Abstract
Integrin αVβ8, which like αVβ6 functions to activate TGF-βs, is atypical. Its β8 subunit binds to a distinctive cytoskeleton adaptor and does not exhibit large changes in conformation upon binding to ligand. Here, crystal structures, hydrogen-deuterium exchange dynamics, and affinity measurements on mutants are used to compare αVβ8 and αVβ6. Lack of a binding site for one of three βI domain divalent cations and a unique β6-α7 loop conformation in β8 facilitate movements of the α1 and α1’ helices at the ligand binding pocket toward the high affinity state, without coupling to β6-α7 loop reshaping and α7-helix pistoning that drive large changes in βI domain-hybrid domain orientation seen in other integrins. Reciprocal swaps between β6 and β8 βI domains increase affinity of αVβ6 and decrease affinity of αVβ8 and define features that regulate affinity of the βI domain and its coupling to the hybrid domain.
Highlights
Integrin αVβ8, which like αVβ6 functions to activate TGF-βs, is atypical
Two integrins, αVβ6 and αVβ8, appear to have evolved primarily to activate transforming growth factor-β1 (TGF-β1) and TGF-β3 and bind with high affinity to a RGDLXX(L/I) motif in the TGF-β prodomain. These integrins activate TGF-β by releasing the growth factor (GF) from the prodomain, which otherwise surrounds the GF and holds it in a latent form in which it cannot bind TGF-β receptors5. αVβ8 is expressed much more highly in the brain than in any other tissues[3] and especially highly on glial cells, where it localizes to synapses with other glia and neurons and within synaptosomes6. αVβ8 in the central nervous system is required to activate TGF-β1 complexed with the milieu molecule LRRC33 on microglia cells and for the maintenance of myelin, axons, and neurons in certain regions of the central nervous system and in motor pathways in the brain and spinal cord[7]
An integrin αVβ8 headpiece fragment with high mannose Nglycans was expressed in GnTI-deficient HEK293 cells, purified, crystallized, and soaked with or without the TGF-β1 ligand peptide G213RRGDLATIHG223 (Table 1)
Summary
Integrin αVβ8, which like αVβ6 functions to activate TGF-βs, is atypical. Its β8 subunit binds to a distinctive cytoskeleton adaptor and does not exhibit large changes in conformation upon binding to ligand. Two integrins, αVβ6 and αVβ8, appear to have evolved primarily to activate transforming growth factor-β1 (TGF-β1) and TGF-β3 and bind with high affinity to a RGDLXX(L/I) motif in the TGF-β prodomain. Among the 24 integrin heterodimers, 22 bridge extracellular ligands to the actin cytoskeleton by binding through specific sites in integrin β-subunit cytoplasmic domains to the adaptors talin and kindlin (Fig. 1a–c)[8]. Higher affinity results from tightening of the ligand-binding site in the integrin β-subunit βI domain at the β1-α1 loop and α1-helix around the metal ion-dependent adhesion site (MIDAS) (Fig. 1b, c)[11,12]. In contrast to other integrins, binding of ligand to αVβ8 fails to induce swing-out of the hybrid domain; that is, the open headpiece conformation (Fig. 1f)[14,15,16].
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