Biology subjects

Sawaya, M. R.

Publications and source records attributed to Sawaya, M. R..

At least 19 recordsLinked to original sources

bioRxiv2026

AlphaFold-Driven Structural Proteomics Reveals Extensive Cellulosome Machinery in Human Ruminococcal Symbionts

Cellulosomes are large, surface-displayed enzyme complexes that enable anaerobic bacteria to degrade recalcitrant plant polysaccharides, yet cellulosome-expressing bacteria are thought to be rare in the human gut. Here we show that extensive sequence divergence obscures the detection of many ruminococcal cellulosomes by conventional sequence homology-based methods. Using proteome-scale AlphaFold2 structural predictions, we uncovered a substantially expanded set of cellulosome-producing Ruminococcus species, including six previously unrecognized human symbionts. Structure-based clustering identifies several novel cohesin families that retain conserved folds despite extreme sequence divergence and define distinct, phylogenetically conserved cellulosome architectures. The analysis reveals R. callidus and related human symbionts encode elaborate cellulosomes that are invisible to sequence-based annotation. Similarly, R. difficilis, a human gut symbiont, is found to produce an atypical cohesin-based assembly enriched in amylases and related starch-binding proteins that may enable this microbe to degrade resistant starches that evade digestion in the upper gastrointestinal tract. Together, these findings reveal that ruminococcal cellulosomes are far more prevalent and diverse than previously appreciated and demonstrate the power of structural proteomics to uncover deeply divergent functional systems in the gut microbiome. Significance StatementPlant cell wall polysaccharides are a major dietary carbon source, yet their degradation relies on rare, highly specialized microbial enzyme assemblies known as cellulosomes, which have long appeared uncommon in the human gut. Using proteome-scale structure prediction combined with experimental validation, we show that cellulosomes are far more widespread and structurally diverse in human-associated Ruminococcus species than previously appreciated. We identify multiple new cohesin families and reveal distinct cellulosome architectures likely adapted to degrade different dietary substrates. Together, these findings redefine the distribution and evolution of cellulosomes in gut microbes and demonstrate the power of structural proteomics to uncover deeply diverged biological systems.

microbiology↗

bioRxiv2025

Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase

A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation. eTOC BlurbLimiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang et al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system. HighlightsO_LIAtomic cryo-EM structure of CRL5-PCMTD1 determined C_LIO_LIArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activity C_LIO_LIPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activity C_LIO_LIRecognition of L-isoaspartyl residues is dependent on cofactor engagement C_LI

biochemistry↗

bioRxiv2025

Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes

With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.

biophysics↗

bioRxiv2024

How short peptides can disassemble ultra-stable tau fibrils extracted from Alzheimers disease brain by a strain-relief mechanism

Reducing fibrous aggregates of protein tau is a possible strategy for halting progression of Alzheimers disease (AD). Previously we found that in vitro the D-peptide D-TLKIVWC disassembles tau fibrils from AD brains (AD-tau) into benign segments with no energy source present beyond ambient thermal agitation. This disassembly by a short peptide was unexpected, given that AD-tau is sufficiently stable to withstand disassembly in boiling SDS detergent. To consider D peptide-mediated disassembly as a potential therapeutic for AD, it is essential to understand the mechanism and energy source of the disassembly action. We find assembly of D-peptides into amyloid-like fibrils is essential for tau fibril disassembly. Cryo-EM and atomic force microscopy reveal that these D-peptide fibrils have a right-handed twist and embrace tau fibrils which have a left-handed twist. In binding to the AD-tau fibril, the oppositely twisted D-peptide fibril produces a strain, which is relieved by disassembly of both fibrils. This strain-relief mechanism appears to operate in other examples of amyloid fibril disassembly and provides a new direction for the development of first-in-class therapeutics for amyloid diseases.

neuroscience↗

bioRxiv2023

A Suite of Designed Protein Cages Using Machine Learning Algorithms and Protein Fragment-Based Protocols

Designed protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, while methods for their creation remain challenging and unpredictable. In the present study, we apply new computational approaches to design a suite of new tetrahedrally symmetric, self-assembling protein cages. For the generation of docked poses, we emphasize a protein fragment-based approach, while for de novo interface design, a comparison of computational protocols highlights the power and increased experimental success achieved using the machine learning program ProteinMPNN. In relating information from docking and design, we observe that agreement between fragment-based sequence preferences and ProteinMPNN sequence inference correlates with experimental success. Additional insights for designing polar interactions are highlighted by experimentally testing larger and more polar interfaces. In all, using X-ray crystallography and cryo-EM, we report five structures for seven protein cages, with atomic resolution in the best case reaching 2.0 [A]. We also report structures of two incompletely assembled protein cages, providing unique insights into one type of assembly failure. The new set of designed cages and their structures add substantially to the body of available protein nanoparticles, and to methodologies for their creation.

