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Betaglycan (BG) is a transmembrane co-receptor of the transforming growth factor-β (TGF-β) family of signaling ligands. It is essential for embryonic development, tissue homeostasis and fertility in adults. It functions by enabling binding of the three TGF-β isoforms to their signaling receptors and is additionally required for inhibin A (InhA) activity. Despite its requirement for the functions of TGF-βs and InhA in vivo, structural information explaining BG ligand selectivity and its mechanism of action is lacking. Here, we determine the structure of TGF-β bound both to BG and the signaling receptors, TGFBR1 and TGFBR2. We identify key regions responsible for ligand engagement, which has revealed binding interfaces that differ from those described for the closely related co-receptor of the TGF-β family, endoglin, thus demonstrating remarkable evolutionary adaptation to enable ligand selectivity. Finally, we provide a structural explanation for the hand-off mechanism underlying TGF-β signal potentiation. Betaglycan is a co-receptor for selective TGF-β family ligands. Here, the authors solve its structure in complex with TGF-β and the signaling receptors, which explains its ligand selectivity and reveals its mechanism in potentiating TGF-β signaling.

Eg5, a member of the kinesin superfamily of microtubule-based motors, is essential for bipolar spindle assembly and maintenance during mitosis, yet little is known about the mechanisms by which it accomplishes these tasks. Here, we used an automated optical trapping apparatus in conjunction with a novel motility assay that employed chemically modified surfaces to probe the mechanochemistry of Eg5. Individual dimers, formed by a recombinant human construct Eg5–513–5His, stepped processively along microtubules in 8-nm increments, with short run lengths averaging approximately eight steps. By varying the applied load (with a force clamp) and the ATP concentration, we found that the velocity of Eg5 was slower and less sensitive to external load than that of conventional kinesin, possibly reflecting the distinct demands of spindle assembly as compared with vesicle transport. The Eg5–513–5His velocity data were described by a minimal, three-state model where a force-dependent transition follows nucleotide binding.

Eg5 or KSP is a homotetrameric Kinesin‐5 involved in centrosome separation and assembly of the bipolar mitotic spindle. Analytical gel filtration of purified protein and cryo‐electron microscopy (cryo‐EM) of unidirectional shadowed microtubule–Eg5 complexes have been used to identify the stable dimer Eg5‐513. The motility assays show that Eg5‐513 promotes robust plus‐end‐directed microtubule gliding at a rate similar to that of homotetrameric Eg5 in vitro . Eg5‐513 exhibits slow ATP turnover, high affinity for ATP, and a weakened affinity for microtubules when compared to monomeric Eg5. We show here that the Eg5‐513 dimer binds microtubules with both heads to two adjacent tubulin heterodimers along the same microtubule protofilament. Under all nucleotide conditions tested, there were no visible structural changes in the monomeric Eg5–microtubule complexes with monastrol treatment. In contrast, there was a substantial monastrol effect on dimeric Eg5‐513, which reduced microtubule lattice decoration. Comparisons between the X‐ray structures of Eg5‐ADP and Eg5‐ADP‐monastrol with rat kinesin‐ADP after docking them into cryo‐EM 3‐D scaffolds revealed structural evidence for the weaker microtubule–Eg5 interaction in the presence of monastrol.

Betaglycan (BG) is a transmembrane co-receptor of the transforming growth factor-β (TGF-β) family of signaling ligands. It is essential for embryonic development and tissue homeostasis and fertility in adults. It functions by enabling binding of the three TGF-β isoforms to their signaling receptors and is additionally required for inhibin A (InhA) activity. Despite its requirement for the functions of TGF-βs and InhA in vivo, structural information explaining BG ligand selectivity and its mechanism of action is lacking. Here, we determine the structure of TGF-β bound both to BG and the signaling receptors, TGFBR1 and TGFBR2. We identify key regions responsible for ligand engagement, which has revealed novel binding interfaces that differ from those described for the closely related co-receptor of the TGF-β family, endoglin, thus demonstrating remarkable evolutionary adaptation to enable ligand selectivity. Finally, we provide a structural explanation for the hand-off mechanism underlying TGF-β signal potentiation.

The Kinesin-5 subfamily of the kinesin superfamily of molecular motors has been shown to play an integral role in the transfer of genetic material from mother-cell to daughter-cell. These homotetrameric kinesins function by crosslinking two microtubules in the mitotic spindle and imparting a force necessary to both assemble and maintain the spindle. The purpose of this dissertation has been to gain a better understanding of how Eg5/KSP, a member of the Kinesin-5 subfamily, coordinates the biochemical activities of its motor domains, to fulfill its cellular role. This dissertation focuses on a truncation of the human Eg5 gene that produces a dimeric motor. Analysis of this motor has indicated that the two motor domains, which interact with the same microtubule, function cooperatively. In some respects, dimeric Eg5 resembles conventional kinesin. Both motors are capable of translocating along the microtubule by taking successive steps before dissociating. To achieve this phenomenon, both motors couple the turnover of a single molecule of ATP to each advancement while maintaining the two motor domains out of phase through alternating catalytic cycles. Also, both motors have their stepping gated by ATP binding. The mechanistic commonalities between dimeric Eg5 and conventional kinesin, however, do not reach beyond a similar mechanism of stepping. This work has uncovered a novel biphasic, microtubule associated mechanochemical cycle. Dimeric Eg5 is the first kinesin known to begin the microtubule associated phase of its ATPase cycle with both motor domains associated with the microtubule. Furthermore, the transition to this two-motor-domain-bound state is the slow step governing steady-state ATP turnover. This slow transition only occurs once in the cycle and prior to processive movement. During processive movement, the catalytic step governs the rate of motor stepping. Dimeric Eg5 is also the first kinesin motor to have a rate-limiting catalytic step.