Recently Designed Multivalent Spike Binders Cannot Bind Multivalently─How Do They Achieve Enhanced Avidity to SARS-CoV-2?

The trimeric spike protein of SARS-CoV-2 has been targeted by antibody mimics that bind near or at the receptor-binding domain to neutralize the virus. Several independent studies have reported enhanced binding avidity for dimers and trimers, where binding domains are connected by short peptides. The enhanced avidity of the multivalent constructs was attributed to their simultaneously binding two or three sites within a single spike trimer. We argue here that the 15-20 amino acid peptide linkers, when considered as worm-like-chains, are too short to span the binding sites within a single spike. The enhanced avidity of the multivalent constructs may be explained by a rebinding mechanism, which does not involve multivalent binding.

The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro.

Bacterial cell and chloroplast division are driven by a contractile "Z ring" composed of the tubulin-like cytoskeletal GTPase FtsZ. Unlike bacterial Z rings, which consist of a single FtsZ, the chloroplast Z ring in plants is composed of two FtsZ proteins, FtsZ1 and FtsZ2. Both are required for chloroplast division in vivo, but their biochemical relationship is poorly understood. We used GTPase assays, light scattering, transmission electron microscopy, and sedimentation assays to investigate the assembly behavior of purified Arabidopsis thaliana (At) FtsZ1 and AtFtsZ2 both individually and together. Both proteins exhibited GTPase activity. AtFtsZ2 assembled relatively quickly, forming protofilament bundles that were exceptionally stable, as indicated by their sustained assembly and slow disassembly. AtFtsZ1 did not form detectable protofilaments on its own. When mixed with AtFtsZ2, AtFtsZ1 reduced the extent and rate of AtFtsZ2 assembly, consistent with its previously demonstrated ability to promote protofilament subunit turnover in living cells. Mixing the two FtsZ proteins did not increase the overall GTPase activity, indicating that the effect of AtFtsZ1 on AtFtsZ2 assembly was not due to a stimulation of GTPase activity. However, the GTPase activity of AtFtsZ1 was required to reduce AtFtsZ2 assembly. Truncated forms of AtFtsZ1 and AtFtsZ2 consisting of only their conserved core regions largely recapitulated the behaviors of the full-length proteins. Our in vitro findings provide evidence that FtsZ1 counterbalances the stability of FtsZ2 filaments in the regulation of chloroplast Z-ring dynamics and suggest that restraining FtsZ2 self-assembly is a critical function of FtsZ1 in chloroplasts.

FtsZ at mid-cell is essential in Escherichia coli until the late stage of constriction.

There has been recent debate as to the source of constriction force during cell division. FtsZ can generate a constriction force on tubular membranes in vitro, suggesting it may generate the constriction force in vivo. However, another study showed that mutants of FtsZ did not affect the rate of constriction, whereas mutants of the PG assembly did, suggesting that PG assembly may push the constriction from the outside. Supporting this model, two groups found that cells that have initiated constriction can complete septation while the Z ring is poisoned with the FtsZ targeting antibiotic PC190723. PC19 arrests treadmilling but leaves FtsZ in place. We sought to determine if a fully assembled Z ring is necessary during constriction. To do this, we used a temperature-sensitive FtsZ mutant, FtsZ84. FtsZ84 supports cell division at 30 °C, but it disassembles from the Z ring within 1 min upon a temperature jump to 42 °C. Following the temperature jump we found that cells in early constriction stop constricting. Cells that had progressed to the later stage of division finished constriction without a Z ring. These results show that in Escherichia coli, an assembled Z ring is essential for constriction except in the final stage, contradicting the simplest interpretation of previous studies using PC19.

A Unified Model for Treadmilling and Nucleation of Single-Stranded FtsZ Protofilaments.

Bacterial cell division is tightly coupled to the dynamic behavior of FtsZ, a tubulin homolog. Recent experimental work in vitro and in vivo has attributed FtsZ's assembly dynamics to treadmilling, in which subunits add to the bottom and dissociate from the top of protofilaments. However, the molecular mechanisms producing treadmilling have yet to be characterized and quantified. We have developed a Monte Carlo model for FtsZ assembly that explains treadmilling, and also explains assembly nucleation by the same mechanisms. A key element of the model is a conformational change from R (relaxed), which is highly favored for monomers, to T (tense), which is favored for subunits in a protofilament. This model was created in MATLAB. Kinetic parameters were converted to probabilities of execution during a single, small time step. These were used to stochastically determine FtsZ dynamics. Our model is able to accurately describe the results of several in vitro and in vivo studies for a variety of FtsZ flavors. With standard conditions, the model FtsZ polymerized and produced protofilaments that treadmilled at 23 nm/s, hydrolyzed GTP at 3.6-3.7 GTP min-1 FtsZ-1, and had an average length of 30-40 subunits, all similar to experimental results. Adding a bottom capper resulted in shorter protofilaments and higher GTPase, similar to the effect of the known bottom capper protein MciZ. The model could match nucleation kinetics of several flavors of FtsZ using the same parameters as treadmilling and varying only the R to T transition of monomers.

