This work probes the role of charge in the oligomeric assembly, toxicity, and membrane destabilization of a series of peptides derived from Aβ and the E22Q and E22K familial mutants. In the mutant Aβ peptides, an acidic residue (E) is replaced with either a neutral or basic residue (Q or K), thus altering the net charge of the peptide. Acetylation at peripheral positions permits modulation of charge of the peptides and allows investigation of the role of charge in their oligomeric assembly, cytotoxicity, and membrane disruption. Peptides with the same net charge generally behave similarly even if the amino acid residue at position 22 differs. As the net charge of the peptide decreases, so does the extent of assembly, cytotoxicity, and membrane destabilization, which were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lactate dehydrogenase (LDH)-release assays with SH-SY5Y cells, and dye leakage assays using liposomes. These findings suggest that the charge of the amino acid side chain, rather than its size or hydrophobicity, accounts for the differences in the oligomeric assembly and toxicity of the E22 familial mutants of Aβ.

Modulators of the plasma membrane protect cells from diverse toxic agents, ranging from protein misfolded oligomers to pore forming agents

Numerous neurodegenerative diseases are characterized by the misfolding and deposition of proteinaceous aggregates in the central nervous system. Intense efforts are underway to target pharmacologically oligomeric assemblies of amyloid proteins, as these heterogenous and transient intermediates are thought to play a seminal role in the onset and development of a range of protein misfolding diseases, including Alzheimer’s and Parkinson’s diseases. We have shown previously that aminosterols can target the toxicity induced by several types of protein misfolded oligomers by displacing these toxins from cell membranes through a generic mechanism.

Subcellular Dynamics of a Conserved Bacterial Polar Scaffold Protein

In order to survive, bacterial cells rely on precise spatiotemporal organization and coordination of essential processes such as cell growth, chromosome segregation, and cell division. Given the general lack of organelles, most bacteria are forced to depend on alternative localization mechanisms, such as, for example, geometrical cues. DivIVA proteins are widely distributed in mainly Gram-positive bacteria and were shown to bind the membrane, typically in regions of strong negative curvature, such as the cell poles and division septa. Here, they have been shown to be involved in a multitude of processes: from apical cell growth and chromosome segregation in actinobacteria to sporulation and inhibition of division re-initiation in firmicutes. Structural analyses revealed that DivIVA proteins can form oligomeric assemblies that constitute a scaffold for recruitment of other proteins. However, it remained unclear whether interaction with partner proteins influences DivIVA dynamics. Using structured illumination microscopy (SIM), single-particle tracking (SPT) microscopy, and fluorescent recovery after photobleaching (FRAP) experiments, we show that DivIVA from Corynebacterium glutamicum is mobilized by its binding partner ParB. In contrast, we show that the interaction between Bacillus subtilis DivIVA and its partner protein MinJ reduces DivIVA mobility. Furthermore, we show that the loss of the rod-shape leads to an increase in DivIVA dynamics in both organisms. Taken together, our study reveals the modulation of the polar scaffold protein by protein interactors and cell morphology. We reason that this leads to a very simple, yet robust way for actinobacteria to maintain polar growth and their rod-shape. In B. subtilis, however, the DivIVA protein is tailored towards a more dynamic function that allows quick relocalization from poles to septa upon division.

Curcumin Inhibits Membrane-Damaging Pore-Forming Function of the β-Barrel Pore-Forming Toxin Vibrio cholerae Cytolysin.

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin (β-PFT). Upon encountering the target cells, VCC forms heptameric β-barrel pores and permeabilizes the cell membranes. Structure-function mechanisms of VCC have been extensively studied in the past. However, the existence of any natural inhibitor for VCC has not been reported yet. In the present study, we show that curcumin can compromise the membrane-damaging activity of VCC. Curcumin is known to modulate a wide variety of biological processes and functions. However, the application of curcumin in the physiological scenario often gets limited due to its extremely poor solubility in the aqueous environment. Interestingly, we find that VCC can associate with the insoluble fraction of curcumin in the aqueous medium and thus gets separated from the solution phase. This, in turn, reduces the availability of VCC to attack the target membranes and thus blocks the membrane-damaging action of the toxin. We also observe that the soluble aqueous extract of curcumin, generated by the heat treatment, compromises the pore-forming activity of VCC. Interestingly, in the presence of such soluble extract of curcumin, VCC binds to the target membranes and forms the oligomeric assembly. However, such oligomers appear to be non-functional, devoid of the pore-forming activity. The ability of curcumin to bind to VCC and neutralize its membrane-damaging activity suggests that curcumin has the potential to act as an inhibitor of this potent bacterial β-PFT.

