Abstract
<jats:p>TGF-β signals through a conserved pathway yet produces diverse outcomes, pointing to regulatory mechanisms upstream of receptor engagement. A family of four latent TGF-β binding proteins (LTBPs) tether pro-TGF-βs and control their localization and activation, but how distinct LTBPs contribute to this regulation is unclear. Here we combine cryo-electron microscopy with functional assays to dissect LTBP-pro-TGF-β interactions. We resolve the LTBP-1/pro-TGF-β1 and LTBP-3/pro-TGF-β3 complex structures, revealing a conserved yet plastic hydrophobic binding interface, and systematically map key residues across all four LTBP and three pro-TGF-β subtypes that determine binding specificity and affinity. Unexpectedly, LTBP-2, previously thought incapable of TGF-β binding, engages pro-TGF-β1 through a covalent linkage via its 16th EGF-like domain, providing a mechanistic link between LTBP-2 function and TGF-β signaling. Beyond tethering, LTBP-3 shifts pro-TGF-β3 from spontaneous activation toward integrin dependence, establishing LTPBs as dynamic modulators that dictate not only where but also how TGF-β is unleashed. Collectively, these findings establish that LTBPs are not redundant ECM scaffolds but a family of functionally distinct regulators that differentially encode TGF-β spatial localization and activation, with implications for isoform-selective therapeutic strategies.</jats:p>