Protein domains biased toward a few amino acid typesare vitalfor the formation of biomolecular condensates in living cells. Thesemembraneless compartments are formed by molecules exhibiting a rangeof molecular motions and structural order. Missense mutations increasecondensate persistence lifetimes or structural order, properties thatare thought to underlie pathological protein aggregation. In the contextof stress granules associated with neurodegenerative diseases, thisprocess involves the rigidification of protein liquid droplets intoβ-strand rich protein fibrils. Here, we characterize the molecularmechanism underlying the rigidification of liquid droplets for thelow complexity domain of the Cytotoxic granule associated RNA bindingprotein TIA1 (TIA1) stress granule protein and the influence of adisease mutation linked to neurodegenerative diseases. A seeding procedureand solid state nuclear magnetic resonance measurements show thatthe low complexity domain converges on a β-strand rich fibrilconformation composed of 21% of the sequence. Additional solid statenuclear magnetic resonance measurements and difference spectroscopyshow that aged liquid droplets of wild type and a proline-to-leucinemutant low complexity domain are composed of fibril assemblies thatare conformationally heterogeneous and structurally distinct fromthe seeded fibril preparation. Regarding low complexity domains, ourdata support the functional template-driven formation of conformationallyhomogeneous structures, that rigidification of liquid droplets intoconformationally heterogenous structures promotes pathological interactions,and that the effect of disease mutations is more nuanced than increasingthermodynamic stability or increasing β-strand structure content. These membraneless organelles facilitate RNA processingand transport, and control proteostasis during cellular stress.7 LC domains are wellrepresented in proteins that form a variety of macroscopic assembliesin living cells8 and are of significantinterest in the context of biomolecular condensation. These sequencesinherently contain the multivalent property required for liquid dropletformation9 and can assemble into more rigidamyloid-like fibrils functionally and pathologically.11 Liquid droplets are normally characterized by significant molecularmotion and disorder.4 The aging of theliquid droplets, a process where the droplets adopt a more rigid structurewith potentially more homogeneous molecular conformations, is particularlypertinent for understanding how RNA granules fail to disassemble,facilitating the buildup of protein aggregates.12A low complexity (LC) domain is a protein segment that is biasedtoward a subset of the 20 amino acid types typically used for naturalprotein synthesis.13 Frontal temporal dementia (FTD) is the second most common form ofdementia after Alzheimer’s disease.14 These two diseases have many genetic links to LC domain proteinsinvolved in the formation of RNA granules.15 The RNA-binding protein TIA1 (Cytotoxic granule associated RNA bindingprotein TIA1) has ALS and FTD missense mutations clustered in a C-terminalLC domain.16 While the buildup of TIA1-richinclusions is not typical of ALS and FTD pathology, TIA1 disease mutationsresult in the persistence and rigidification of stress-related RNAgranules and are associated with the pathological deposition of otherLC domain proteins.16The misassembly of LC domain proteins is intricatelylinked toneurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) isa progressive neurodegenerative disease of the spinal cord and brainthat results in the loss of muscle movement.17 TheTIA1 LC domain is a 96 amino acid sequence biased toward Gln, Gly,Tyr, and Pro residues that assembles into amyloid-like fibrils andliquid droplets in vitro.19 Organized and reversible fibrillar self-assemblyis possibly a functional activity for the domain.20 Yet, despite significant interest in TIA1-mediated condensationprocesses, there are few structural characterizations of TIA1 fibrilformation. The P362L LC domain mutation delays the disassembly offunctional full-length TIA1 assemblies.18 Several Pro-to-hydrophobic mutations in the TIA1 LC domain leadto increased aggregation rates for the protein and suggest an antipathogenicrole for Pro residues.18 However, the residue-specificeffects of LC domain mutations on the rigidification of TIA1 liquiddroplets remain uncharacterized at high resolution. The biologicalfunction and pathogenicity of TIA1 involves LC domain self-assemblyprocesses for which the molecular mechanisms remain unknown.20Functionalactivity of the TIA1 protein includes the regulationof RNA splicing and translation.Here, we report the results from solidstate nuclear magnetic resonance(NMR) measurements, electron and bright field microscopy imaging,and fluorescence assays that characterize condensed states of wild-typeand P362L mutant forms of the TIA1 LC domain. High-resolution measurementsof seeded fibrils report on the precise location of the most fibril-proneregion of the protein. Analysis of aged liquid droplets provide insightsinto the structural changes underlying liquid droplet rigidificationand how an ALS and FTD mutation affects the process. Our results providea basis for understanding the sequence context of disease mutationsin the TIA1 LC domain as it relates liquid droplet aging and contributeto our understanding of the broader conformational space sampled byLC domains during proper biological function and disease pathology.E.coli and purified under denaturing conditions usingNi2+ affinity chromatography. The P362L mutant was obtainedusing standard polymerase chain reaction-based methods. The completedetails of these procedures are provided in the Supporting Information.His-tagged wild-type and P362L mutant TIA1 LC domains (residuesI291–Q386) were recombinantly expressed in g for 20min at ∼20 °C.TIA1 LC domainprotein in 500 mM sodium chloride, 6 M urea, ∼60 mM imidazole,and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),pH 7.5 at 355 μM, was buffer-exchanged into 20 mM Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol),200 mM sodium chloride, pH 7.5, using a 500 μL centrifugal concentratingdevice . The protein was diluted to 70 μMin 20 mM Tris, 40 mM sodium chloride, pH 7.5, tip-sonicated, and incubatedovernight at ∼20 °C. The protein was next diluted to 18μM in 20 mM HEPES, pH 7.5, and tip-sonicated to make seeds.A solution of 70 μM pure TIA1 LC domain was dialyzed into 20mM HEPES, pH 7.5, overnight and centrifuged prior to adding the seedsin a 1% mass ratio. The mixture was incubated with rotation at ∼20°C for 7 d and tip-sonicated twice during the incubation. Next,the solution was diluted to 17 μM in 20 mM HEPES, pH 7.5, andtip-sonicated. This solution was added at 5% by mass ratio to a centrifugedsolution of pure TIA1 LC domain dialyzed into 20 mM HEPES, pH 7.5.The solution was then stored at 4 °C for 25 d before warmingto ∼20 °C and diluting to half with a centrifuged 12 μMsolution of pure TIA1 LC domain dialyzed into 20 mM HEPES, pH 7.5.The dilution was tip-sonicated before adding to a larger volume ofthe 12 μM protein at a 5% mass ratio. The protein was incubatedquiescently for 13 d. Aliquots of the fibrils were prepared and frozenat −80 °C for later use as seeds. Tip-sonication was performedusing a Branson 250 Sonifier equipped with a 1/8-inch microtip operatedat 10% power for 1 min total on time in cycles of 0.1 s on and 1 soff. All centrifguation steps were 20,00013C and 15N labeled TIA1 LC domain protein was dialyzed from denaturing conditionsinto 20 mM HEPES, pH 7.5, overnight at a concentration of 180 μMin 6 mL volume and centrifuged. TIA1 LC domain seeds amounting to1% by mass of the isotopically labeled material were removed fromthe −80 °C storage, warmed to ∼20 °C, tip-sonicated,and added to the dialyzed TIA1 LC domain protein. The solution wasmixed with a pipette and incubated quiescently at ∼20 °Cfor 7 d. These isotopically labeled fibrils were diluted to 90 μMand tip-sonicated to form seeds. The seeds were added at a 2% massratio to centrifuged pure 13C and 15N labeledTIA1 LC domain dialyzed into 20 mM HEPES, pH 7.5 . The solution was mixed with a pipette and incubated quiescentlyfor 1 d. The presence of visually homogenous fibrils and the absenceof amorphous aggregates was confirmed by negative stain transmissionelectron microscopy (TEM). Tip-sonication was performed using a Branson250 Sonifier equipped with a 1/8-inch microtip operated at 10% powerfor 1 min total on time in cycles of 0.1 s on and 1 s off. All centrifguationsteps were 20,000g for 20 min at ∼20 °C.Pure For both wild-typeand P362L mutant TIA1 LC domain, a total of 15 mg purified proteinwas diluted to 83 μM using 500 mM sodium chloride, 6 M urea,200 mM imidazole, and 20 mM HEPES, pH 7.5, and dialyzed against 1L of 150 mM sodium chloride and 20 mM HEPES, pH 7.5. 