α-synuclein-assisted oligomerization of β-amyloid (1–42)

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative disorders, characterized by aggregation of amyloid polypeptides, β-amyloid (Aβ) and α-synuclein (αS), respectively. Aβ and αS follow similar aggregation pathways, starting from monomers, to soluble toxic oligomeric assemblies, and to insoluble fibrils. Various studies have suggested overlaps in the pathologies of AD and PD, and have shown Aβ-αS interactions. Unfortunately, whether these protein-protein interactions lead to self- and co-assembly of Aβ and αS into oligomers – a potentially toxic synergistic mechanism – is poorly understood. Among the various Aβ isoforms, interactions of Aβ containing 42 amino acids (Aβ (1–42), referred to as Aβ42) with αS are of most direct relevance due to the high aggregation propensity and the strong toxic effect of this Aβ isoform. In this study, we carefully determined molecular consequences of interactions between Aβ42 and αS in their respective monomeric, oligomeric, and fibrillar forms using a comprehensive set of experimental tools. We show that the three αS conformers, namely, monomers, oligomers and fibrils interfered with fibrillization of Aβ42. Specifically, αS monomers and oligomers promoted oligomerization and stabilization of soluble Aβ42, possibly via direct binding or co-assembly, while αS fibrils hindered soluble Aβ42 species from converting into insoluble aggregates by the formation of large oligomers. We also provide evidence that the interactions with αS were mediated by various parts of Aβ42, depending on Aβ42 and αS conformers. Furthermore, we compared similarities and dissimilarities between Aβ42-αS and Aβ40-αS interactions. Overall, the present study provides a comprehensive depiction of the molecular interplay between Aβ42 and αS, providing insight into its synergistic toxic mechanism.

The Effect of Ethanol on Disassembly of Amyloid-β1-42 Pentamer Revealed by Atomic Force Microscopy and Gel Electrophoresis.

The most common type of dementia, Alzheimer’s disease, is associated with senile plaques formed by the filamentous aggregation of hydrophobic amyloid-β (Aβ) in the brains of patients. Small oligomeric assemblies also occur and drugs and chemical compounds that can interact with such assemblies have attracted much attention. However, these compounds need to be solubilized in appropriate solvents, such as ethanol, which may also destabilize their protein structures. As the impact of ethanol on oligomeric Aβ assembly is unknown, we investigated the effect of various concentrations of ethanol (0 to 7.2 M) on Aβ pentameric assemblies (Aβp) by combining blue native-PAGE (BN-PAGE) and ambient air atomic force microscopy (AFM). This approach was proven to be very convenient and reliable for the quantitative analysis of Aβ assembly. The Gaussian analysis of the height histogram obtained from the AFM images was correlated with band intensity on BN-PAGE for the quantitative estimation of Aβp. Our observations indicated up to 1.4 M (8.3%) of added ethanol can be used as a solvent/vehicle without quantitatively affecting Aβ pentamer stability. Higher concentration induced significant destabilization of Aβp and eventually resulted in the complete disassembly of Aβp.

Protection against Amyloid-β Oligomer Neurotoxicity by Small Molecules with Antioxidative Properties: Potential for the Prevention of Alzheimer's Disease Dementia.

Soluble oligomeric assemblies of amyloid β-protein (Aβ), called Aβ oligomers (AβOs), have been recognized as primary pathogenetic factors in the molecular pathology of Alzheimer’s disease (AD). AβOs exert neurotoxicity and synaptotoxicity and play a critical role in the pathological progression of AD by aggravating oxidative and synaptic disturbances and tau abnormalities. As such, they are important therapeutic targets. From a therapeutic standpoint, it is not only important to clear AβOs or prevent their formation, it is also beneficial to reduce their neurotoxicity. In this regard, recent studies have reported that small molecules, most with antioxidative properties, show promise as therapeutic agents for reducing the neurotoxicity of AβOs. In this mini-review, we briefly review the significance of AβOs and oxidative stress in AD and summarize studies on small molecules with AβO-neurotoxicity-reducing effects. We also discuss mechanisms underlying the effects of these compounds against AβO neurotoxicity as well as their potential as drug candidates for the prevention and treatment of AD.

Structures and functions of the membrane-damaging pore-forming proteins.