biochemistry↗

bioRxiv2023

Cryo-EM Structure of a Human LECT2 Amyloid Fibril Reveals a Network of Polar Ladders at its Core

ALECT2 is a type of systemic amyloidosis caused by deposition of the leukocyte cell-derived chemotaxin-2 (LECT2) protein in the form of fibrils. In ALECT2, LECT2 fibril deposits can be found in the glomerulus, resulting in renal failure. Affected patients lack effective treatment options outside of renal transplant or dialysis. While the structure of LECT2 in its globular form has been determined by X-ray crystallography, structures of LECT2 amyloid fibrils remain unknown. Using single particle cryo-EM, we now find that human LECT2 forms robust twisting fibrils with canonical amyloid features. At their core, LECT2 fibrils contain two mating protofilaments, the ordered core of each protofilament spans residues 55-75 of the LECT2 sequence. The overall geometry of the LECT2 fibril displays features in line with other pathogenic amyloids. Its core is tightly packed and stabilized by a network of hydrophobic contacts and hydrogen-bonded uncharged polar residues, while its outer surface displays several charged residues. The robustness of LECT2 fibril cores is illustrated by their limited dissolution in 3M urea and their persistence after treatment with proteinase K. As such, the LECT2 fibril structure presents a potential new target for treatments against ALECT2.

biochemistry↗

bioRxiv2022

Structure of a reversible amyloid fibril formed by the CPEB3 prion-like domain reveals a core sequence involved in translational regulation

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is a prion-like RNA-binding polypeptide. As a functional prion, CPEB3 is thought to modulate protein synthesis at synapses and enable consolidation of long-term memory in neurons. Here, we report that the prion-like domain 1 of CPEB3 self-assembles into labile amyloid fibrils in vitro. A cryoEM structure of these fibrils reveals an ordered 48-residue core, spanning L103 to F151. CPEB3 constructs lacking this amyloidogenic segment form abnormal puncta in cells when compared to wild type CPEB3, with reduced localization in dormant p-bodies and increased localization in stress granules. Removal of the amyloid core segment in CPEB3 also abolishes its ability to regulate protein synthesis in neurons. Collectively, this evidence suggests that the newly identified amyloidogenic segment within the CPEB3 prion domain is important for its regulated aggregation in cells and suggest its involvement in regulating translational activity and potentially long-term memory formation.

biochemistry↗

bioRxiv2022

Rigidified Scaffolds for 3-Angstrom Resolution Cryo-EM of Small Therapeutic Protein Targets

Numerous technical advances have made cryo-EM an attractive method for atomic structure determination. Cryo-EM is ideally suited for large macromolecular structures, while problems of low signal-to-noise prevent routine structure determination of proteins smaller than about 50 kDa. This size limitation excludes large numbers of important cellular proteins from structural characterization by this powerful technique, including many cell-signaling proteins of high therapeutic interest. In the present work, we use molecular engineering techniques to rigidify an imaging scaffold, based on a designed protein cage, to the point where 3 [A] resolution can be achieved, even for very small proteins. After optimizing the design of the rigidified scaffold on test proteins, we apply this imaging system to the key oncogenic signaling protein KRAS, which represents an outstanding challenge in the area of structure-based drug design. Despite its 19 kDa size, we show that the structure of KRAS, in multiple mutant forms, and bound to its GDP ligand, can be readily interpreted at a resolution slightly better than 3.0 [A]. This advance further expands the capability of cryo-EM to become an essentially universal method for protein structure determination, including for applications to small therapeutic protein targets.

biochemistry↗

bioRxiv2022

Cryo-EM structure of amyloid fibril formed by α-synuclein familial A53E mutation

Synucleinopathies, including Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA) have the same hallmark pathologic feature of misfolded -synuclein protein accumulation in the brain. PD patients who carry -syn hereditary mutations tend to have an earlier onset and more severe clinical symptoms and pathology than sporadic PD patients who carry wild-type (WT) -syn. Therefore, revealing the structural effect of -syn hereditary mutations on the wild-type fibril structure can help us understand synucleinopathies structural basis. Here, we present a 3.38 [A] cryo-electron microscopy structure of -synuclein fibrils containing the hereditary A53E mutation. The A53E fibril is symmetrically composed of two protofilaments, as are many other synucleopathic structures - including WT. Interestingly, the interface between the protofilaments in A53E has significantly less buried surface area than all other documented fibril structures of -syn and its other mutants. The A53E fibril also exhibits slower formation/growth in in vitro fibrillation experiment compared to other mutants. This implies that the structural differences - both in the protofilament and between each protofilament of A53E - change the aggregation mechanism, or in the least, its kinetics of formation. These differences influence the molecular characteristics of each fibril mutant and likely plays a macro-scale role in progressing one clinical pathology over another.