Microtubule Assembly from Single Flared Protofilaments-Forget the Cozy Corner?

A paradigm shift for models of MT assembly is suggested by a recent cryo-electron microscopy study of microtubules (MTs). Previous assembly models have been based on the two-dimensional lattice of the MT wall, where incoming subunits can add with longitudinal and lateral bonds. The new study of McIntosh et al. concludes that the growing ends of MTs separate into flared single protofilaments. This means that incoming subunits must add onto the end of single protofilaments, forming only a longitudinal bond. How can growth of single-stranded protofilaments exhibit cooperative assembly with a critical concentration? An answer is suggested by FtsZ, the bacterial tubulin homolog, which assembles into single-stranded protofilaments. Cooperative assembly of FtsZ is thought to be based on conformational changes that switch the longitudinal bond from low to high affinity when the subunit is incorporated in a protofilament. This novel mechanism may also apply to tubulin assembly and could be the primary mechanism for assembly onto single flared protofilaments.

The cell division protein MinD from Pseudomonas aeruginosa dominates the assembly of the MinC-MinD copolymers.

Cell division of rod-shaped bacteria requires the Z ring, a ring of FtsZ filaments associated with the inner-membrane wall. The MinCDE proteins help localize the Z ring to the center of the Escherichia coli cell. MinC, which inhibits Z-ring assembly, is a passenger on MinD. Previous studies have shown that MinC-MinD from E. coli and Aquifex aeolicus assemble in vitro into extended filaments with a 1:1 stoichiometry. However, a recent study has raised questions about the function of the MinC-MinD copolymer in vivo, because its assembly appears to require a high concentration of these two proteins and has a long lag time, and its blockade does not affect in vivo activities. Here, we found that MinC and MinD from Pseudomonas aeruginosa coassemble into filaments with a 1:1 stoichiometry. We also found that the minimal concentration of ∼4 µm required for assembly applies only to MinD because above 4 µm MinD, even very low MinC concentrations sustained coassembly. As previously reported, the MinC-MinD coassembly exhibited a long lag of ∼100 s when initiated by ATP. Premixing MinD with ATP eliminated this lag, suggesting that it may be due to slow MinD dimerization following ATP activation. We also discovered that MinC-MinD copolymers quickly bound FtsZ filaments and formed huge bundles. Our results resolve previous questions about the low concentration of MinC and the lag time, insights that may inform future investigations into the exact role of the MinC-MinD copolymer in vivo.

L form bacteria growth in low-osmolality medium.

L form bacteria do not have a cell wall and are thought to require medium of high osmolality for survival and growth. In this study we tested whether L forms can adapt to growth in lower osmolality medium. We first tested the Escherichia coli L form NC-7, generated in 1987 by Onoda following heavy mutagenesis. We started with growth in osmoprotective medium (~ 764 mOsm kg-1) and diluted it stepwise into medium of lower osmolality. At each step the cells were given up to 10 days to adapt and begin growing, during which they apparently acquired multiple new mutations. We eventually obtained a strain that could grow in LB containing only 34 mM NaCl, 137 mOsm kg-1 total. NC-7 showed a variety of morphologies including spherical, angular and cylindrical cells. Some cells extruded a bud that appeared to be the outer membrane enclosing an enlarged periplasm. Additional evidence for an outer membrane was sensitivity of the cells to the compound CHIR-090, which blocks the LPS pathway, and to EDTA which chelates Mg that may stabilize and rigidify the LPS in the outer membrane. We suggest that the mechanical rigidity of the outer membrane enables the angular shapes and provides some resistance to turgor in the low-osmolality media. Interestingly, cells that had an elongated shape underwent division shortly after addition of EDTA, suggesting that reducing the rigidity of the outer membrane under some turgor pressure induces division before lysis occurs. We then tested a well-characterized L form from Bacillus subtilis. L form strain LR-2L grew well with sucrose at 1246 and 791 mOsm kg-1. It survived when diluted directly into 440 mOsm kg-1 but grew poorly, achieving only 1/10 to 1/5 the density. The B. subtilis L form apparently adapted to this direct dilution by rapidly reducing cytoplasmic osmolality.