Pore-forming proteins (PFPs) of the diverse life forms have emerged as the potent cell-killing entities owing to their specialized membrane-damaging properties. PFPs have the unique ability to perforate the plasma membranes of their target cells, and they exert this functionality by creating oligomeric pores in the membrane lipid bilayer. Pathogenic bacteria employ PFPs as toxins to execute their virulence mechanisms, whereas in the higher vertebrates PFPs are deployed as the part of the immune system and to generate inflammatory responses. PFPs are the unique dimorphic proteins that are generally synthesized as water-soluble molecules, and transform into membrane-inserted oligomeric pore assemblies upon interacting with the target membranes. In spite of sharing very little sequence similarity, PFPs from diverse organisms display incredible structural similarity. Yet, at the same time, structure-function mechanisms of the PFPs document remarkable versatility. Such notions establish PFPs as the fascinating model system to explore variety of unsolved issues pertaining to the structure-function paradigm of the proteins that interact and act in the membrane environment. In this article, we discuss our current understanding regarding the structural basis of the pore-forming functions of the diverse class of PFPs. We attempt to highlight the similarities and differences in their structures, membrane pore-formation mechanisms, and their implications for the various biological processes, ranging from the bacterial virulence mechanisms to the inflammatory immune response generation in the higher animals.

Abstract 9749: High-Avidity Binding Drives Nucleation of Amyloidogenic Transthyretin Monomer in Cardiac Amyloidosis

Amyloidosis involves stepwise growth of fibrils assembled from soluble precursor proteins. Mutations of plasma carrier protein transthyretin (TTR) are associated with hereditary transthyretin amyloidosis (ATTR). TTR naturally folds into a stable tetramer for its function, whereas conditions and mutations that foster aberrant monomer formation facilitate TTR oligomeric aggregation and subsequent fibril extension. We investigated the dynamics of early assembly of oligomers by wild-type TTR, in comparison to its TTR V30M and TTR V122I disease mutants. We selected buffer conditions for monitoring time-dependent redistribution among monomer, dimer, tetramer, and oligomer contents in the presence and absence of multimeric TTR seeds. The seeds were artificially constructed recombinant multimers that contained 20-40 TTR subunits via engineered biotin-streptavidin interactions. As expected, these multimer seeds rapidly nucleated TTR monomers and dimers into larger complexes, while having no effect on tetramers. In vivo , streptavidin-induced multimers formed ATTR-like deposits in the heart and the kidney following i.v. injection in mice. While all three variants prominently deposited in the kidney glomerulus, only TTR V30M resulted in extensive deposition in the heart. The cardiac TTR deposits varied in size and shape and were localized to the intermyofibrillar space along the capillaries. These results are consistent with the notion of monomeric TTR to engage high-avidity interactions with tissue amyloids. Our multimeric induction approach therefore provides a new model for studying the initiation of TTR deposition in the heart.

Precision Mapping of Amyloid-β Binding Reveals Perisynaptic Localization and Spatially Restricted Plasticity Deficits.

Secreted amyloid-β (Aβ) peptide forms neurotoxic oligomeric assemblies thought to cause synaptic deficits associated with Alzheimer’s disease (AD). Soluble Aβ oligomers (Aβo) directly bind to neurons with high affinity and block plasticity mechanisms related to learning and memory, trigger loss of excitatory synapses and eventually cause cell death. While Aβo toxicity has been intensely investigated, it remains unclear precisely where Aβo initially binds to the surface of neurons and whether sites of binding relate to synaptic deficits. Here, we used a combination of live cell, super-resolution and ultrastructural imaging techniques to investigate the kinetics, reversibility and nanoscale location of Aβo binding. Surprisingly, Aβo does not bind directly at the synaptic cleft as previously thought but, instead, forms distinct nanoscale clusters encircling the postsynaptic membrane with a significant fraction also binding presynaptic axon terminals. Synaptic plasticity deficits were observed at Aβo-bound synapses but not closely neighboring Aβo-free synapses. Thus, perisynaptic Aβo binding triggers spatially restricted signaling mechanisms to disrupt synaptic function. These data provide new insight into the earliest steps of Aβo pathology and lay the groundwork for future studies evaluating potential surface receptor(s) and local signaling mechanisms responsible for Aβo binding and synapse dysfunction.

Chemical Tuning of Exciton versus Charge-Transfer Excited States in Conformationally Restricted Arylene Cages.