65 μL aliquotswere taken out of the dialysis tubing after gently pipetting to homogenizeand mix the sample, at 90 min, 3 h, and 4 h for fluorescence measurementsand microscopy imaging. The protein was transferred from the dialysisbag after 4 h to a 50 mL conical tube and incubated on the benchtopat room temperature to age quiescently. Additional 65 μL aliquotswere removed for analysis after gently pipetting to homogenize thesample at 24 h, 48 h, and 1 week. The TIA1 LC domain aged liquid dropletsamples were harvested for the NMR measurements by centrifugationafter 9 d for the wild-type and at 14 d for the P362L mutant.Supporting Information.Details of the bright field, TEM, atomicforce microscopy, and confocal fluorescence microscopy are providedin the g and 12 °C for 1 h. The ratio of the 412 nm absorbance of thesupernatant to that of a 20 μM ThT control sample in identicalbuffer conditions was used to determine the amount of bound ThT inthe aged liquid droplet samples. Additional details are in the Supporting Information.Trp fluorescencewas measured on neat protein solutions. Thioflavin-T (ThT) fluorescencewas measured on samples containing equal parts neat protein solutionand 40 μM ThT. The reported values are the average of threemeasurements with an uncertainty of ±1 standard deviation. BoundThT was quantified by centrifuging the samples at 233,000g at 25 °C for30 min and aged liquid droplet wild-type and P362L mutant TIA1 LCdomain were harvested at 233,000g at 12 °C for∼20 h. For all samples, hydrated pellets containing 10–15mg of TIA1 LC domain protein were transferred into 3.2 mm thin-walledpencil-style zirconia rotors (revolution NMR) with a spatula and packedby centrifugation. To compact the samples, the seeded TIA1 LC domainsample was centrifuged at 25,000g for ∼30h at 8 °C and the aged liquid droplet samples were centrifugedat 25,000g for 20 min at 12 °C. The rotor drivetip and top cap were sealed with cyanoacrylate gel. Based on averageprotein density (1.35 g/cm3) and the volume of the NMRrotor (36 μL), the hydrated samples are approximately 30% protein.Residual soluble protein concentration was determined after the initialcentrifugation step using absorbance measurements at 280 nm recordedwith a 1 mm pathlength and a calculated extinction coefficient of46,870 M–1 cm–1 (ProtParam21).Seeded TIA1 LC domainfibrils were pelleted at 30,000Table S1. Unless otherwise specified,the sample temperature was ∼10 °C. For all cross-polarizationexperiments, 83.3 kHz 1H decoupling was used. SPINAL-64was used for the direct and indirect acquisition periods, and CW wasused for the 15N–13C cross-polarizationsteps. For the INEPT experiments, 10 kHz WALTZ-16 1H decouplingwas used. The observed chemical shifts were externally referencedto the DSS scale with the 13C downfield peak of Adamantaneat 40.48 ppm using a dehydrated sample of 1–13Clabeled Ala powder. 13C T2 measurementswere made by inserting a variable length spin-echo period immediatelyafter the cross-polarization step in a 1H–13C 1D experiment. 15N T2 measurementswere made by inserting a variable length spin-echo period betweenthe 1H–15N and 15N–13CA cross-polarization steps in a 1D NCA experiment. For site-resolved 15N T2 measurements, a 2D experimentwas used. Echo periods were varied up to 10.2 ms for 13C and 15.2 ms for 15N and utilized high power 83.3 kHz 1H decoupling. The integrated 1D signal intensity (non-glycylCA for 13C) was fit to single exponentials in TopSpin 3.6software. The T2 values from the 2D spectrawere extracted using NMRPipe and fit to single exponentials usingPython scripts. For all spectra, carbon–carbon correlationspectra were plotted with contours increasing by a factor of 1.4.Nitrogen–carbon correlation spectra were plotted with contoursincreasing by a factor of 1.25.Experiments were performed on 18.8 Tmagnets at the UC Davis NMR Campus Core Facility and the National High Magnetic Field Laboratory .BlackFox NMR and Low-E triple resonance 3.2 mm MAS probes were usedfor all experiments. A table of data acquisition parameters is providedin 22 recorded on the seeded TIA1 LC domain fibrilsample. A 2D TEDOR-NCACX experiment23 wasused to identify the NCACX signals for proline residues. Signal assignmentwas achieved using the MCASSSIGN algorithm24 with a procedure that generally followed our previous assignmentsfor LC domain samples.28 The input signal tables are presented in Tables S2–S4 and do not include weak signals, such as thosefrom the Gly residues highlighted in Figures S1A and S4, that do not have matching signalsin the 3D NCOCX spectrum. These signals account for much of the broadsignal intensity observed in the 2D NCACX and NCOCX spectra in 7 iterations per step. The GOOD, BAD, EDGE, and USEDweights were ramped from (0–10), (10–60), (0–8),and (0–6) during the annealing calculation, and a differentrandom number seed was used for each calculation. After the firstround of calculations, signals were assigned for residues T347–N357in 100% of the calculations. These assignments were fixed for a secondround of calculations, which resulted in the same signals being assignedto residues A338–Q346 in 100% of the calculations. Subsequentcalculations failed to converge on a unique result due to the largeCB/CG uncertainties assigned for some signals and the pseudo-repetitivenature of the TIA1 LC domain sequence. The calculation run with theonly His-tag as the input sequence resulted in no signals being assignedconsistently to any stretch of residues greater than three, rulingout the possibility of the His-tag being significantly ordered inthe fibril structure. A calculation run such that signals could notbe assigned to residues A338–N357 resulted in no significantassignment of signals outside this region, ruling out the possibilitythat some of the chemical shifts derived from multiple residues insimilar chemical environments and the possibility of a longer stretchof the TIA1 LC domain immobilized in the fibril core. Finally, a calculationrun on the complete TIA1 LC domain with the NCOCX signals assignedto A338–N357 omitted but complete signal tables for the NCACXand CANCO data did not result in any significant assignments, rulingout the possibility of two different strongly ordered conformationsfor residues A338–N357. There are two sets of observed Thrsignals that only differ significantly in their CG2 chemical shifts.The assignment process was unable to distinguish these sites due totheir identical CA and CB chemical shifts and nearly identical amideN chemical shifts. Neither of these signals was ever consistentlyassigned to the very C-terminal Thr site or the two Thr sites in theHis-tag.Chemical shift peak tablesrepresenting NCACX, NCOCX, and CANCO chemical shift correlations wereconstructed from the 3D cross-polarization-based data sets16 These mutationsare dispersed along the length of the TIA1 LC domain rather than localizedto a specific region of the primary sequence. Ala, Ile, Leu, Met,Val, Trp, Ser, and Thr residues are less common in the TIA1 LC domain.Ala and Val residues are found throughout the LC domain, and Trp isspread along the first two-thirds of the LC domain. The Ile residuesare confined to the N-terminal region, and Ser and Thr residues arefound in the C-terminal region. The distribution of the less commonamino acids makes them highly informative reporters on structure anddynamics in the LC domain.29 produced homogenous preparationsof TIA1 LCdomain fibrils. Pure TIA1 LC domain protein was allowed to aggregateat near-neutral pH and moderate ionic strength. Then, the proteinwas transferred to a low ionic strength solution and sonicated toproduce seeds. The seeds were then added to soluble TIA1 LC domainprotein at near-neutral pH and low ionic strength, and templated growthwas allowed to proceed. The process was repeated sequentially, usingthe fibrils resulting from templated growth as seeds in the subsequentround, a total of five times with seed to soluble protein mass ratiosbetween 1 and 5%. The negatively stained TEM image in 18 although differences in staining and image quality preventany further comparison. The average fibril width in the TEM micrographsis 12.8 ± 2.4 nm. Additional fibril TEM images and an illustrationof the fibril width measurement are shown in Figure S1. The residual soluble protein concentration for the seededpreparation is 1.4 μM. Using a model where fibrils do not fragment,30 this concentration corresponds to a Gibbs energyof dissociation (ΔG) for a TIA1 LC domain monomerfrom the fibril of ∼32 kJ/mol at ∼283 K.A seeding procedure like the one used for the β-amyloidpeptide32 in Figure S2A shows at least two sharp Gly signals and a regionof broad Gly signal intensity, although an analysis of this regionis complicated by the presence of a spinning sideband. The aromatic–aliphaticregion and aromatic–aromatic region of the spectrum in Figure S2A′–A″ show broadand weak signals and consistent backbone-sidechain correlations forTrp, Tyr, and Phe residues but lack any strong signals arising fromHis residues. The weak signal intensity in the aromatic region issimilar to the other LC domain fibrils we have studied using solidstate NMR.28 Weak aromatic side-chain signals are due to eitherpoor internuclear magnetization transfers due to motionally averageddipolar couplings or the aromatic side chains acting as a magnetization“sink” leading to a loss of magnetization in Phe andTyr residues.3322 in 23 ensures accurate reporting of all rigid Proresidues in nitrogen–carbon spectra, as it uses magnetizationtransfers originating from the CA proton rather than the amide proton.The TEDOR spectrum of the TIA1 LC domain fibrils in Figure S2B contains two sharp and distinct Pro signals with 15N chemical shift values of ∼130 ppm that arise fromsites that are in well-defined and rigid conformations. These areconsistent with weak Pro signal intensities in the cross-polarization-basedspectrum in 15N chemical shift values of ∼135–140 ppm consistentwith Pro residues that are either loosely ordered or sample heterogeneousconformations. There is no signal intensity corresponding to theseheterogeneous Pro residues in the cross-polarization-based spectrumin The 2D cross-polarization-based nitrogen–carbonspectrumFigure S2C uses scalar magnetization transfers34 that arise from highly mobile regions of the fibril structure.There is a signal in the spectrum with random coil NMR chemical shiftsthat can be unambiguously assigned to a Thr CB site. Signals fromaromatic atoms in His sidechains are also present in the spectrum.The remainder of the signals in the spectrum arise from other aliphaticcarbons that cannot be unambiguously assigned.The proton-carbonspectrum shown in Figure S2B with the protein sequence in Figure S1C indicates that very few sites in the TIA1 LC domainare highly mobile. In our TIA1 LC construct, the only His residuesand two additional Thr residues are present in the His-tag  used to expressand isolate the TIA1 LC domain. The lack of strong signals from Hisresidues in the cross polarization-based spectra indicate that thesesites are not part of the rigid fibril core. Furthermore, the unambiguoussignals from His sidechain sites and a Thr CB site in the spectrumin Figure S1B reinforce the interpretationthat the tag does not influence the structure of the rigid fibrilfor the TIA1 LC domain. The 2D spectra presented in S2B, and S2C are therefore consistent with a TIA1 LC domain fibrilstructure formed by a strongly immobilized and well-ordered core composedof but not limited to Ala, Thr, Ser, and Asn or Asp residues, withthe remainder of the TIA1 LC domain exhibiting structural heterogeneityand limited motion, and the His-tag undergoing