How bacterial cell division might cheat turgor pressure - a unified mechanism of septal division in Gram-positive and Gram-negative bacteria.

An important question for bacterial cell division is how the invaginating septum can overcome the turgor force generated by the high osmolarity of the cytoplasm. I suggest that it may not need to. Several studies in Gram-negative bacteria have shown that the periplasm is isoosmolar with the cytoplasm. Indirect evidence suggests that this is also true for Gram-positive bacteria. In this case the invagination of the septum takes place within the uniformly high osmotic pressure environment, and does not have to fight turgor pressure. A related question is how the V-shaped constriction of Gram-negative bacteria relates to the plate-like septum of Gram-positive bacteria. I collected evidence that Gram-negative bacteria have a latent capability of forming plate-like septa, and present a model in which septal division is the basic mechanism in both Gram-positive and Gram-negative bacteria.

Spontaneous Unfolding-Refolding of Fibronectin Type III Domains Assayed by Thiol Exchange: THERMODYNAMIC STABILITY CORRELATES WITH RATES OF UNFOLDING RATHER THAN FOLDING.

Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand ß-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1-1 s-1, for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils.

The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments.

FtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in Escherichia coli Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.

FtsZ Constriction Force - Curved Protofilaments Bending Membranes.

FtsZ assembles in vitro into protofilaments (pfs) that are one subunit thick and ~50 subunits long. In vivo these pfs assemble further into the Z ring, which, along with accessory division proteins, constricts to divide the cell. We have reconstituted Z rings in liposomes in vitro, using pure FtsZ that was modified with a membrane targeting sequence to directly bind the membrane. This FtsZ-mts assembled Z rings and constricted the liposomes without any accessory proteins. We proposed that the force for constriction was generated by a conformational change from straight to curved pfs. Evidence supporting this mechanism came from switching the membrane tether to the opposite side of the pf. These switched-tether pfs assembled "inside-out" Z rings, and squeezed the liposomes from the outside, as expected for the bending model. We propose three steps for the full process of cytokinesis: (a) pf bending generates a constriction force on the inner membrane, but the rigid peptidoglycan wall initially prevents any invagination; (b) downstream proteins associate to the Z ring and remodel the peptidoglycan, permitting it to follow the constricting FtsZ to a diameter of ~250 nm; the final steps of closure of the septum and membrane fusion are achieved by excess membrane synthesis and membrane fluctuations.

FtsZ filament capping by MciZ, a developmental regulator of bacterial division.

Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.

Probing for Binding Regions of the FtsZ Protein Surface through Site-Directed Insertions: Discovery of Fully Functional FtsZ-Fluorescent Proteins.

FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a "Z ring" at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE: One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.

Fibronectin Conformation and Assembly: Analysis of Fibronectin Deletion Mutants and Fibronectin Glomerulopathy (GFND) Mutants.

To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FNΔI1-5, FNΔIII1-3, FNΔIII4-8, and FNΔIII11-14. A monomeric form, FNmono, which lacked the C-terminal dimerization region, was also created. FNtnA-D was generated by swapping FNIII domains 1-8 in FNΔIII11-14 with seven FNIII domains from tenascin-C. The conformations of these mutants were analyzed by glycerol gradient sedimentation under low-salt (20 mM NaCl) and high-salt (200 mM NaCl) conditions. Surprisingly, most of the mutants showed a compact conformation under low-salt conditions, except for FNtnA-D. When we tested these mutants in cell culture, FNΔI1-5, FNΔIII1-3, and FNtnA-D were unable to form a matrix. Interestingly, FNΔIII1-3 and FNtnA-D were capable of co-assembly with full-length FN, while FNΔI1-5 was not. This indicates that the segment I1-5 is crucial for matrix assembly and segment III1-3 is also important. Mutations in FN are associated with glomerulopathy, but when we studied mutant proteins, the single-nucleotide mutations had only minor effects on conformation and matrix assembly. The mutations may destabilize their FNIII domains or generate dimers of dimers by disulfide cross-linking.

FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings.

FtsZ protofilaments (pfs) form the bacterial cytokinetic Z ring. Previous work suggested that a conformational change from straight to curved pfs generated the constriction force. In the simplest model, the C-terminal membrane tether is on the outside of the curved pf, facing the membrane. Tubulin, a homologue of FtsZ, also forms pfs with a curved conformation. However, it is well-established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In this study, we explored the FtsZ curvature direction by fusing large protein tags to the FtsZ termini. Thin section electron microscopy showed that the C-terminal tag was on the outside, consistent with the bending pf model. This has interesting implications for the evolution of tubulin. Tubulin likely began with the curvature of FtsZ, but evolution managed to reverse direction to produce outward-curving rings, which are useful for pulling chromosomes.