Covalent assemblies of conjugated organic chromophores provide the opportunity to engineer new excited states with novel properties. In this work, a newly developed triple-stranded cage architecture, in which meta-substituted aromatic caps serve as covalent linking groups that attach to both top and bottom of the conjugated molecule walls, is used to tune the properties of thiophene oligomer assemblies. Benzene-capped and triazine-capped 5,5'-(2,2-bithiophene)-containing arylene cages are synthesized and characterized using steady-state and time-resolved spectroscopic methods. The conformational freedom and electronic states are analyzed using time-dependent density functional theory. The benzene cap acts as a passive spacer whose electronic states do not mix with those of the chromophore walls. The excited state properties are dominated by through-space interactions between the chromophore subunits, generating a neutral Frenkel H-type exciton state. This excitonic state undergoes intersystem crossing on a 200 ps time scale while the fluorescence output is suppressed by a factor of 2 due to a decreased radiative rate. Switching to a triazine cap enables electron transfer from the chromophore-linker after the initial excitation to the exciton state, leading to the formation of a charge-transfer state within 10 ps. This state can avoid intersystem crossing and exhibits red-shifted fluorescence with enhanced quantum yield. The ability to interchange structural modules with different electronic properties while retaining the overall cage morphology provides a new approach for tuning the properties of discrete chromophore assemblies.

Visualization of Sparsely-populated Lower-order Oligomeric States of Human Mitochondrial Hsp60 by Cryo-electron Microscopy

Human mitochondrial Hsp60 (mtHsp60) is a class I chaperonin, 51% identical in sequence to the prototypical E. coli chaperonin GroEL. mtHsp60 maintains the proteome within the mitochondrion and is associated with various neurodegenerative diseases and cancers. The oligomeric assembly of mtHsp60 into heptameric ring structures that enclose a folding chamber only occurs upon addition of ATP and is significantly more labile than that of GroEL, where the only oligomeric species is a tetradecamer. The lability of the mtHsp60 heptamer provides an opportunity to detect and visualize lower-order oligomeric states that may represent intermediates along the assembly/disassembly pathway. Using cryo-electron microscopy we show that, in addition to the fully-formed heptamer and an “inverted” tetradecamer in which the two heptamers associate via their apical domains, thereby blocking protein substrate access, well-defined lower-order oligomeric species, populated at less than 6% of the total particles, are observed. Specifically, we observe open trimers, tetramers, pentamers and hexamers (comprising ∼4% of the total particles) with rigid body rotations from one subunit to the next within ∼1.5–3.5° of that for the heptamer, indicating that these may lie directly on the assembly/disassembly pathway. We also observe a closed-ring hexamer (∼2% of the particles) which may represent an off-pathway species in the assembly/disassembly process in so far that conversion to the mature heptamer would require the closed-ring hexamer to open to accept an additional subunit. Lastly, we observe several classes of tetramers where additional subunits characterized by fuzzy electron density are caught in the act of oligomer extension.

Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-crystallin on Chaperone Function and Anti-Apoptotic Activity.

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

BthTX-II from Bothrops jararacussu venom has variants with different oligomeric assemblies: An example of snake venom phospholipases A2 versatility

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.

Protein–Small Molecule Interactions in Native Mass Spectrometry

Small molecule drug discovery has been propelled by the continual development of novel scientific methodologies to occasion therapeutic advances. Although established biophysical methods can be used to obtain information regarding the molecular mechanisms underlying drug action, these approaches are often inefficient, low throughput, and ineffective in the analysis of heterogeneous systems including dynamic oligomeric assemblies and proteins that have undergone extensive post-translational modification. Native mass spectrometry can be used to probe protein-small molecule interactions with unprecedented speed and sensitivity, providing unique insights into polydisperse biomolecular systems that are commonly encountered during the drug discovery process. In this review, we describe potential and proven applications of native MS in the study of interactions between small, drug-like molecules and proteins, including large multiprotein complexes and membrane proteins. Approaches to quantify the thermodynamic and kinetic properties of ligand binding are discussed, alongside a summary of gas-phase ion activation techniques that have been used to interrogate the structure of protein-small molecule complexes. We additionally highlight some of the key areas in modern drug design for which native mass spectrometry has elicited significant advances. Future developments and applications of native mass spectrometry in drug discovery workflows are identified, including potential pathways toward studying protein-small molecule interactions on a whole-proteome scale.

Identification of key stabilizing interactions of amyloid-β oligomers based on fragment molecular orbital calculations on macrocyclic β-hairpin peptides.