more rapid motion ina random coil-like configuration.A comparisonof the amino acid types observed in the spectra in Figure S3. Almost all spectral regions exhibit well-resolved signalintensities in the 3D spectra, although there is some overlap in theregion reporting on CB and CG sites from Gln, Glu, Phe, Tyr, Trp,His, Met, Arg, and Leu residues. However, there are enough featuresto identify signals from 15 distinct residues. Additionally, signalsunambiguously attributable to two Ala, three Gly, two Pro, five Asnor Asp, two Thr, and four Ser residues are clearly identified in thesespectra. Signals for Leu, Val, or Ile are not observed in the 3D spectra. Figure S4 shows representative planes from theGly region of the 3D NCACX spectrum relative to their position inthe 2D NCACX spectrum in Tables S2–S4. These tables do not include weak andbroad signals that are not consistent with significant ordering ofneighboring residues, such as the many weak NCACX Gly signals shownin Figures S2A and S4 that do not havematching crosspeaks in the NCOCX and CANCO spectra.A total of 33 residues wereresolved using 3D cross-polarizationbased spectra. The signal to noise and resolution in these spectraare shown using representative 2D planes in 24 The procedureused is similar to our work on other LC domain protein fibrils.28 A complete description of the assignment calculations is presentedin the Figure S5. The signals for F344are relatively weak and the correlations for this residue are notobserved in the CANCO spectrum, which is consistent with either motionallyaveraged dipolar coupling strengths or the Phe side chain acting asa magnetization “sink”.33 As expected due to the lack of an amide proton for Pro residues,the NCOCX signals preceding P352 and P356 are missing, as are theNCACX signals for these residues. The aromatic-aliphatic sidechaincorrelations consistent with our assignments for W339 and W353 areshown in Figure S2A. Signals representingan amino acid sequence of XGXX (consistent with residues W309–W312or I319–Y322) could not be unambiguously assigned due to thelow signal to noise of the flanking residues, suggesting an orderedregion surrounded by more mobile segments of the protein. 35 torsion angle predictions from the assignedcarbon and nitrogen chemical shifts. The large positive φ andlarge negative ψ values indicate the immobilized residues arein β-strand conformations. The spectra in S2–S5 therefore are consistent with a β-strand rich fibril structureformed by a 20-residue core composed of residues A338–N357,flanked regions that are not highly mobile and lack well-defined structure.Unambiguousand statistically significant sequence-specific assignmentsfor residues 338–357 were obtained using the MCASSIGN Monte-Carlosimulated annealing algorithm.28 andpersist for up to 4 h. After 3 h the denaturant concentration is lessthan 200 mM. Movie S1 shows that at 1.5h, when the denaturant concentration is ∼0.8 M, the liquiddroplets fuse with one another, consistent with a condensed liquiddroplet phase of mobile TIA1 LC domain protein. As the liquid dropletsare allowed to age over 24 h, they clump together. Figure S1. The atomic force microscopy(AFM) images in Figure S6 also show thatthe large aggregates observed in the bright field images of wild typeand P362L mutant aged liquid droplets contain large masses of highlybundled fibril-like structures. The residual soluble protein concentrationsfor the wild-type and P362L liquid droplet preparations were 1.4 μM,indicating the aged liquid droplets have similar thermodynamic stabilitiesas the seeded fibrils.The bright field microscope imagesin 36 The confocal fluorescence microscopyimages in Figure S7 show that at 1.5 hthe ThT fluorescence signal arises from the liquid droplet species,while at 4 h, the images show that ThT fluorescence arises from hardeneddroplets and fibril-like protrusions extending from them. The confocalfluorescence microscopy images in Figure S6 show that the large masses of protein observed after 1 week forboth wild type and P362L mutant samples all exhibit increased ThTfluorescence. Both the wild-type and P362L mutant TIA1 LC domain followa similar ThT time course, suggesting the liquid droplet to fibriltransition is similar for both samples. The total increase in theThT intensity is larger for the P362L mutant sample than the wild-typesample and is consistent with previous ThT measurements on fibrilsof full-length TIA1.16The ThT fluorescence spectra in Figure S8 shows that the magnitude ofthe ThT fluorescence signal from liquid droplet samples aged for atleast 1 week is primarily dependent on the amount of bound ThT. Acrossmultiple preparations of aged liquid droplets, we have observed variableabsolute ThT intensities. Comparing the wild-type and P362L mutantaged liquid droplet samples we find: the fluorescence signal per boundThT molecule is similar, our TEM and AFM images show predominantlyfibril structures, our solid state NMR spectra show no differencein the molecular conformations sampled by the TIA1 LC domain (videinfra), and the residual soluble protein concentrations are similar.Therefore, the differences in ThT signal between the wild type andP362L mutant preparations are most likely due to variable degreesof fibril bundling and access of the ThT to the TIA1 LC domain moleculesin a given sample rather than an increased amount of fibrils or differingfibril types.37 The large suppression of Trp fluorescence and distribution of theTrp residues in the TIA1 LC domain suggest that significant portionsof the protein are protected from the solvent in the fibrils and toa lesser extent in the liquid droplets. The similar time-dependentTrp fluorescence signatures for both the wild-type and P362L mutantTIA1 LC domain indicate these processes are similar. The spectra at4 h do not suggest an intermediate state with highly solvent exposedTrp residues as the liquid droplets age.The intrinsic tryptophan fluorescence spectrain The ThT and Trp fluorescencemeasurements in Figure S9 shows overlay and difference spectra for the wild-type and P362Lmutant aged liquid droplets, revealing minimal conformational differencesin the TIA1 LC domains forming rigid structures in these samples.Since these experiments report on all rigid molecules in the samples,these spectra indicate that there is no significant difference inthe amount of fibrils present in the wild-type and P362L mutant agedliquid droplets. Overall, the aged liquid droplet spectra are similarto those of the seeded wild-type TIA1 LC domain fibrils in Figure S9.Figure S9. TEDOR-based spectra are also shown in Figure S9 to probe the presence of rigid Pro residues. These spectrahighlight the conformational similarity of the wild-type and P362Lmutant aged liquid droplets. However, like the carbon–carbonspectra, the nitrogen–carbon difference spectrum for the wild-typeseeded fibril and aged liquid droplet spectra in Figure S9.The nitrogen–carbon cross-polarization-basedspectra in Figure S10 show thatlowering the sample temperature for the aged liquid droplet samplesdoes not significantly change the appearance of the cross-polarization-and TEDOR-based spectra. The 1H spectra of these samplesin Figure S10 show that water in the samplesis not frozen at the −20 °C sample temperature, consistentwith freezing point depression caused by the presence of salt andprotein. Reducing the temperature probes the presence of loosely orderedbut conformationally homogenous structure in these samples.25 As molecular motions decrease with temperature,cross-polarization and TEDOR based magnetization transfers shouldbe more efficient due to stronger dipolar coupling interactions. Notably,the signals reporting on the seeded wild-type fibril conformationdo not appear in these spectra. These low temperature data are thereforenot consistent with the presence of a loosely ordered structure similarto the seeded wild-type conformation and further support that thereare significant structural differences in molecular conformationsbetween the seeded fibril and aged liquid droplet TIA1 LC domain samples.The low temperature spectra of seeded TIA1 LC domain fibrils shownin Figure S10 exhibits slightly broaderlineshapes than the spectra recorded at 10 °C, but have otherwisesimilar appearances, indicating that there is no additional orderingof the seeded TIA1 LC domain fibrils at −20 °C.The spectra in Scalar-based experiments were performed on the wild-type and P362Lmutant TIA1 LC domain aged liquid droplets. No signals were observedin the spectra recorded under the same conditions as the seeded TIA1LC domain fibrils. Lack of signal intensity in these spectra indicatethat there are no residues with significant molecular motion in eitheraged liquid droplet sample. The absence of signals from the His-tagin these spectra suggest that the His-tag has reduced mobility relativeto the seeded sample. However, the absence of strong or sharp Hissignals in the cross-polarization-based spectra of the aged liquiddroplets indicate that the His-tag has not adopted a rigid conformationeither.15N and 13C relaxationmeasurements for these samples probe the presence of structural heterogeneityand molecular motion in the samples. 