Liposome division by a simple bacterial division machinery.

We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed patches that produced concave distortions when viewed at the equator of the liposome. When viewed en face at the top of the liposome, many of the patches were seen to be small Z rings, which still maintained the concave depressions. We also succeeded in reconstituting the more natural, two-protein system, with FtsA and FtsZ-YFP (having the FtsA-binding peptide instead of the mts). Unilamellar liposomes incorporating FtsA and FtsZ-YFP showed a variety of distributions, including foci and linear arrays. A small fraction of liposomes had obvious Z rings. These Z rings could constrict the liposomes and in some cases appeared to complete the division, leaving a clear septum between the two daughter liposomes. Because complete liposome divisions were not seen with FtsZ-mts, FtsA may be critical for the final membrane scission event. We demonstrate that reconstituted cell division machinery apparently divides the liposome in vitro.

Gene product 0.4 increases bacteriophage T7 competitiveness by inhibiting host cell division.

Bacteriophages take over host resources primarily via the activity of proteins expressed early in infection. One of these proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. Here, we show that Gp0.4 is a direct inhibitor of the E. coli filamenting temperature-sensitive mutant Z division protein. A chemically synthesized Gp0.4 binds to purified filamenting temperature-sensitive mutant Z protein and directly inhibits its assembly in vitro. Consequently, expression of Gp0.4 in vivo is lethal to E. coli and results in bacteria that are morphologically elongated. We further show that this inhibition of cell division by Gp0.4 enhances the bacteriophage's competitive ability. This division inhibition is thus a fascinating example of a strategy in bacteriophages to maximize utilization of their hosts' cell resources.

Tenascin-C is an innate broad-spectrum, HIV-1-neutralizing protein in breast milk.

Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1-neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1-neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1-exposed breastfed infants are protected against mucosal HIV-1 transmission.

Rapid in vitro assembly of Caulobacter crescentus FtsZ protein at pH 6.5 and 7.2.

FtsZ from most bacteria assembles rapidly in vitro, reaching a steady-state plateau in 5-10 s after addition of GTP. A recent study used a novel dynamic light-scattering technique to assay the assembly of FtsZ from Caulobacter crescentus (CcFtsZ) and reported that assembly required 10 min, ∼100 times slower than for related bacteria. Previous studies had indicated normal, rapid assembly of CcFtsZ. We have reinvestigated the assembly kinetics using a mutant L72W, where assembly of subunits into protofilaments results in a significant increase in tryptophan fluorescence. We found that assembly reached a plateau in 5-10 s and showed no change in the following 10 min. This was confirmed by 90° light scattering and negative-stain electron microscopy. The very slow kinetics in the dynamic light-scattering study may be related to a refractory state induced when the FtsZ protein is stored without nucleotide, a phenomenon that we had observed in a previous study of EcFtsZ. We conclude that CcFtsZ is not an outlier, but shows rapid assembly kinetics similar to FtsZ from related bacteria.

The structure of irisin reveals a novel intersubunit ß-sheet fibronectin type III (FNIII) dimer: implications for receptor activation.

Irisin was recently identified as a putative myokine that is induced by exercise. Studies suggest that it is produced by cleavage of the FNDC5 (fibronectin domain-containing protein 5) receptor; irisin corresponds to the extracellular receptor ectodomain. Data suggesting that irisin stimulates white-to-brown fat conversion have led to the hypothesis that it does so by binding an unknown receptor, thus functioning as a myokine. As brown fat promotes energy dissipation, myokines that elicit the transformation of white to brown fat have potentially profound benefits in the treatment of obesity and metabolic disorders. Understanding the molecular basis for such exercise-induced phenomena is thus of considerable interest. Moreover, FNDC5-like receptors are highly conserved and have been shown to be critical for neuronal development. However, the structural and molecular mechanisms utilized by these proteins are currently unknown. Here, we describe the crystal structure and biochemical characterization of the FNDC5 ectodomain, corresponding to the irisin myokine. The 2.28 Å structure shows that irisin consists of an N-terminal fibronectin III (FNIII)-like domain attached to a flexible C-terminal tail. Strikingly, the FNIII-like domain forms a continuous intersubunit ß-sheet dimer, previously unobserved for any FNIII protein. Biochemical data confirm that irisin is a dimer and that dimerization is unaffected by glycosylation. This finding suggests a possible mechanism for receptor activation by the irisin domain as a preformed myokine dimer ligand or as a paracrine or autocrine dimerization module on FNDC5-like receptors.