biophysics↗

bioRxiv2022

Amyloid fibrils in frontotemporal lobar degeneration with TDP-43 inclusions are composed of TMEM106B, rather than TDP-43

FTLD is the third most common neurodegenerative condition, following only Alzheimers and Parkinsons diseases. FTLD typically presents in 45-64-year-olds with behavioral changes or progressive decline of language skills. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions detected by TDP-43 immunoreactivity. Here, we extracted amyloid fibrils from brains of four patients, representing four out of five FTLD-TDP subclasses and determined their near-atomic resolution structures by cryo-EM. Unexpectedly, all amyloid fibrils examined are composed of a 135-residue C-terminal fragment of TMEM106B, a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP. In addition to TMEM106B fibrils, abundant non-fibrillar aggregated TDP-43 is present, as revealed by immunogold labeling. Our observations confirm that FTLD-TDP is an amyloid-involved disease and suggest that amyloid involvement in FTLD-TDP is of protein TMEM106B, rather than of TDP-43.

molecular biology↗

bioRxiv2022

Cryo-EM structure of RNA-induced tau fibrils reveals a small C-terminal core that may nucleate fibril formation

In neurodegenerative diseases including Alzheimers and ALS, proteins that bind RNA are found in aggregated forms in autopsied brains. Evidence suggests that RNA aids nucleation of these pathological aggregates; however, the mechanism has not been investigated at the level of atomic structure. Here we present the 3.4 [A] resolution structure of fibrils of full-length recombinant tau protein in the presence of RNA, determined by electron cryo-microscopy (cryoEM). The structure reveals the familiar in-register cross-{beta} amyloid scaffold, but with a small fibril core spanning residues Glu391 to Ala426, a region disordered in the fuzzy coat in all previously studied tau polymorphs. RNA is bound on the fibril surface to the positively charged residues Arg406 and His407 and runs parallel to the fibril axis. The fibrils dissolve when RNAse is added, showing that RNA is necessary for fibril integrity. While this structure cannot exist simultaneously with the tau fibril structures extracted from patients brains, it could conceivably account for the nucleating effects of RNA cofactors followed by remodeling as fibrils mature. Significance statementApplication of cryoEM has greatly expanded our understanding of atomic structures of mature pathological amyloid fibrils, but little is known at the molecular level of the initiation of fibril formation. RNA has been shown to be one cofactor for formation of fibrils of tau protein, and is known also to bind to other proteins, including TDP-43, FUS, and HNRNPA2, which form pathological inclusions. Our cryoEM structure of recombinant tau protein with RNA reveals a 36 residue, C-terminal fibril core bound to RNA which runs parallel to the fibril axis. We speculate that this structure could represent an early step in the formation of tau fibrils.

biophysics↗

bioRxiv2021

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

bioRxiv2021

Fragment-based ab initio phasing of peptidic nanocrystals by MicroED

Microcrystal electron diffraction (MicroED) is transforming the visualization of molecules from nanocrystals, rendering their three-dimensional atomic structures from previously unamenable samples. Peptidic structures determined by MicroED include naturally occurring peptides, synthetic protein fragments and peptide-based natural products. However, as a diffraction method, MicroED is beholden to the phase problem, and its de novo determination of structures remains a challenge. ARCIMBOLDO, an automated, fragment-based approach to structure determination. It eliminates the need for atomic resolution, instead enforcing stereochemical constraints through libraries of small model fragments, and discerning congruent motifs in solution space to ensure validation. This approach expands the reach of MicroED to presently inaccessible peptidic structures including segments of human amyloids, and yeast and mammalian prions, and portends a more general phasing solution while limiting model bias for a wider set of chemical structures.