15N and 13C T2 values measured with 1D experimentsfor the wild-type and P362L mutant aged liquid droplets, and the seededwild-type fibrils. Also shown in Figure S11 are 15N T2 measurements for resolved,sharp signals and regions of weak, broad signal intensity in a 2DNCACX spectrum. The bulk measurements indicate that the intrinsic 15N and 13C linewidths for the aged liquid droplet and seeded samples are samples are onthe order of 0.3–0.5 ppm. The measurements from the 2D NCACXspectra show that the sharp signals have even narrower 15N intrinsic linewidths, while the broad signals have similar valuesas those in the aged liquid droplet samples. In the context of the2D solid state NMR spectra shown in Cross-polarization-based S1), (ii) the seededfibril core is formed by residues A338–N357 in β-strandconformations (S2–S5), (iii) aging of TIA1LC domain liquid droplets results in β-strand rich fibrils thatare conformationally heterogeneous and structurally distinct fromthe seeded fibrils , and (iv) the P362L mutation lies outside of the core forming regionfor the seeded fibrils and does not alter the structural characteristicsof aged droplets (S9).Here we have shown, (i) a seeded preparationof the TIA1 LC domainyields uniform fibrils 1 and S1,droplets , and S9.38 However, the detailed conformation of the HP1αprotein in the aged liquid droplet sample was not characterized inthe study. Aging of the fused in sarcoma (FUS) LC domain showed asimilar increase of rigid structure in the presence of highly mobileand disordered regions of the protein.39 In this case, the aged liquid droplets were predominantly composedof a single rigid molecular conformation with NMR chemical shiftsindicative of β-strand structures, results that were supportedby TEM images and ThT positive fluorescence measurements.39 Although the molecular conformation of the FUSLC domain in the aged liquid droplets was similar to seeded FUS LCdomain fibrils,27 significant differencesin the solid state NMR spectra suggest the structural conversion wasincomplete. Our recent study of aged liquid droplets of the TAR DNA-bindingprotein 43 (TDP43) LC domain revealed the protein converges on a singularβ-strand rich molecular conformation without seeding.28 In these aged liquid droplets however, therewas evidence for conformational heterogeneity and only a small fractionof the LC domain exhibited rapid molecular motion.28Solid state NMR studiesof condensate rigidification are currently limited in number but arecapable of providing highly informative atomic-resolution or residue-specificcharacterizations of molecular conformation and motion. Heterochromatinprotein 1α (HP1α) was shown to undergo a liquid dropletrigidification characterized by increased uniform and rigid structurecoexisting with highly mobile and disordered structure.39Our measurements show the TIA1 LC domain aged liquiddroplets exhibit strong ThT fluorescence relative to the newly formedliquid droplets, which is consistent with an increased amount of β-strandstructure. However, the individual molecules in the aged liquid dropletsare conformationally heterogeneous given the lack of strong and sharppeaks in the solid state NMR spectra, in contrast to the more conformationallyhomogenous aged liquid droplets of the FUS and TDP43 LC domains. Furthermore,significant portions of the TIA1 LC domain in both the seeded fibrilsand aged liquid droplets do not participate in the rigid core of thestructure but have limited molecular motion and are likely looselyordered, characteristics that differ from the HP1α protein andFUS LC domain but are similar to the TDP43 LC domain. We also notethe result that the ThT and Trp fluorescence spectra and TEM imagesof the TIA1 LC domain liquid droplet aging process suggest a non-fibrillaraggregate intermediate between the liquid droplet and β-strandrich fibril states, which was not observed for the HP1α, FUS-LCdomain, or TDP43-LC domain proteins.42 The fibril conformation that has the fastest propagation rate isselected, which may also be the most thermodynamically stable molecularconformation.29 However, different Aβfibril polymorphs exhibit similar thermodynamic stabilities.43 While the TEM images and fluorescence data shownhere indicate that the TIA1 LC domain liquid droplets age into predominantlyfibril structures visually similar to the seeded TIA1 LC domain, oursolid state NMR measurements show the aged liquid droplets exhibita significant degree of conformational heterogeneity, contain a limitedamount of the molecular conformation selected through seeding, andare inconsistent with the presence of another dominant conformation.These results suggest that the TIA1 LC domain is polymorphic regardingthe formation of fibril structures, and our residual concentrationmeasurements indicate that these conformations all have similar thermodynamicstabilities. Our observations suggest the intriguing possibility thatthe TIA1 LC domain conformations in aged liquid droplets might bestructurally compatible and could coexist within the same fibril.Fibril seeding propagates specific fibril conformationsfor amyloidproteins like β-amyloid (Aβ), Tau, and α-synuclein.27 and heterogeneousribonucleoprotein A2 (hnRNPA2)11 LC domainfibrils are formed by longer 57-residue sequences. It has been proposedthat monomorphic fibrils are functional.48 The amino acid content of these three LC domains is presented in 46 is a defining feature for theFUS LC domain. While these residues do not seem to play the same dominantrole in the hnRNPA2 fibrils, these residues are abundant in the TIA1LC domain fibril core. Aromatic residues that interact through pi–piinteractions44 are similarly present inall three LC domain fibril cores. Regarding the hydrophobic aliphaticresidues that stabilize the cores of many pathological amyloid fibrils,these residues are scarce in the FUS and hnRNPA2 fibrils. The TIA1LC domain fibril core is enriched in these residues and is more likethe TDP43 protein in this regard. The TDP43 LC domain is polymorphic,51 which is a feature of pathogenic amyloid fibrils.47 The higher prevalence of hydrophobic residuesin the TIA1 LC domain fibril core compared to the FUS, hnRNPA2, andother similar LC domains may suggest that the propensity for liquiddroplets to mature into polymorphic fibrils is likely to be more pathogenicthan functional. However, the lack of TIA1 positive inclusions inpatient tissues16 points to a more complicatedrole for TIA1 than simply forming pathogenic fibrils. One point worthyof further investigation is the lack of highly mobile regions outsidethe rigid core for the TIA1 LC domain fibrils. Longer rigid core regions,such as those in the FUS LC domain fibrils, sometimes have highlymobile flanking regions. It could be that the loosely ordered regionsin the TIA1 LC domain fibrils provide extra stability for a shorterrigid core.ResiduesA338–N357 of the TIA1 LC domain have both differences and similaritieswith other fibril forming LC domain proteins. For the monomorphicLC domains, both the FUS18 The overall persistence time of the wild-type and P362L mutant liquiddroplets is shorter in our study of the TIA1 LC domain alone, butthe rate of liquid droplet assembly is similar to the full-lengthTIA1 protein.16 The increased rate andmagnitude of ThT fluorescence for the P362L mutant are also consistentwith the full length TIA1 protein.16 Pro-to-hydrophobicmutations in the TIA1 LC domain have been proposed to promote extensionof β-strand structures.18 While ourstudy does not directly address the structure of seeded TIA1 LC domainP362L mutant fibrils, our results clearly show that the seeded wild-typeTIA1 LC domain fibrils can accommodate uniform and rigid conformationsfor Pro residues (P352 and P356). Although Pro residues typicallyterminate β-strand structures, as observed in the FUS LC domainfibril structure,27 Pro residues can beaccommodated in β-strand structures as observed in a fragmentof Transthyretin (TTR).52 The observedchemical shifts for the TTR, and FUS and hnRNPA2 LC domain fibrilsare compared to those observed for the TIA1 LC domain in 13CG chemicalshifts are particularly sensitive to these conformations with valuesbetween ∼24–25 ppm indicative of cis conformations.57 Based on this metric, none of the Pro residueslisted in According to the results of our measurements, the P362L mutationin the TIA1 LC domain resides outside the seeded fibril core and doesnot appreciably alter the molecular conformations present in the agedliquid droplets. In the full-length TIA1 protein, the primary effectof the Pro-to-Leu mutations and other disease associated LC domainmutations are an increased persistence time of stress-induced granulesand more rapid increases in ThT fluorescence.20 Our detailed characterizationof the seeded fibril core may therefore provide a starting point fora more precise dissection of functional TIA1 assembly. Furthermore, the heterogeneous molecular conformationswe observefor the aged wild-type and P362L mutant liquid droplets are not appreciablydifferent. Given the similarities in maturation kinetics for our TIA1LC domain liquid droplets with the full-length TIA1 protein, we proposeour data support that the P362L mutation mainly affects LC domainprotein–protein interactions in the dynamic liquid dropletstate rather thermodynamically favoring a specific rigid fibrillarconformation. The increased persistence time and altered molecularinteractions inside the liquid droplets caused by mutations may thereforebe responsible for nucleating aggregation of other proteins like TDP43.However, our study of the isolated LC domain does not unambiguouslyrule out that the P362L mutation in the full-length protein couldalso disrupt interactions between the LC domain and the N-terminalRNA-binding domains. We also note that considerable discussion hasbeen devoted to the functional role of limited seeded TIA1 fibrilformation.in vitro to form micellar structuresmediated by interactions with the N-terminal RNA-binding domains,53 rather than the LC domain studied here. Whileit is probable that all potential interactions for the TIA1 proteinare relevant in living cells, the LC domain is a key component of in vivo behavior, as observed in altered stress granuledynamics due to LC domain mutations.16 Determiningthe balance of N-terminal oligomerization and C-terminal fibril formationis a key next step in characterizing TIA1 function and pathology.Finally, it is important to note that the full-lengthTIA1 proteinhas been observed 8 The amino acid biases of these proteins vary greatly. Guidingprinciples are emerging for the role of amino acid types and distributionsfor liquid droplet formation.55 However, functionalamyloid-like fibril formation is also a key component of organizingcellular activity.56 The large number ofmissense mutations in LC domains linked to disease15 make it clear that the precise sequences of these proteinsare important. Since functional LC domains differ in amino acid contentfrom those of traditional and purely pathological amyloids, furthercharacterizations of these fascinating and ubiquitous protein domainsare needed.Characterizing TIA1 LC domain assemblyis an important componentof a broad search for understanding other functional and pathologicalactivities governed by LC domain protein self-assembly. 30% of proteinsin the human proteome have at least one LC domain.


