Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides.

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state.

Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1.

Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants.

Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster.

Integrin alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+) + Mg(2+), and firm adhesion in Mg(2+) and Mn(2+), mimicking the two key steps in leukocyte accumulation in inflamed vasculature. We mutated an interlinked linear array of three divalent cation-binding sites present in integrin beta-subunit I-like domains. The middle, metal ion-dependent adhesion site (MIDAS) is required for both rolling and firm adhesion.

Integrin avidity regulation: are changes in affinity and conformation underemphasized?

Integrins play critical roles in development, wound healing, immunity and cancer. Central to their function is their unique ability to modulate dynamically their adhesiveness through both affinity- and valency-based mechanisms. Recent advances have shed light on the structural basis for affinity regulation and on the signaling mechanisms responsible for both affinity and valency modes of regulation.

Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.

Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 (alphaLbeta2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein-fused and yellow fluorescent protein-fused alphaL and beta2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling).

Small molecule integrin antagonists that bind to the beta2 subunit I-like domain and activate signals in one direction and block them in the other.

Leukocyte integrins contain an inserted (I) domain in their alpha subunits and an I-like domain in their beta(2) subunit, which directly bind ligand and regulate ligand binding, respectively. We describe a novel mechanistic class of integrin inhibitors that bind to the metal ion-dependent adhesion site of the beta(2) I-like domain and prevent its interaction with and activation of the alpha(L) I domain. The inhibitors do not bind to the alpha(L) I domain but stabilize alpha/beta subunit association and can show selectivity for alpha(L)beta(2) compared to alpha(M)beta(2).

Structure of integrin alpha5beta1 in complex with fibronectin.

The membrane-distal headpiece of integrins has evolved to specifically bind large extracellular protein ligands, but the molecular architecture of the resulting complexes has not been determined. We used molecular electron microscopy to determine the three-dimensional structure of the ligand-binding headpiece of integrin alpha5beta1 complexed with fragments of its physiological ligand fibronectin. The density map for the unliganded alpha5beta1 headpiece shows a 'closed' conformation similar to that seen in the alphaVbeta3 crystal structure.

Therapeutic antagonists and conformational regulation of integrin function.

Integrins are a structurally elaborate family of adhesion molecules that transmit signals bi-directionally across the plasma membrane by undergoing large-scale structural rearrangements. By regulating cell-cell and cell-matrix contacts, integrins participate in a wide range of biological processes, including development, tissue repair, angiogenesis, inflammation and haemostasis. From a therapeutic standpoint, integrins are probably the most important class of cell-adhesion receptors.

Complex between nidogen and laminin fragments reveals a paradigmatic beta-propeller interface.

Basement membranes are fundamental to tissue organization and physiology in all metazoans. The interaction between laminin and nidogen is crucial to the assembly of basement membranes. The structure of the interacting domains reveals a six-bladed Tyr-Trp-Thr-Asp (YWTD) beta-propeller domain in nidogen bound to laminin epidermal-growth-factor-like (LE) modules III3-5 in laminin (LE3-5).

Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking.

The integrin LFA-1 interacts with a variety of ligands termed ICAMs. ICAM-1 and ICAM-2 are both expressed on endothelium and serve as counterreceptors during lymphocyte trafficking. In this study, we analyzed their relative contribution to lymphocyte recirculation through lymph nodes and to recruitment into lung and inflamed skin by blocking mAbs against ICAM-1 and ICAM-2 and mice deficient for ICAM-1.

The critical cytoplasmic regions of the alphaL/beta2 integrin in Rap1-induced adhesion and migration.

Rap1 is a potent inside-out signal that increases LFA-1 adhesive activity. In this study, we have defined the cytoplasmic region of the alphaL and beta2 integrin that are required for Rap1-stimulated adhesion and subsequent migration on ICAM-1. Human LFA-1 bearing truncated and point-mutated alphaL and beta2 cytoplasmic regions were reconstituted in mouse IL-3-dependent proB cells, BAF/3.

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand.

The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligand-binding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aV beta 3 show two different conformations.

High affinity ligand binding by integrins does not involve head separation.

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain.

Structure and allosteric regulation of the alpha X beta 2 integrin I domain.

The integrin alpha X beta 2 (CD11c/CD18, p150,95) binds ligands through the I domain of the alpha X subunit. Ligands include the complement factor fragment iC3b, a key component in the innate immune defense, which, together with the expression of alpha X beta 2 on dendritic cells and on other leukocytes, suggests a role in the immune response. We now report the structure of the alpha X I domain resolved at 1.

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation.

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding.

Predicted and experimental structures of integrins and beta-propellers.

Integrins and other cell surface receptors have been fertile grounds for structure prediction experiments. Recently determined structures show remarkable successes, especially with beta-propeller domain predictions, and also reveal how ligand binding by integrins is conformationally regulated.

Continuous time transfer using GPS carrier phase.

The Astronomical Institute of the University of Berne is hosting one of the Analysis Centers (AC) of the International GPS Service (IGS). A network of a few GPS stations in Europe and North America is routinely analyzed for time transfer purposes, using the carrier phase observations. This work is done in the framework of a joint project with the Swiss Federal Office of Metrology and Accreditation (METAS).

Stabilizing the integrin alpha M inserted domain in alternative conformations with a range of engineered disulfide bonds.

Conformational movement of the C-terminal alpha7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an alpha/beta Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the alpha7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cbeta atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation.

Archaeal surface layer proteins contain beta propeller, PKD, and beta helix domains and are related to metazoan cell surface proteins.

The surface layer of archaeobacteria protects cells from extreme environments and, in Methanosarcina, may regulate cell adhesion. We identify three domain types that account for the complete architecture of numerous Methanosarcina surface layer proteins (SLPs). We solve the crystal structure for two of these domains, which correspond to the two N-terminal domains of an M.

Transition from rolling to firm adhesion is regulated by the conformation of the I domain of the integrin lymphocyte function-associated antigen-1.

The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation.

Integrin activation and structural rearrangement.

Among adhesion receptor families, integrins are particularly important in biological processes that require rapid modulation of adhesion and de-adhesion. Activation on a timescale of < 1 s of beta2 integrins on leukocytes and beta3 integrins on platelets enables deposition of these cells at sites of inflammation or vessel wall injury. Recent crystal, nuclear magnetic resonance (NMR), and electron microscope (EM) structures of integrins and their domains lead to a unifying mechanism of activation for both integrins that contain and those that lack an inserted (I) domain.

Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling.

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands.