Anthony W. Partridge

Structural Basis of Integrin Activation by Talin

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 talins 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.

Structure of the integrin beta3 transmembrane segment in phospholipid bicelles and detergent micelles.

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 high-resolution structure of an integrin transmembrane domain. To assess the influence of the membrane model system, structure determinations of the beta3 integrin transmembrane segment and flanking sequences were carried out in both phospholipid bicelles and detergent micelles. In bicelles, a 30-residue linear alpha-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 K716s 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 beta3 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 beta3 transmembrane segment. In the micelle, residues L717-I721 are also embedded but deviate from linear alpha-helical conformation in contrast to I693-K716, which closely resemble the bicelle structure.

The antithrombotic potential of selective blockade of talin-dependent integrin αIIbβ3 (platelet GPIIb–IIIa) activation

Abstract In vitro studies indicate that binding of talin to the beta(3) integrin cytoplasmic domain (tail) results in integrin alpha(IIb)beta(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 beta(3) tail. We introduced a beta(3)(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a beta(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 alpha(IIb)beta(3) require binding of talin to the beta(3) tail. beta(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 beta(3)(Y747A) and virtually all beta(3)-null animals examined. Remarkably, less than 5% of beta(3)(L746A) animals exhibited this form of bleeding. These results establish that alpha(IIb)beta(3) activation in vivo is dependent on the interaction of talin with the beta(3) integrin cytoplasmic domain. Furthermore, they suggest that modulation of beta(3) integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.

Missense mutations in transmembrane domains of proteins: phenotypic propensity of polar residues for human disease.

Previous experiments on the cystic fibrosis transmembrane conductance regulator suggested that non‐native polar residues within membrane domains can compromise protein structure/function. However, depending on context, replacement of a native residue by a non‐native residue can result either in genetic disease or in benign effects (e.g., polymorphisms). Knowledge of missense mutations that frequently cause protein malfunction and subsequent disease can accordingly reveal information as to the impact of these residues in local protein environments. We exploited this concept by performing a statistical comparison of disease‐causing mutations in protein membrane‐spanning domains versus soluble domains. Using the Human Gene Mutation Database of 240 proteins (including 80 membrane proteins) associated with human disease, we compared the relative phenotypic propensity to cause disease of the 20 naturally occurring amino acids when removed from—or inserted into—native protein sequences. We found that in transmembrane domains (TMDs), mutations involving polar residues, and ionizable residues in particular (notably arginine), are more often associated with protein malfunction than soluble proteins. To further test the hypothesis that interhelical cross‐links formed by membrane‐embedded polar residues stabilize TMDs, we compared the occurrence of such residues in the TMDs of mesophilic and thermophilic prokaryotes. Results showed a significantly higher proportion of ionizable residues in thermophilic organisms, reinforcing the notion that membrane‐embedded electrostatic interactions play critical roles in TMD stability. Proteins 2004;54:000.000.

Integrin activation by talin.

Summary.  The development and integrity of the cardiovascular system depends on integrins, a family of adhesion receptors, vitally important for homeostasis of animal species from fruit fly to man. Integrins are critical players in cell migration, cell adhesion, cell cycle progression, differentiation, and apoptosis. Consequently, integrins have a major impact on the patterning and functions of the blood and cardiovascular system. Integrins undergo conformational changes, which alter their affinity for ligands through a process operationally defined as integrin activation. Integrin activation is important for platelet aggregation, leukocyte extravasation, and cell adhesion and migration, thus influencing such processes as hemostasis, inflammation and angiogenesis. Recently, a series of studies have begun to define the mechanism of integrin activation by demonstrating that binding of a cytoskeletal protein, talin, to integrin β subunit cytoplasmic tail is a last common step in integrin activation. These findings indicate that talin is likely to be at the center of converging signaling pathways regulating integrin activation.

Resorufin Butyrate as a Soluble and Monomeric High-Throughput Substrate for a Triglyceride Lipase

Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay (Z′ > 0.8). Critically, using parallel approaches, they show that resorufin butyrate is soluble and monomeric under assay conditions. The presented assay should be useful as a simple and inexpensive primary or secondary screen for the discovery of therapeutic lipase modulators.