Besides dietary sources of energy, roots and tuber crops can also serve as functional foods and nutraceutical ingredients to be explored in disease risk reduction and wellness. However, they are easily spoiled because of their high moisture contents and enzymatic reactions. Therefore, this review aimed at gathering information on the effects of various pretreatment methods on the quality of tuber crops before converting them into flour. Studies reported by different scholars showed that there were significant differences in physicochemical and functional properties between untreated tuber and the treated tuber samples. The review also highlighted that the chemical treatment methods, particularly sulfite treatment, could increase the lightness value of the flour. In addition, blanching could induce a decrease in protein, ash, and fat. Despite this, blanching pretreatment techniques increased moisture and carbohydrate content. Chemical treatment increases the ash content, which might be responsible for chemical diffusion into the sample. The reviewed results showed that the application of different pretreatments on tuber crops significantly improves many quality characteristics such as color, fiber content, carbohydrate, and the functional properties used for value addition during food product development in the industry. Therefore, application of pretreatment methods particularly chemical treatments could enhance nutritional value, and functional and physical properties of tuber flours. Solanum tuberosum Lam.), Oromo dinnich (Plectranthus edulis), sweet potato , anchote , cassava (Manihot esculenta), and yam  are widely cultivated in Ethiopia.Root and tuber crops are grown in hot and humid regions worldwide . In devePotato is a starch crop, which is consumed by more than one billion people in the world and the third most important food crop in the world after maize, wheat, and rice . CassavaThe tuber crops indicated above were produced in huge quantities due to their adaptability to various climatic conditions, grown in different types of soil, and disease resistance. However, the perishable characteristics of the tuber crops limit the production interest of the growers. Additionally, the high moisture contents and ease of bruising reduce the shelf life of tuber crops during storage and production. Capotorto et al.  also repTo enhance the shelf life of tuber crops, it is essential to inactivate enzymes and reduce the water activity through various pretreatment techniques including chemical, thermal, and physical methods . BlanchiAfter pretreatment, it is imperative to process the tuber crops into flour for further utilization as a value addition during new food product development. The food products made from tuber crop flour are major parts of the staple diet for most developing countries; hence, the ability to form composite flour and the availability of the flour are major economic issues . IncorpoIn this review, factors that determine the quality of tuber flours after pretreatments such as physical properties, proximate composition, and functional properties are highlighted in the following paragraphs:Data collected from different scholars regarding the physicochemical properties are summarized in µm (nonblanched) to 17–38 µm (blanched). The reduction of starch granules is advantageous because it increases its digestibility [Particle size is an important property that may influence the physicochemical properties of tuber flours such as swelling, water binding capacity, and pasting properties . Harijontibility .μm) compared to the control and sodium metabisulfite-treated flours (70% of the control and sodium metabisulfite-treated samples were below 100 μm) [µm), mainly those treated with sodium metabisulfite, were suitable in applications of bakery such as baking bread, biscuits, and other pastry products [However, yam blanched using hot water relatively produced greater particle sizes (50% of the blanched flour particles were less than 100  100 μm) . This co 100 μm) . Generalproducts .Pretreatment and drying methods had a significant effect on the pH of sweet potato flour samples, which ranged from 4.5 to 7.0 . Among tCommonly, low pH value imparts a sour taste to the flour, which makes it less preferable for consumption . HoweverL∗a∗b∗color space in 1976 as an international standard for color measurement. The L∗a∗b∗color is made up of two chromatic components  and a luminance or brightness component  [Color is an essential physical quality factor directly related to the acceptability of food products and helps the consumers to make purchase decisions of bakery products. The Commission Internationale d'Eclairage (CIE) created the r white) . Table 1L∗ Value(1) . L∗ is an important characteristic for drying treatments since it is often the first quality feature that consumers evaluate when determining brand sensory acceptability [L∗ values. This suggests that the sodium metabisulfite pretreatment resulted in some bleaching, which was responsible for the flour's resistance to enzymatic browning. A similar study was reported by Desalegn and Kibr [L∗) (75.48–84.29), compared to the blanching method and untreated flour. The potassium metabisulfite-treated anchote flour had the greatest whiteness index in this investigation, which could be due to the method's ability to slow both enzymatic and nonenzymatic processes in the anchote flour.tability . Buckmantability  investigand Kibr , who notL∗ value increase (which ranged from 54.68 to 50.14) was reported for purple-fleshed sweet potato pretreatment by soaking chips in sodium metabisulfite solution [L∗ value of the pretreated potato samples. Similarly, Nascimento and Canteri [L∗ value of unblanched potato flour was darker than the blanched flour. The presence of PPO and peroxidase (POD) enzymes, which are responsible for the enzymatic browning response, results in the creation of dark chemicals contributing to the low L∗ value of the potato sample. However, Rita et al. [L∗ value than the boiled, grated, and steamed yam samples.Chemical pretreatments, particularly those involving sulfite and salts, are important to preserve the color of fruits and vegetables due to their potential to retard both enzymatic and nonenzymatic reactions . Furthersolution . Sun et solution  also ind Canteri  indicateA∗ and B∗Values(2) . The pretreatment methods could decrease the redness/green (a∗) values of the samples. For instance, the untreated anchote flour had a higher value than the blanched and potassium metabisulfite-treated anchote flour dried under different drying methods. The a∗value for anchote flour ranges from 3.02 to 6.34 [a∗ value) than the CaCl2- and NaHSO3-treated sweet potato samples [2 treatment, on the other hand, was the most effective in stabilizing the surface, freshness, and color, had no effect on human health, and was preferred as an alternative chemical treatment method [ to 6.34 . Similar samples . However samples . CaCl2 tt method .∗ value compared to other treatments including blanching and untreated samples. The metabisulfite-treated Pachyrhizus erosus flour (jicama) was less yellow than the other samples. The b∗ values (18.26–22.41) for the pretreated sweet potato flour were higher than the control sample at all pretreated concentrations [According to Buckman et al. , sodium trations . These htrations . Accorditrations . The coltrations .The moisture content of all the flours was below the 14% specified standard of the revised regulation of the Standard Organization of Nigeria . The preThe application of pretreatment helps to restrict the moisture losses and improves the drying characteristics of the tuber crops . The autFood proteins are essential nutrients for structural and functional purposes in the human body, and they provide the essential amino acids required for metabolism. Tubers are low in protein content . PretreaDesalegn and Kibr  found thThe total ash content of the samples indicates the minerals present . Ngoma eFibers are nondigestible carbohydrates, which provide fecal bulkiness and less intestinal transit, and play a great role in cholesterol-level reduction , 50. DesFats are used as an alternative energy source and are essential to the structure and biological processes of cells. Ngoma et al.  reportedCarbohydrates are among the most important constituents of tuber crops as sources of dietary energy . It was On the other hand, chemical treatment increased the protein, fat, and ash contents of the samples, whereas during blanching these nutrition elements were reduced due to leaching , 21, 23.The bulk density is a measurement of the relative volume of packaging material that is used to determine the storage, transportation, marketing, and wet food processing industries , 58. It A different bulk density was observed for various tuber crops due to the difference in variety, pretreatment, blanching time and temperature, and types of drying method . The finp > 0.05) difference between the blanching method and potassium metabisulfite-treated samples (p > 0.05) variation between the blanched and potassium metabisulfite-treated samples. Bikila et al. [Desalegn and Kibr  also com samples . Accordia et al.  also conIncreased water absorption capacity (WAC) during the preparation of food products could boost yield, uniformity, and food body . As indiFana Haile and Fisseha  determinAs reported by Ngoma et al. , sweet pOil absorption capacity (OAC) is an important flour quality characteristic to maintain flavor and improve the mouth feel of the food products . The phySwelling power (SP) is a measure of the granules' absorption index after heating and determines the capacity of starch molecules to hold water via hydrogen bonding, which is related to amino acid and starch concentration . Zhu 6969 indicaAccording to Rosida et al.  findingsNgoma et al.  indicateIn this review, impacts of pretreatment techniques on the quality of tuber flour particularly on the physical, functional, and proximate composition were summarized. The reviewed results showed that the application of pretreatments, particularly blanching, can enhance the flour lightness color that is attractive for the consumer. In addition, the pretreatment method can also enhance the dietary fiber, ash, and carbohydrate content of tuber flours. The functional properties of the tuber crop flour were also improved by the pretreatment methods, which is important for the production of valuable food products.