Analyzing the electronic states and inter-/intra-molecular interactions of amyloid oligomers expand our understanding of the molecular basis of Alzheimer's disease and other amyloid diseases. In the current study, several high-resolution crystal structures of oligomeric assemblies of Aβ-derived peptides have been studied by the ab initio fragment molecular orbital (FMO) method. The FMO method provides comprehensive details of the molecular interactions between the residues of the amyloid oligomers at the quantum mechanical level. Based on the calculations, two sequential aromatic residues (F19 and F20) and negatively charged E22 on the central region of Aβ have been identified as key residues in oligomer stabilization and potential interesting pharmacophores for preventing oligomer formation.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

Topology evaluation of models for difficult targets in the 14th round of the critical assessment of protein structure prediction (CASP14).

This report describes the tertiary structure prediction assessment of difficult modeling targets in the 14th round of the Critical Assessment of Structure Prediction (CASP14). We implemented an official ranking scheme that used the same scores as the previous CASP topology-based assessment, but combined these scores with one that emphasized physically realistic models. The top performing AlphaFold2 group outperformed the rest of the prediction community on all but two of the difficult targets considered in this assessment. They provided high quality models for most of the targets (86% over GDT_TS 70), including larger targets above 150 residues, and they correctly predicted the topology of almost all the rest. AlphaFold2 performance was followed by two manual Baker methods, a Feig method that refined Zhang-server models, two notable automated Zhang server methods (QUARK and Zhang-server), and a Zhang manual group. Despite the remarkable progress in protein structure prediction of difficult targets, both the prediction community and AlphaFold2, to a lesser extent, faced challenges with flexible regions and obligate oligomeric assemblies. The official ranking of top-performing methods was supported by performance generated PCA and heatmap clusters that gave insight into target difficulties and the most successful state-of-the-art structure prediction methodologies.

Assembly-Line Catalysis in Bifunctional Terpene Synthases.

The magnificent chemodiversity of more than 95 000 terpenoid natural products identified to date largely originates from catalysis by two types of terpene synthases, prenyltransferases and cyclases. Prenyltransferases utilize 5-carbon building blocks in processive chain elongation reactions to generate linear C5n isoprenoid diphosphates (n ≥ 2), which in turn serve as substrates for terpene cyclases that convert these linear precursors into structurally complex hydrocarbon products containing multiple rings and stereocenters. Terpene cyclization reactions are the most complex organic transformations found in nature in that more than half of the substrate carbon atoms undergo changes in chemical bonding during a multistep reaction sequence proceeding through several carbocation intermediates. Two general classes of cyclases are established on the basis of the chemistry of initial carbocation formation, and structural studies from our laboratory and others show that three fundamental protein folds designated α, β, and γ govern this chemistry. Catalysis by a class I cyclase occurs in an α domain, where a trinuclear metal cluster activates the substrate diphosphate leaving group to generate an allylic cation. Catalysis by a class II cyclase occurs in a β domain or at the interface of β and γ domains, where an aspartic acid protonates the terminal π bond of the substrate to yield a tertiary carbocation. Crystal structures reveal domain architectures of α, αβ, αβγ, βγ, and β.In some terpene synthases, these domains are combined to yield bifunctional enzymes that catalyze successive biosynthetic steps in assembly line fashion. Structurally characterized examples include bacterial geosmin synthase, an αα domain enzyme that catalyzes a class I cyclization reaction of C15 farnesyl diphosphate in one active site and a transannulation-fragmentation reaction in the other to yield C12 geosmin and C3 acetone products. In comparison, plant abietadiene synthase is an αβγ domain enzyme in which C20 geranylgeranyl diphosphate undergoes tandem class II-class I cyclization reactions to yield the tricyclic product. Recent structural studies from our laboratory show that bifunctional fungal cyclases form oligomeric complexes for assembly line catalysis. Bifunctional (+)-copalyl diphosphate synthase adopts (αβγ)6 architecture in which the α domain generates geranylgeranyl diphosphate, which then undergoes class II cyclization in the βγ domains to yield the bicyclic product. Bifunctional fusicoccadiene synthase adopts (αα)6 or (αα)8 architecture in which one α domain generates geranylgeranyl diphosphate, which then undergoes class I cyclization in the other α domain to yield the tricyclic product. The prenyltransferase α domain mediates oligomerization in these systems. Attached by flexible polypeptide linkers, cyclase domains splay out from oligomeric prenyltransferase cores.In this Account, we review structure-function relationships for these bifunctional terpene synthases, with a focus on the oligomeric systems studied in our laboratory. The observation of substrate channeling for fusicoccadiene synthase suggests a model for dynamic cluster channeling in catalysis by oligomeric assembly line terpenoid synthases. Resulting efficiencies in carbon management suggest that such systems could be particularly attractive for use in synthetic biology approaches to generate high-value terpenoid natural products.