biochemistry↗

bioRxiv2021

Why amyloid fibrils have a limited width

Amyloid fibrils can grow indefinitely long by adding protein chains to the tips of the fibril through {beta}-sheet hydrogen bonding; however, they do not grow laterally beyond [~]10-20 nm. This prevents amyloid fibrils from growing into two-dimensional or three-dimensional arrays. The forces that restrict lateral association of {beta}-sheets in amyloid fibrils are not immediately apparent. We hypothesize that it is the helical symmetry of amyloid fibrils that imposes the limit on fibril width by incurring an increasing separation between helically related molecules as a function of radial distance from the helical axis. The unavoidable consequence is that backbone hydrogen bonds that connect symmetrically related layers of the fibril become weaker towards the edge of the fibril, ultimately becoming too weak to remain ordered. To test our hypothesis, we examined 57 available cryo-EM amyloid fibril structures for trends in interstrand distance and {beta}-sheet hydrogen bonding as a function of radial distance from the helical axis. We find that all fibril structures display an increase in interstrand distance as a function of radius and that most fibril structures have a discernible increase in {beta}-sheet hydrogen bond distances as a function of radius. In addition, we identify a high resolution cryo-EM structure that does not follow our predicted hydrogen bonding trends and perform real space refinement with hydrogen bond distance and angle restraints to restore predicted hydrogen bond trends. This highlights the potential to use our analysis to ensure realistic hydrogen bonding in amyloid fibrils when atomic resolution cryo-EM maps are not available. Significance StatementThe number of amyloid fibril structures determined has exploded in recent years due to advances in structural biology techniques. However, we are still at the beginning stages of understanding amyloid fibril assembly. One important property that is critical to fibril formation and mechanical properties is the fibril width. Despite the diversity of fibril folds discovered, all amyloid fibrils are constrained to a width of 10-20 nm. Here, we use simple geometry and structural analysis to identify that the limited width of amyloid fibrils arises from the helical twist of {beta}-sheets in amyloid fibrils. Our findings provide important considerations for the accurate modeling of hydrogen bonds in amyloid fibrils as well as for the possible prediction and design of amyloid-based nanomaterials.

biochemistry↗

bioRxiv2021

Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19. One Sentence SummaryAtomic structures of amyloid-driving peptide segments from SARS-CoV-2 Nucleoprotein inform the development of Covid-19 therapeutics.

biochemistry↗

bioRxiv2020

CryoEM reveals how the small molecule EGCG binds to AD brain-derived tau fibrils and initiates fibril disaggregation

EGCG, the most abundant favanol in green tea, is one of the few natural compounds known to inhibit amyloid fibril formation of proteins associated with neurodegeneration, and to disaggregate amyloid fibrils. Little is known of the mechanism of molecular action of EGCG, or how it or other small molecules interact with amyloid fibrils. Here we present a 3.9 [A] resolution cryoEM structure that reveals the site of EGCG binding to Alzheimers disease (AD) brain-derived tau fibrils. The structure suggests that EGCG disaggregates fibrils of AD-tau by wedging into a cleft that is at the interface of two protofilaments of the paired helical filament, and by causing charge repulsions between tau layers of the fibril. In support of this, we observe separation of the protofilaments that EGCG wedges between, and accompanying displacement of the adjacent {beta}-helix domain. By resolving the site of EGCG binding, our structure defines a pharmacophore-like cleft in the AD-tau fibril that will be of use for the discovery of surrogate compounds with more desirable drug-like properties.

biochemistry↗

bioRxiv2020

Cryo-EM structure and inhibitor design of human IAPP (amylin) fibrils

Human islet amyloid polypeptide (hIAPP, or amylin) is a 37 amino acid hormone secreted by pancreatic islet {beta}-cells. Aggregation of hIAPP into amyloid fibrils is found in more than 90% of Type-II Diabetes (T2D) patients and is considered to be associated with T2D pathology. Although different models have been proposed, the high resolution structure of hIAPP fibrils is unknown. Here we report the cryo-EM structure of recombinant full-length hIAPP fibrils. The fibril is composed of two symmetrically-related protofilaments with ordered residues 14-37 that meet at a 14-residue central hydrophobic core. Our hIAPP fibril structure (i) supports the previous hypothesis that residues 20-29, especially 23-29 are the primary amyloid core of hIAPP, (ii) suggests a molecular mechanism for the action of the hIAPP hereditary mutation S20G, (iii) explains why the 6 residue substitutions in rodent IAPP prevent aggregation, and (iv) suggests possible regions responsible for the observed hIAPP cross-seeding with {beta}-amyloid. Furthermore, we performed structure-based inhibitor design to generate potential hIAPP aggregation inhibitors via a capping strategy. Four of the designed candidates delay hIAPP aggregation in vitro, providing a starting point for the development of T2D therapeutics and proof-of-concept that the capping strategy can be used on full-length cryo-EM fibril structures.

biochemistry↗

bioRxiv2020

The cryoEM structure of the fibril-forming low-complexity domain of hnRNPA2 reveals distinct differences from pathogenic amyloid and shows how mutation converts it to the pathogenic form

hnRNPA2 is one of a group of human ribonucleoproteins (RNPs) involved in RNA metabolism which form fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and that missense mutations in this domain are found in the disease multisystem proteinopathy (MSP) with symptoms indistinguishable from ALS and FTD. However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into {beta}-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both their reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to pathogenic form.

molecular biology↗