Streptococcus thermophilus, one of the main lactic acid bacteria used in dairy fermentations. The predictions highlight the mosaic nature of these devices that share functional domains for which active sites and residues could be unambiguously identified. Such AlphaFold2 analyses of phage-encoded host adhesion devices should become a standard method to characterize phage-host interaction machineries and to reliably annotate phage genomes.Successful bacteriophage infection starts with specific recognition and adhesion to the host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limits their structural analyses by experimental approaches such as X-ray crystallography and electron microscopy. However, the protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of whole adhesion devices of five representative siphophages infecting  Oenococcus oeni phages OE33PA and Vinitor 162, using a preliminary version of AF2 that did not include the multimer option (The release of the powerful AlphaFold2 (AF2) software in mid-2021 revolutionised structural biology . AF2 makr option .Streptococcus thermophilus have been the focus of significant research attention in recent years due to the threat they pose to global industrial dairy fermentations  using HHpred and identified several carbohydrate-binding modules (CBMs) in two conserved siphophage tail components termed the distal tail protein (Dit) and the tail associated lysin   . Host biin  . UnexpecMoineauvirus and Brussowvirus as a whole is beyond the possibilities of these techniques. Indeed, many of these phages possess large Tal proteins  as well as long and flexible extensions, which have been observed by negative staining electron microscopy (nsEM)  . Furtherin phago . Therefol domain  with parl domain .Moineauvirus and Brussowvirus genera with the latest version of AlphaFold2 multimer (as of January 2022)   . In thise (CWPS) . Notewors HHpred .S. thermophilus phages were selected for analysis in this study that represent both the Moineauvirus and Brussowvirus genera. Moineauviruses DT1  to perform the predictions. To note, this notebook does not use PDB templates (as do “true” AlphaFold2 servers), thereby providing a totally naive structure prediction. Furthermore, this ColabFold allows the modeling of homo-multimers. Due to memory limitations, long sequences had to be split in sequence stretches with considerable overlap for later assembly. In a first pass, we ran structure predictions for monomers in order to determine sensible stretch boundaries to be assembled in trimers  or hexamers (Dit). The number of residues in the multimeric stretch predictions had to be less than 1,400 residues. Moreover, we predicted structures of stretches with overlapping segments to allow full-length assembly of the full-length multimers using Coot  they have been isolated in geographically distinct locations, 2) they possess unique host ranges, and 3) they exhibit different adhesion devices according to their sequences. Furthermore, based on a recent phylogenetic analysis of dairy streptococcal phages , and two are members of the Brussowvirus genus . Phage 9851 was isolated from a dairy fermentation in France, and it infects S. thermophilus strain ST64985   , the Tal N-terminal domains resemble that of prophage MuSo2 from eidensis . Their weidensis . Howevereidensis . Sequenceidensis , and thege EGD-e , using tesidues) . These mesidues) . Therefoesidues) . A Dali Lmo1076) , an N-acLmo1076) . TherefoLmo1076) . Of noteLmo1076) . The disLmo1076) . Module Lmo1076) . Dali reLmo1076) , hence wLmo1076) , a cysteLmo1076) . NotablyLmo1076) .L. lactis phage Tuc2009 BppA protein, a CBM-containing component of its adhesion device  to ∼1,200 residues (TP-778L). In the five phages, a short α-helix immediately follows the N-terminal domain and abuts to a ∼90-residue immunoglobulin (Ig)-like domain . Notablyn device . The nexn device . Our desvia a tandem of the collagen-like linker and 3β domain   Table 1. POMGnT1β domain . Noteworing Dali , a domaicum ManA . Overallcum ManA .E. coli phage K1F endo-sialidase CIMCD, the B. subtilis phage GA-1 neck appendage protein CIMCD, or the receptor-binding C-terminal domain of phage T5 L-Shaped Tail Fibre, are known to interact with host cell wall receptors, like lipopolysaccharides  abutting to a β-prism domain formed by 3 × 10 β-strands, except for phage TP-778L in which this domain contains only 3 × 5 β-strands. These domains returned low-confidence and non-functionally relevant hits, using Dali, with various proteins containing anti-parallel β-sheets . Howevercharides . It is ns phages .S. thermophilus Moineauviruses and Brussowviruses, besides the Dit and Tal pair, we previously identified a third ORF that exhibits the characteristics of a bona fide CWPS-specific receptor binding protein (RBP). Structural predictions of the five phage RBPs as monomers identified a linear assembly of seven β-stranded domains , and between one and three CBMs. It is noteworthy that the last three 3β domains of TP-778L (3β_3−5) resemble a linker domain of phage Tuc2009 adhesion device BppA, and that the CBMs belonging to STP1, SW13, 9851 and TP-778L share the same fold as BppA’s linker and CBM. In contrast with the significant sequence variability associated with Dit CBMs, Tal CBMs are often more conserved at sequence level. While the three Tal CBMs of phages 9851 and TP-778L possess quasi identical sequences, the CBMs of the three other phages differ significantly. This variability is not surprising since these phages bind to different hosts with different saccharide motifs. At the C-terminal end, the five phages share a common structural motif involving a long α-helix and a β-prism. These β-prisms exhibit conserved sequences, with the exception of TP-778L β-prism that is shorter than those of the other four. In contrast with all the other domains of these adhesion devices to which we could assign a function, the functional purpose of these β-prism domains remains mysterious. They likely play a structural role, for example keeping together the Tal trimeric assembly, and/or a host binding function that is observed in some other β-prism domains from phages infecting E. coli (The Tals vary very considerably in their length and in the number of modules they possess. Phages DT1, STP1 and 9851 share a common gp27-like N-terminal domain, present in most  E. coli .The third ORF, which we named RBP due to the structural similarity with lactococcal and listerial RBPs, are formed by a chain of four structural Ig-like domains, followed by three domains found in canonical siphophage RBP. These domains include a tandem of β-prisms followed by a β-sandwich domain resembling closely that of the RBP head domain of lactococcal phage p2  or listeO. oeni, highlights the likely advantages of these CBMs for phage infection (Phages that prevail in dairy fermentation facilities and products are presented with an abundance of potential host cells that facilitate their proliferation. However, the abundance of phages in these fermentations also creates a highly competitive environment and dairy phages have evolved to incorporate multiple CBMs to enhance their ability to initiate contact with potential host strains. The identification of several CBMs within various adhesion device proteins of dairy lactococcal, lactobacilli and streptococcal phages, as well as of phages infecting the wine-making lactic acid bacteria nfection . TherefoMoineauvirus and Brussowvirus genera. Thanks to the easy access and user friendliness of AF2 Colab’s NoteBooks, such structural predictions and analyses of phage ORFs constitute a potent and reliable method of phage genomic and functional annotation, and particularly of their often under-annotated adhesion devices.These AF2 predictions made it possible to perform a precise assignment and analysis of the adhesion device of representative streptococcal phages of the


Forcepia sp., with nanomolar growth inhibitory activity and a unique cytotoxicity profile against the National Cancer Institute 60-cell-line screen. Here, we identified the putative biosynthetic pathway for LSA. Genomic binning of the Forcepia sponge metagenome revealed a Gram-negative bacterium belonging to the phylum Verrucomicrobia as the candidate producer of LSA. Phylogenetic analysis showed that this bacterium, here named “Candidatus Thermopylae lasonolidus,” only has 88.78% 16S rRNA identity with the closest relative, Pedosphaera parvula Ellin514, indicating that it represents a new genus. The lasonolide A (las) biosynthetic gene cluster (BGC) was identified as a trans-acyltransferase (AT) polyketide synthase (PKS) pathway. Compared with its host genome, the las BGC exhibits a significantly different GC content and pentanucleotide frequency, suggesting a potential horizontal acquisition of the gene cluster. Furthermore, three copies of the putative las pathway were identified in the candidate producer genome. Differences between the three las repeats were observed, including the presence of three insertions, two single-nucleotide polymorphisms, and the absence of a stand-alone acyl carrier protein in one of the repeats. Even though the verrucomicrobial producer shows signs of genome reduction, its genome size is still fairly large (about 5 Mbp), and, compared to its closest free-living relative, it contains most of the primary metabolic pathways, suggesting that it is in the early stages of reduction.Invertebrates, particularly sponges, have been a dominant source of new marine natural products. For example, lasonolide A (LSA) is a potential anticancer molecule isolated from the marine sponge  Forcepia sp. (50) values in the nanomolar range against certain cell lines in the National Cancer Institute 60-cell-line screen (–6–Lasonolide A (LSA) is a cytotoxic polyketide derived from the marine sponge epia sp.  and B  PKS pathway (las) that is