Helix-helix interactions between transmembrane α-helices 3 and 4 within the cystic fibrosis transmembrane conductance regulator protein

Cystic fibrosis (CF) is an autosomal recessive disease for which the associated defective protein is the cystic fibrosis transmembrane conductance regulator (CFTR) [1]. While the three dimensional structure of CFTR is currently unknown, its main role has been shown to act as a chloride ion channel at the apical membrane of epithelial cells [2]. Amongst the many mutations that cause CF, some lie within the transmembrane (TM) which are thought to constitute the bulk of the chloride ion pore. In order to understand how these mutations result in CF disease, it is necessary to understand, at the molecular level, how the individual TM pack together within the TM domain. This information could be obtained by determining which helices have affinity for each other. We are currently undertaking this task by synthesizing the individual TM helices and using fluorescence resonance energy transfer (FRET) to detect helix–helix interactions within membrane–mimetic environments. Thus far, we have synthesized peptides TM-3 ( K K K M G L A L A H F V W I A P L Q V A L L M G L I W G K K K ) and T M – 4 (KKKLQASAFCGLGFLIVLALFQAGLGRMKKK). The two native tryptophans (Trp) in TM–3 can be used as FRET donors. During synthesis, an AEDANS group was added to the N–terminus of TM–4 to act as a FRET acceptor. Initial FRET results suggest that TM-3 and TM–4 have a helix–helix interaction.

Transmembrane Segment Peptides of the Ff Phage Major Coat Protein Form Parallel Homodimers

The Ff filamentous phage major coat protein (MCP) is located in the inner membrane of host cell Escherichia coli prior to assembly into the lipid-free virion. The 50-residue MCP consists of a mobile N-terminal amphipathic helix that is connected by a helical hinge region to a ca 20-residue transmembrane helix. In membrane environments the MCP has been shown to specifically self-associate into dimers in both in vitro [1] and in vivo [2] studies. In the present work, peptide versions of the MCP lacking the N-terminal arm were synthesized with the sequence KKKC-Y21IGYA-WAMVVVIVGATIGIKLFKKFTSK48-amide in order to investigate the orientation of the MCP transmembrane domains in detergent micelles. Peptides were labeled with pyrene fluorophores at the N-terminal Cys residue to establish the topology (ie. parallel vs anti-parallel) of the homodimers in detergent micelles using excimer fluorescence experiments. Experiments were carried out in two detergents, sodium dodecyl sulfate (SDS) and perfluorooctanoate (PFO) to investigate whether there was any correlation between the degree of excimer fluorescence and type of membrane mimetic environment used.

Covalent and Non-Covalent Oligomerization of the Transmembrane α-Helix 4 from the Cystic Fibrosis Conductance Regulator

Cystic fibrosis (CF) is an autosomal recessive disease that affects approximately 1 in 2000 people in Canada and the United States [1,2]. The disease arises through mutations to the cystic fibrosis transmembrane conductance regulator protein (CFTR). Although the three-dimensional structure for this protein has not been solved, its major function has been shown to act as a chloride ion channel in the apical membranes of epithelial cells. Amongst the identified mutations that result in CF, over 80 of these involve amino acid changes within the transmembrane domains (TMDs) [3]. The TMDs are predicted to consist of 12 α-helices that are believed to form the majority of the chloride ion pore. We hypothesized that many of the mutations that occur in the TMDs result in CF disease by altering the packing of the TM helices. Here, we investigated whether the CF phenotypic mutation V232D, occurring in TM helix 4, could result in altered helical packing. Our approach involved the synthesis of two peptides; one containing the wild-type sequence of TM4 (KKKLQASAFCGLGFLIV232LALFQAGLGRMKKK, TM4-wt), while the other contained the same sequence with the V232D mutation (KKKLQASAFCGLGFLID232LALFQAGLGRMKKK, TM4-VD).