TGF-β is synthesized as a proprotein that dimerizes in the endoplasmic reticulum. After processing in the Golgi to cleave the N-terminal prodomain from the C-terminal growth factor (GF) domain in each monomer, pro-TGF-β is secreted and stored in latent complexes. It is unclear which prodomain and GF monomer are linked before proprotein convertase cleavage and how much conformational change occurs following cleavage. We have determined a structure of pro-TGF-β1 with the proprotein convertase cleavage site mutated to mimic the structure of the TGF-β1 proprotein. Structure, mutation, and model building demonstrate that the prodomain arm domain in one monomer is linked to the GF that interacts with the arm domain in the other monomer in the dimeric structure (i.e. the prodomain arm domain and GF domain in each monomer are swapped). Swapping has important implications for the mechanism of biosynthesis in the TGF-β family and is relevant to the mechanism for preferential formation of heterodimers over homodimers for some members of the TGF-β family. Our structure, together with two previous ones, also provides insights into which regions of the prodomain–GF complex are highly structurally conserved and which are perturbed by crystal lattice contacts.

Significance: Integrins are cell-surface molecules that link extracellular ligands to the cytoskeleton. Force exerted by the cytoskeleton that is resisted by the ligand is thought to be important in activating the integrin by stabilizing an extended-open conformational state with high affinity for ligand. However, integrin α _V β ₈ does not interact with the cytoskeleton in the same way. Here, we show that, although the closely related integrins α _V β ₈ and α _V β ₆ bind the same ligand, pro-TGF-β1, their conformational responses to ligand binding and regulation by metal ions are quite different. These differences correlate with their distinct linkage to the cytoskeleton.

Myeloid lineage cells present the latent form of transforming growth factor-β1 (L-TGF-β1) to the membrane using an anchor protein LRRC33. Integrin α_Vβ₈ activates extracellular L-TGF-β1 to trigger the downstream signaling functions. However, the mechanism designating the specificity of TGF-β1 presentation and activation remains incompletely understood. Here, we report cryo-EM structures of human L-TGF-β1/LRRC33 and integrin α_Vβ₈/L-TGF-β1 complexes. Combined with biochemical and cell-based analyses, we demonstrate that LRRC33 only presents L-TGF-β1 but not the -β2 or -β3 isoforms due to difference of key residues on the growth factor domains. Moreover, we reveal a 2:2 binding mode of integrin α_Vβ₈ and L-TGF-β1, which shows higher avidity and more efficient L-TGF-β1 activation than previously reported 1:2 binding mode. We also uncover that the disulfide-linked loop of the integrin subunit β₈ determines its exquisite affinity to L-TGF-β1. Together, our findings provide important insights into the specificity of TGF-β1 signaling achieved by LRRC33 and integrin α_Vβ₈.