likely responsible for the biosynthesis of LSA. Furthermore, the entire las biosynthetic gene cluster (BGC) has been captured on five overlapping fosmids and reassembled for future heterologous expression. We propose that the las BGC is present in a yet-uncultivated bacterium belonging to a novel genus under the phylum Verrucomicrobia. Additionally, evidence suggests las BGC is repeated thrice within the symbiont with minor sequence variations between them. We also suggest that the las BGC has been horizontally acquired and has a codon adaptation index comparable to that of highly expressed genes. Finally, we show that the Verrucomicrobia symbiont is in the very early stages of genome reduction and is likely to further reduce its size.Here, we describe a Forcepia sp. sponges collected from the Gulf of Mexico  synthase, free-standing ketosynthase (KS), acyl carrier protein (ACP), and two enoyl-CoA hydratases (ECH) (see las BGC at its 3′ end (Forcepia sp. and searched for trans-AT PKS BGCs. DNA was extracted from two different regions (referred to as Forcepia_v1 and Forcepia_v2) of the same sponge and subjected to shotgun metagenomic sequencing. The reads were trimmed, assembled, and then binned into metagenome-assembled genomes (MAGs). The metagenomes were found to be abundant in Acidobacteria, Proteobacteria, and Chloroflexota , whereas fosmids captured after WGS are colored blue . (B) Relative abundance of different phyla (GTDB taxonomy) in the sequenced Forcepia_v2 metagenome. Each block shows the relative abundance of each metagenome-assembled genome (MAG), with different colors representing the phylum they belong to. The las BGC-carrying bin is marked with a star. (C) Assembly graph of las BGC_v1 and contigs connected to it visualized in BANDAGE . (D) Mapping of paired-end reads to contigs connected to las BGC_v1. Contigs in green boxes represent the las BGC, red boxes represent the 5′ end of las BGC, and blue boxes represent the 3′ end of las BGC. (E) Assembly of the seven contigs making up las BGC_v2. (F) Assembly graph of las BGC_v2 and contigs connected to it visualized in BANDAGE . (G) Mapping of paired-end reads to contigs connected to las BGC_v2. Contigs in green boxes represent the las BGC, red boxes represent the 5′ end of las BGC, and blue boxes represent the 3′ end of las BGC. Panels C, D, F, and G were edited for clarity by removing contigs which had either very few paired-end read connections, were mapping to themselves, or were very small. (H) BGC distribution in the Forcepia_v2 metagenome. AntiSMASH  annotations of bacterial contigs greater than 3,000 bp are shown. Each bar indicates a MAG, grouped by phylum (GTDB taxonomy). The star marks the MAG containing the las BGC. BGC annotations have been simplified into polyketide synthase (PKS); type 1 PKS; type 3 PKS; trans-AT PKS; nonribosomal peptide synthetase (NRPS); ribosomally synthesized, posttranslationally modified peptide (RiPP); hgIE-KS; hgIE-KS-T1PKS; terpenes; and others. (I) Phylogenetic tree of 51 different Verrucomicrobia genomes. Bootstrap values were calculated using RaxML with 1,000 bootstrap replicates. Download FIG S1, EPS file, 2.9 MB.(A) Alignment of fosmids to the Copyright © 2022 Uppal et al.2022Uppal et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01524-22.6TABLE S1Forcepia sp. fosmid library before WGS. (B) Primers used for confirming the presence of terminal connections with the las BGC. (C) Primers used for screening the Forcepia sp. fosmid library after WGS. (D) Primers used for DNA assembly and validation of transformants. Download Table S1, XLSX file, 0.1 MB.List of oligonucleotide primers used for different purposes. (A) Primers used for screening the Copyright © 2022 Uppal et al.2022Uppal et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01524-22.7TABLE S2Table S2, XLSX file, 0.06 MB.Metadata and taxonomic classification of all the MAGs. Download Copyright © 2022 Uppal et al.2022Uppal et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the trans-AT PKS pathways against our assembled metagenome was performed. In the case of Forcepia_v1, the top hits were all to a contig of length 98 kbp labeled gnl|UoN|bin5_1_edit_8, strongly suggesting that this contig contains trans-AT PKS genes and may possess the potential LSA pathway. Contig gnl|UoN|bin5_1_edit_8 was manually inspected and corrected for sequence gaps (trans-AT PKS pathway with a truncated condensation domain (in bin3674_131), analysis of the metagenome using AntiSMASH (las BGC” below); it encoded a stand-alone ACP domain and about 47 amino acid residues, which completed the terminal KS domain of gnl|UoN|bin5_1_edit_8. Both of these contigs were assembled together, and annotation of genes and biosynthetic domains within this assembly reaffirmed that they are likely involved in LSA synthesis, through the gene cluster we termed las BGC_v1. The sequence of las BGC_v1 was also in alignment with previously sequenced fosmids identified from the metagenomic library.A tBLASTN  search once gaps . With thntiSMASH  . Furthermore, mapping paired-end reads onto bacterial contigs  (las BGC_v2). Similar to las BGC_v1, inspection of the assembly graph (las BGC_v2 have been binned incorrectly and should be part of bin4_1 (phylum Verrucomicrobia). As a result, the contigs comprising las BGC_v2, as well as additional contigs  . Except ly graph  and mapply graph  revealed contigs , were matagenome .las BGC from both Forcepia_v1 and Forcepia_v2 using clinker  for future heterologous expression of the las BGC.In order to capture the whole of the and 2-13 , which elas BGC (Forcepia_v1 bin5_1 and Forcepia_v2 bin4_1) was identified to belong to phylum Verrucomicrobiota, order Pedosphaerales, and genus UBA2970 by GTDB-TK v1.5.0 (database r202)  of Forcepia_v1 bin5_1 and Forcepia_v2 bin4_1 is 99.9%, suggesting little strain heterogeneity between the sites in the sponge beyond a small amount perhaps attributable to sequencing errors. To our knowledge, this is the first time a trans-AT PKS BGC has been reported in an organism belonging to the order Pedosphaerales. A phylogenetic tree of 51 different Verrucomicrobia genomes (Pedosphaera parvula Ellin514 (NCBI assembly accession no. GCA_000172555), with 88.78% identity to the 16S rRNA sequence. As per the 16S rRNA gene identity cutoffs proposed by Yarza et al. , is proposed to be protecting the host sponge from predators. Lasonolidus suggests the bacterium is associated with lasonolide A and also rhymes with the Spartan king of the 300 hoplites, Leonidas. Despite being the putative producer of LSA, “Ca. Thermopylae lasonolidus” is not highly abundant in the metagenome, having a relative abundance of just over 2.65% in Forcepia_v1 and 1.78% in Forcepia_v2 (The putative symbiont genome carrying the se r202) . Excludi genomes  placed ta et al. , this recepia_v2 .las BGC is shown in las BGC consists of 6 trans-AT PKS proteins (lasHJLMNO), 10 accessory genes (lasCDEFIKPQRS), and 5 genes with no or an unknown role in LSA synthesis (lasABGTU). Phylogenetic analysis of 944 different KS domains  contains a condensation domain, similar to those found in nonribosomal peptide synthetase pathways, as its terminal domain. We propose this terminal condensation domain is responsible for cyclizing and cleaving the final PKS product  domain is often used in ed sites , 35. AHs domains , 36. Las domains  and phyl domains . The acc domains . The ACP domains , 38. Las domains   have been marked. (B) Phylogenetic tree of AT and AH domains. The different types of domain separate into different clades . (C) Phylogenetic tree of ECH1 and ECH2 domains. Both the domains separate into different clades . (D and E) Alignment of las ECH1 (D) and ECH2 (E) domains with respective ECH domains from other PKS pathways. Sequence that is required for the formation of the oxyanion hole, which stabilizes the enolate anions, is marked. LasH_a and LasO_a are proposed to be inactive, as they are truncated and show poor homology to the rest of the ECH domains . (F) Alignment of las KS domains with active site residues (CHH) marked. “i” and “c” in the LasD KS indicate the incomplete and complete KS domain in different repeats of LasD, respectively. LasD is a decarboxylating KS; they are known to lack the active site cysteine . (G) Alignment of las KR domains with two from the erythromycin BGC to allow comparison. Active site residues and conserved motifs are marked. The presence or absence of the second aspartate in the LDD motif is supposed to predict the stereochemistry of the hydroxyl group . Figures have been truncated for clarity and to show only the relevant sites. In phylogenetic trees, las BGC domains are highlighted in white. Download Data Set S2, PDF file, 8.0 MB.