Structure of an Extended β Integrin

Abstract

Integrins are a family of 24 α/β heterodimeric adhesion receptors that transmit bidirectional signals across the cell membrane. Each integrin subunit contains a large extracellular domain composed of headpiece and leg piece, a transmembrane domain and usually a short cytoplasmic tail. The α headpiece consists of the common β-propeller and thigh domains, and an extra αI domain inserted into the β-propeller domain in a subset of α subunits such as αL. The α leg piece includes calf-1 and -2 domains. The β headpiece contains βI, which is a structural homolog of αI domain, hybrid, PSI, and I-EGF1 domains. The β leg piece contains I-EGF2-4 and β-tail domains. Structural studies in the last 15 years, largely based on the two β3 integrins, αIIbβ3 and αVβ3, greatly advanced our understanding of integrin conformational regulation upon activation. The platelet specific αIIbβ3 plays an essential role in hemostasis and thrombosis, while the αVβ3 is important for the growth and survival of many cell types. In the resting state, integrin adopts a bent conformation with the headpiece folded onto the leg piece. Crystallographic and EM studies of αIIbβ3 and αVβ3 revealed two major conformational rearrangements, the swing-out motion of the hybrid domain, resulting in headpiece opening, and the switchblade-like movement of the headpiece from the leg domains, leading to integrin extension and leg separation. Despite the visualization of β3 extension by EM at very low resolution, a high-resolution extended β3 structure has been missing.A Leu33Pro polymorphism at the β3 PSI domain defines the human platelet alloantigen 1 (HPA-1) system. The mismatched HPA-1a/b (Leu33/Pro33) is a major cause of two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP) due to the generation of anti-HPA-1 alloantibodies. In addition, it has been controversial whether the Pro33 variant can enhance β3 activity, being a risk factor for thrombosis. It is also unknown whether the Leu33Pro substitution affects the conformation of PSI domain, which undergoes outward movement along with the headpiece opening and extension.It has been difficult to obtain the crystal structure of a full-length extended integrin largely due to the conformational heterogeneity and flexibility, especially in the lower leg domains. In this study, we engineered a chimeric β3 fragment, denoted as cβ3-E2, including the domains of PSI, hybrid, I-EGF1, I-EGF2, and the βI replaced with the αL αI domain, which enables the expression of β3 in the absence of α subunit. We determined the crystal structures of cβ3-E2 in both Leu33 and Pro33 forms. Strikingly, two conformations of cβ3-E2, an intermediate state and a fully extended state are simultaneously present in one crystal. The β3 extension occurs at the I-EGF1 and I-EGF2 interface, denoted as β-knee. Our structure revealed the structural changes of the flexible loop, C473-C486 or C1-C2 loop of I-EGF2 in the β-knee, which has been shown to modulate integrin activation by mutagenesis studies. No significant structural differences were observed between the PSI domains having Leu33 and Pro33. We next compared the ligand binding of β3-Leu33 and β3-Pro33 induced by Mn2+ or talin head domain. No significant differences were observed either in αIIbβ3 or αVβ3, indicating that the Leu33Pro substitution may not affect β3 integrin activation. However, the PSI C26-C38 loop harboring the Leu33Pro polymorphism moves closer to the I-EGF1 domain in the extended conformation. In addition, we found that a completely conserved arginine (Arg93 in β3) at the PSI/hybrid domain interface is important for maintaining the resting integrin state since the alanine or glutamine substitution renders β1, β2 and β3 integrin hyperactive. Interestingly, a naturally occurring β3-Arg93Gln substitution has been shown to disrupt the HPA-1a epitope. Finally, based on our cβ3-E2 structure, we built the extended full-length β3 structures with either closed or open headpiece, which can perfectly fit into the EM structures of extended αIIbβ3 with either closed or open headpiece, respectively. In summary, our study provides structural details of an extended β3 integrin, showing the conformational changes at the PSI, I-EGF1 and I-EGF2 domains, demonstrating their importance in regulating integrin activation and alloimmune response. DisclosuresNo relevant conflicts of interest to declare.