Patrik Nygren

Affinity of talin-1 for the β3-integrin cytosolic domain is modulated by its phospholipid bilayer environment

Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the β3 cytosolic tail causes activation of the integrin αIIbβ3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the β3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the β3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the β3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the β3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.

Anomalous settlement behavior of Ulva linza zoospores on cationic oligopeptide surfaces

Identification of settlement cues for marine fouling organisms opens up new strategies and methods for biofouling prevention, and enables the development of more effective antifouling materials. To this end, the settlement behaviour of zoospores of the green alga Ulva linza onto cationic oligopeptide self-assembled monolayers (SAMs) has been investigated. The spores interact strongly with lysine- and arginine-rich SAMs, and their settlement appears to be stimulated by these surfaces. Of particular interest is an arginine-rich oligopeptide, which is effective in attracting spores to the surface, but in a way which leaves a large fraction of the settled spores attached to the surface in an anomalous fashion. These ‘pseudo-settled’ spores are relatively easily detached from the surface and do not undergo the full range of cellular responses associated with normal commitment to settlement. This is a hitherto undocumented mode of settlement, and surface dilution of the arginine-rich peptide with a neutral triglycine peptide demonstrates that both normal and anomalous settlement is proportional to the surface density of the arginine-rich peptide. The settlement experiments are complemented with physical studies of the oligopeptide SAMs, before and after extended immersion in artificial seawater, using infrared spectroscopy, null ellipsometry and contact angle measurements.

Fundamental Design Principles That Guide Induction of Helix upon Formation of Stable Peptide−Nanoparticle Complexes

We have shown that it is possible to design a peptide that has a very low helical content when free in solution but that adopts a well-defined helix when interacting with silica nanoparticles. From a systematic variation of the amino acid composition and distribution in designed peptides, it has been shown that the ability to form helical structure upon binding to the silica surface is dominated by two factors. First, the helical content is strongly correlated with the net positive charge on the side of the helix that interacts with the silica, and arginine residues are strongly favored over lysine residues in these positions. The second important factor is to have a high net negative charge on the side of the helix that faces the solution. Apparently, both attractive and repulsive electrostatic forces dominate the induction and stabilization of a bound helix. It is also evident that using amino acids that have high propensity to form helix in solution are also advantageous for the formation of helix on surfaces.

The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization

ABSTRACT Induction of cell cycle arrest in lymphocytes following exposure to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.

The Tyrosine Kinase c-Src Specifically Binds to the Active Integrin αIIbβ3 to Initiate Outside-in Signaling in Platelets

Background: β3-bound c-Src initiates outside-in signaling in platelets. Results: After platelet stimulation, β3 binds to the c-Src SH3 domain at a site overlapping with its PPII helix binding site. Conclusion: The interaction of c-Src with β3 requires c-Src activation to vacate the PPII helix binding site. Significance: The mode of c-Src binding to β3 prevents c-Src-mediated signaling in circulating platelets. It is currently believed that inactive tyrosine kinase c-Src in platelets binds to the cytoplasmic tail of the β3 integrin subunit via its SH3 domain. Although a recent NMR study supports this contention, it is likely that such binding would be precluded in inactive c-Src because an auto-inhibitory linker physically occludes the β3 tail binding site. Accordingly, we have re-examined c-Src binding to β3 by immunoprecipitation as well as NMR spectroscopy. In unstimulated platelets, we detected little to no interaction between c-Src and β3. Following platelet activation, however, c-Src was co-immunoprecipitated with β3 in a time-dependent manner and underwent progressive activation as well. We then measured chemical shift perturbations in the 15N-labeled SH3 domain induced by the C-terminal β3 tail peptide NITYRGT and found that the peptide interacted with the SH3 domain RT-loop and surrounding residues. A control peptide whose last three residues where replaced with those of the β1 cytoplasmic tail induced only small chemical shift perturbations on the opposite face of the SH3 domain. Next, to mimic inactive c-Src, we found that the canonical polyproline peptide RPLPPLP prevented binding of the β3 peptide to the RT- loop. Under these conditions, the β3 peptide induced chemical shift perturbations similar to the negative control. We conclude that the primary interaction of c-Src with the β3 tail occurs in its activated state and at a site that overlaps with PPII binding site in its SH3 domain. Interactions of inactive c-Src with β3 are weak and insensitive to β3 tail mutations.

Immobilized chemoattractant peptides mediate adhesion and distinct calcium-dependent cell signaling in human neutrophils.

Chemotaxis is the stimulated directional migration of cells in response to chemotactic factors, manifested for instance during leukocyte interaction with chemoattractants in inflammation. The N-formyl-Met-Leu-Phe (fMLF) bacterial peptide family is particularly potent in attracting and activating neutrophilic granulocytes. To accomplish defined circumstances for recruitment and activation of cells, we fabricated semitransparent gold-coated glass coverslips functionalized with chemoattractant fMLF receptor peptide agonist analogues. Peptides based on a common leading four-amino-acid sequence Gly-Gly-Gly-Cys were thus coupled to two potent fMLF receptor agonists, N-formyl-Tyr-Nle-Phe-Leu-Nle-Gly-Gly-Gly-Cys and N-formyl-Met-Leu-Phe-Gly-Gly-Gly-Cys, and a formylated control peptide, N-formyl-Gly-Gly-Gly-Cys. They were anchored via the SH group of Cys either directly to the gold surface or a mixed self-assembled monolayer composed of maleimide- and hydroxyl-terminated oligo(ethylene glycol) alkyldisulfides. The overall peptide immobilization procedure was characterized with ellipsometry, contact angle measurement, and infrared spectroscopy. When exposed to granulocytes, the agonist surface rapidly recruited neutrophils and the cells responded with extensive spreading and intracellular calcium transients within minutes. The reference peptide generated no such activation, and the cells maintained a more spherical morphology, suggesting that we have been able to immobilize chemoattractant receptor agonist peptides with retained bioactivity. This is a crucial step in designing surfaces with specific effects on cellular behavior.