(A) Alignment of Copyright © 2022 Uppal et al.2022Uppal et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the trans-AT PKS pathways featuring a monooxygenases (MOX) carrying out Baeyer-Villager (BV) oxidations, such as oocydin and sesbanimide , free-lynthesis ), as welynthesis ). Simila oocydin  have con oocydin –49; in ocusperin , nospericusperin , pederincusperin , onnamidcusperin , and labcusperin  have thecusperin , we suggcusperin , is propgriamide , patellagriamide , and bacgriamide , 56.las BGC matched the configuration of the equivalent moieties within the LSA structure produced by total synthesis  domains (in module 7 and module 13) based on their phylogeny  and aligpathways . As showe alkene . Howevere alkene , and as e alkene  59). Mo. Molas Be alkene . LasL DHe alkene . The maj10.1128/mbio.01524-22.5DATA SET S3https://doi.org/10.1021/acschembio.8b00049). Las BGC DH/PS domains are highlighted in white. (B) Alignment of PS domains identified in the las BGC with PS domains from other trans-AT PKS pathways. The DH domain from the erythromycin BGC is used for comparison. LasO DH2 and LasM DH4 are annotated as putative PS domains. Generally, PS domains have an Hx4P motif instead of an Hx8P, and they lack the catalytic aspartate at the DxxxQ/H motif . This was found to be true only for LasM DH4 and not LasO DH2. However, identical variations from a traditional PS domain architecture are also seen in PS domains found in the mandelalide pathway (MndC DH3 and MndD DH3) . (C) Alignment of double bond-shifting DH domains identified in las BGC with similar domains found in other trans-AT PKS pathways. The DH domain from the erythromycin BGC is used for comparison. LasM DH1 and LasN DH1 are annotated as putative double bond-shifting DH domains. Generally, in DH-shifting domains, the conserved proline (P) in the Hx8P motif is often replaced by either valine (V) or leucine (L). In the case of LasM DH1, a methionine (M) instead of V or L appears in the place of P, which is in line with DH sequences observed in the difficidin BGC . Furthermore, DH-shifting domains are sometimes characterized by the replacement of the conserved aspartic acid (D) with asparagine (N) and substitution of glutamine (Q) or histidine (H) with V or L in the DxxxQ/H motif. Even though LasN DH1 has an N in place of D in the DxxxQ/H motif, it substitutes Q/H with a serine (S). This is unusual and not found in any other double bond-shifting DH. (D) Alignment of DH domains present in the las BGC with the DH domain from the erythromycin BGC. Putative PS and double bond-shifting DH domains have been excluded. LasH DH1 and LasM DH2 are annotated as inactive domains due to disrupted catalytic motifs Hx8P and DxxxQ/H. Even though in LasL DH1, the catalytic aspartic acid is replaced by glutamic acid (DxxxQ/H motif), we propose it is active, as a similar mutation is observed in the palmerolide BGC . (E) Alignment of LasL DH3 with other DH domains having a serine in place of proline in the Hx8P motif. The DH domain from the erythromycin BGC is used for comparison. Sequence headers in blue represent DH domains annotated as active, while those in red are annotated as inactive. Download Data Set S3, PDF file, 4.1 MB.(A) Phylogenetic tree of DH and PS domains, which separate into different clades 10COOH] to produce lasonolide G. We suggest that lasonolide E is biosynthesized by a trans-esterification reaction by reacting with an ethanol molecule. We suggest that the biosynthesis of lasonolide D is similar to that of LSA except that it starts with acetate as the starter unit loaded onto the ACP of LasJ, with LasH and LasI being inactive.For the biosynthesis of other LSA analogs, we propose that lasonolide B results from an alternate starter unit, and all of them except for lasonolide D are modified post-PKS . The cytlas BGC (400.165× for las BGC_v1 and 159.02× for las BGC_v2) is roughly three times that of “Ca. Thermopylae lasonolidus” (135.16× in Forcepia_v1 and 48.24× in Forcepia_v2). The 3× coverage suggests three repeats of the putative las BGC. Visual inspection of the assembly graph, as well as mapping of the paired-end reads onto “Ca. Thermopylae lasonolidus,” allowed us to identify three connections on the 3′ end of las BGC but only two connections on the 5′ end of the pathway (contigs 7 and 8) (las BGC about 3 kbp (3.6 kbp for las BGC_v1 and 3.7 kbp for las BGC_v2) from the 5′ end of las BGC. This suggests that the majority of las BGC (about 98 kbp) is repeated thrice, with a 3-kbp segment of the pathway (contig 6) being repeated twice  . Anothered twice . The twoed twice , as welle dosage .10.1128/mbio.01524-22.8TABLE S3las BGC in “Ca. Thermopylae lasonolidus” in Forcepia_v1 and Forcepia_v2. Contig IDs represent the labels in Table S3, XLSX file, 0.07 MB.Contigs making up the three repeats of the Copyright © 2022 Uppal et al.2022Uppal et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the las BGC repeat connected to contig 5 lacks lasC . Although this KS domain has the catalytic active site residues SHH, characteristic of decarboxylating KSs (las BGC_v1)  and thus do not cause any frameshift mutations. Moreover, all three insertions lie between domains within trans-AT PKS proteins, suggesting they do not contribute to functional differences. Change in one base from C to T at 93,995 bp does not result in a change in the amino acid sequence, as both codons (TAC and TAT) encode tyrosine. Finally, a change in the base from A to G at 95,154 bp lies just outside lasS, i.e., in the noncoding region. The above-mentioned differences in the three repeats of las BGC indicate that the repeats have been present long enough to allow divergence. However, the differences between them are not predicted to affect the function of the las BGC.On comparing the three repeats, it was observed that the ting KSs , it lackting KSs , 64, we  BGC_v1) . This walas BGC contigs significantly differ in their 5-mer frequency from “Ca. Thermopylae lasonolidus,” suggesting that the las BGC could have been recently horizontally acquired. Furthermore, the GC percentage of the las BGC is significantly different  from annotated, hypothetical, and pseudogenes  values (BH-tSNE x and BH-tSNE y). (B) Three-dimensional visualization of contigs present in the “Ca. Thermopylae lasonolidus” genome. Contigs making up the las BGC are colored red. (C) GC percentage of different sets of genes in Forcepia_v2 “Ca. Thermopylae lasonolidus.” Las BGC genes are colored in red. (D and E) Codon adaptation index (CAI) of different categories of genes present in Forcepia_v1 “Ca. Thermopylae lasonolidus” and Forcepia_v2 “Ca. Thermopylae lasonolidus,” respectively. Las BGC genes are colored in red. P values for pairwise comparison between different categories of genes are shown in the matrix below their respective plots. P values of <0.05 are considered significant. Other nonsignificant P values are colored red. Annotated and hypothetical genes represent the genes annotated with a function and genes annotated as hypothetical, respectively, by Prokka . Download FIG S2, PDF file, 1.9 MB.(A) Two-dimensional visualization of the initial Autometa binning of Forcepia_v2. The “Copyright © 2022 Uppal et al.2022Uppal et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the las BGC was significantly different  from the annotated, hypothetical, and pseudogenes, but it matched that of highly expressed genes  (Ca. Thermopylae lasonolidus” genome.The codon adaptation index (CAI) compares the synonymous codon usage of a gene and that of a reference set along with measuring the synonymous codon usage bias . The CAIroteins) D and E. Ca. Thermopylae lasonolidus,” with multiple las BGC repeats, represents an important addition to the growing collection of symbiotic Verrucomicrobia  being identified with repeated trans-AT PKS BGCs (trans-AT PKS BGC for pateamine in a bacterium (“Candidatus Patea custodiens”) belonging to phylum Kiritimatiellaeota  are known to be present in genomes of sponge symbionts and have been found to play an important role in regulating their interaction with the host sponge –78. It i–nolidus” , thus su10.1128/mbio.01524-22.9TABLE S4Ca. Thermopylae lasonolidus.” (A) Nonpseudogenes annotated as Eukaryotic-like proteins. (B) Genes forming the PV BMC cluster. (C) Nonpseudogenes annotated by dbCAN2. Download Table S4, XLSX file, 0.2 MB.Gene annotation in Forcepia_v1 and Forcepia_v2 “Copyright © 2022 Uppal et al.2022Uppal et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Planctomycetes and Verrucomicrobia have a unique BMC gene cluster called the Planctomycetes-Verrucomicrobia bacterial microcompartment (PV BMC), which is responsible for production of microcompartment shell proteins BMC-P and BMC-H as well as degradation of l-rhamnose, l-fucose, and fucoidans  are organelles that enclose enzymes within a selectively permeable proteinaceous shell , and theucoidans , 81, 82.nolidus” , and the clinker . One int clinker . The predetected , indicatmicrobia –85.Ca Thermopylae lasonolidus” with its closest free-living relative, Pedosphaera parvula Ellin514 (assembly accession no. GCA_000172555). The draft genome of P. paruva Ellin514 is 7.41 Mbp long, about 2.2 Mbp longer than “Ca. Thermopylae lasonolidus.” Furthermore, in P. paruva Ellin514 only 0.5% of total open reading frames (ORFs) were found to be pseudogenes , relative to the average coding density of 85 to 90% for free-living bacteria  was collected in August of 2005 using the Harbor Branch Oceanographic Institute (HBOI) Johnson Sea Link submersible. Samples were collected at a depth of 70 m from the Gulf of Mexico  on the Pulley Ridge . The sponge samples were immediately frozen at −80°C. The sample ID was 12-VIII-05-1-006 200508121006 2005-08-12 JSL I-4837 (HBOI) Forcepia sp. strain 131921.Forcepia species sponge. For the first round (referred to as Forcepia_v1), Illumina TruSeq DNA libraries were prepared and sequenced by RTL Genomics using an Illumina MiSeq sequencer, giving us 108 million paired-end reads with length of 151 bp. For the second round of sequencing (referred to as Forcepia_v2), Illumina Nextera libraries were prepared and sequenced using a NovaSeq 6000 sequencer, giving us 303 million paired-end reads with length of 150 bp. Fosmids were sequenced by RTL Genomics and Genewiz.The sponge hologenome was extracted using a modified cetyltrimethylammonium bromide (CTAB) DNA extraction method  and thenlas BGC was done using tBLASTN , and genes were called and annotated using Prokka v1  reads have been deposited in the Sequence Read Archive (SRA) with accession nos. SRR18966768 (Forcepia_v1) and SRR18966767 (Forcepia_v2). Sequences for bin5_1 and bin4_1 were deposited under the BioSample accession nos. SAMN27962571 and SAMN27962572, respectively. Las BGC v1 and v2 have been deposited to GenBank with accession numbers ON409579 and ON409580, respectively. Las BGC (las BGC_v1) have been submitted to MIBiG with accession no. BGC0002153.The data associated with this study were deposited under BioProject accession no.