Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins.

Integrin signaling is bidirectional. 'Inside-out' signals regulate integrin affinity for adhesive ligands, and ligand-dependent 'outside-in' signals regulate cellular responses to adhesion. Integrin extracellular domains are yielding to high-resolution structural analyses, and intracellular proteins involved in integrin signaling are being identified. However, a key unresolved question is how integrins propagate signals across the plasma membrane.

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In vitro studies indicate that binding of talin to the β 3 integrin cytoplasmic domain (tail) results in integrin α IIb β 3 (GPIIb-IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the β 3 tail. We introduced a β 3 (Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a β 3 (L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate α IIb β 3 require binding of talin to the β 3 tail. β 3 (L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride-induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of β 3 (Y747A) and virtually all β 3 -null animals examined. Remarkably, less than 5% of β 3 (L746A) animals exhibited this form of bleeding. These results establish that α IIb β 3 activation in vivo is dependent on the interaction of talin with the β 3 integrin cytoplasmic domain. Furthermore, they suggest that modulation of β 3 integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.

Integrin adhesion receptors transduce bidirectional signals across the plasma membrane, with the integrin transmembrane domains acting as conduits in this process. Here, we report the first highresolution structure of an integrin transmembrane domain. To assess the influence of the membrane model system, structure determinations of the 3 integrin transmembrane segment and flanking sequences were carried out in both phospholipid bicelles and detergent micelles. In bicelles, a 30-residue linear R-helix, encompassing residues I693-H772, is adopted, of which I693-I721 appear embedded in the hydrophobic bicelle core. This relatively long transmembrane helix implies a pronounced helix tilt within a typical lipid bilayer, which facilitates the snorkeling of K716's charged side chain out of the lipid core while simultaneously immersing hydrophobic L717-I721 in the membrane. A shortening of bicelle lipid hydrocarbon tails does not lead to the transfer of L717-I721 into the aqueous phase, suggesting that the reported embedding represents the preferred 3 state. The nature of the lipid headgroup affected only the intracellular part of the transmembrane helix, indicating that an asymmetric lipid distribution is not required for studying the 3 transmembrane segment. In the micelle, residues L717-I721 are also embedded but deviate from linear R-helical conformation in contrast to I693-K716, which closely resemble the bicelle structure.

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the ␣ and ␤ transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to ␣/␤ interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the ␤3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the ␣ and ␤ TM domains. Therefore, we propose that helical packing of the ␣ and ␤ TM domains forms a clasp that regulates integrin activation.Integrin heterodimers are essential for the development and functioning of multicellular animals, because they mediate cell migration and cell adhesion and can influence gene expression and cell proliferation (1). All of the integrin heterodimers are composed of single pass Type I transmembrane (TM) 1 protein subunits ␣ and ␤. A central feature of these receptors is their capacity for rapid changes in their adhesive function mediated by changes in their ligand binding affinity, operationally defined here as "activation." The prototypical integrin, platelet ␣IIb␤3, is activated through interactions of the cytoplasmic integrin tails (ϳ20 and 47 residues for ␣ and ␤ tails, respectively) with intracellular proteins such as talin (2). These interactions initiate a long-range conformational change in the large extracellular domains (Ͼ700 residues each), resulting in high affinity binding of fibrinogen, von Willebrand factor, and fibronectin and consequently platelet aggregation and adherence to the vessel wall (1).Initial mutational studies suggested that a salt bridge between ␣IIbArg 995 and ␤3Asp 723 helps maintain the integrin in the low affinity state by forming part of an interactive face between ␣ and ␤ subunit cytoplasmic domains (3). Protein engineering studies from Springer laboratory have further advanced the idea that specific integrin ␣/␤ interactions maintain the low affinity conform...

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)—engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.