High-resolution mapping of the polyclonal immune response to the human platelet alloantigen HPA-1a (PlA1).

Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for ∼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a-specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents.

Autonomous conformational regulation of ß3 integrin and the conformation-dependent property of HPA-1a alloantibodies.

Integrin α/ß heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of ß subunit induce the α/ß separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The ß3 integrins, including αIIbß3 and αVß3, are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the ß3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti-HPA-1a alloantibodies can distinguish the αIIbß3 from αVß3 and affect their functions with unknown mechanisms. Here we designed a single-chain ß3 subunit that mimics a separation of α/ß heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain ß3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the ß3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain ß3 integrin, we identified the conformation-dependent property of anti-HPA-1a alloantibodies, which enables them to differently recognize the ß3 in the bent state vs. the extended state and in the complex with αIIb vs. αV This study provides deeper understandings of integrin conformational activation on the cell surface.

Structure of an extended ß3 integrin.

Cells use adhesion receptor integrins to communicate with their surroundings. Integrin activation and cellular signaling are coupled with change from bent to extended conformation. ß3 integrins, including αIIbß3, which is essential for the function of platelets in hemostasis and thrombosis, and αVß3, which plays multiple roles in diverse cell types, have been prototypes in understanding integrin structure and function. Despite extensive structural studies, a high-resolution integrin structure in an extended conformation remains to be determined. The human ß3 Leu33Pro polymorphism, located at the PSI domain, defines human platelet-specific alloantigens 1a and 1b (HPA-1a/b), immune response to which is a cause of posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia. Leu33Pro substitution has also been suggested to be a risk factor for thrombosis. Here we report the crystal structure of the ß3 headpiece in either Leu33 or Pro33 form, both of which reveal intermediate and fully extended conformations coexisting in 1 crystal. These were used to build high-resolution structures of full-length ß3 integrin in the intermediate and fully extended states, agreeing well with the corresponding conformations observed by electron microscopy. Our structures reveal how ß3 integrin becomes extended at its ß-knee region and how the flexibility of ß-leg domains is determined. In addition, our structures reveal conformational changes of the PSI and I-EGF1 domains upon ß3 extension, which may affect the binding of conformation-dependent anti-HPA-1a alloantibodies. Our structural and functional data show that Leu33Pro substitution does not directly alter the conformation or ligand binding of ß3 integrin.

The membrane-distal regions of integrin α cytoplasmic domains contribute differently to integrin inside-out activation.

Functioning as signal receivers and transmitters, the integrin α/ß cytoplasmic tails (CT) are pivotal in integrin activation and signaling. 18 α integrin subunits share a conserved membrane-proximal region but have a highly diverse membrane-distal (MD) region at their CTs. Recent studies demonstrated that the presence of α CTMD region is essential for talin-induced integrin inside-out activation. However, it remains unknown whether the non-conserved α CTMD regions differently regulate the inside-out activation of integrin. Using αIIbß3, αLß2, and α5ß1 as model integrins and by replacing their α CTMD regions with those of α subunits that pair with ß3, ß2, and ß1 subunits, we analyzed the function of CTMD regions of 17 α subunits in talin-mediated integrin activation. We found that the α CTMD regions play two roles on integrin, which are activation-supportive and activation-regulatory. The regulatory but not the supportive function depends on the sequence identity of α CTMD region. A membrane-proximal tyrosine residue present in the CTMD regions of a subset of α integrins was identified to negatively regulate integrin inside-out activation. Our study provides a useful resource for investigating the function of α integrin CTMD regions.

A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding.

The ligand-binding ßI and αI domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both ßI and αI domains, the α1- and α7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the α1-helix of ßI helps maintain the resting ßI conformation, whereas the homologous position in the αI α1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using αLß2 integrin as a model, we found that the residue volume at the Phe position in the α1-helix is critical for αLß2 activation because trimming the Phe by small amino acid substitutions abolished αLß2 binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for αMß2 integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of ß6-α7 loop and α7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between αL αI and ß2 ßI could be uncoupled because the ß2 extension and headpiece opening could occur independently of the αI activation. Reciprocally, the αI activation does not inevitably lead to the conformational changes of the ß2 subunit. Such loose linkage between the αI and ßI is attributed to the αI flexibility and could accommodate the αLß2-mediated rolling adhesion of leukocytes.

The importance of N-glycosylation on ß3 integrin ligand binding and conformational regulation.

N-glycosylations can regulate the adhesive function of integrins. Great variations in both the number and distribution of N-glycosylation sites are found in the 18 α and 8 ß integrin subunits. Crystal structures of αIIbß3 and αVß3 have resolved the precise structural location of each N-glycan site, but the structural consequences of individual N-glycan site on integrin activation remain unclear. By site-directed mutagenesis and structure-guided analyses, we dissected the function of individual N-glycan sites in ß3 integrin activation. We found that the N-glycan site, ß3-N320 at the headpiece and leg domain interface positively regulates αIIbß3 but not αVß3 activation. The ß3-N559 N-glycan at the ß3-I-EGF3 and αIIb-calf-1 domain interface, and the ß3-N654 N-glycan at the ß3-ß-tail and αIIb-calf-2 domain interface positively regulate the activation of both αIIbß3 and αVß3 integrins. In contrast, removal of the ß3-N371 N-glycan near the ß3 hybrid and I-EGF3 interface, or the ß3-N452 N-glycan at the I-EGF1 domain rendered ß3 integrin more active than the wild type. We identified one unique N-glycan at the ßI domain of ß1 subunit that negatively regulates α5ß1 activation. Our study suggests that the bulky N-glycans influence the large-scale conformational rearrangement by potentially stabilizing or destabilizing the domain interfaces of integrin.

The dual structural roles of the membrane distal region of the α-integrin cytoplasmic tail during integrin inside-out activation.

Studies on the mechanism of integrin inside-out activation have been focused on the role of ß-integrin cytoplasmic tails, which are relatively conserved and bear binding sites for the intracellular activators including talin and kindlin. Cytoplasmic tails for α-integrins share a conserved GFFKR motif at the membrane-proximal region and this forms a specific interface with the ß-integrin membrane-proximal region to keep the integrin inactive. The α-integrin membrane-distal regions, after the GFFKR motif, are diverse both in length and sequence and their roles in integrin activation have not been well-defined. In this study, we report that the α-integrin cytoplasmic membrane-distal region contributes to maintaining integrin in the resting state and to integrin inside-out activation. Complete deletion of the α-integrin membrane-distal region diminished talin- and kindlin-mediated integrin ligand binding and conformational change. A proper length and suitable amino acids in α-integrin membrane-distal region was found to be important for integrin inside-out activation. Our data establish an essential role for the α-integrin cytoplasmic membrane-distal region in integrin activation and provide new insights into how talin and kindlin induce the high-affinity integrin conformation that is required for fully functional integrins.