Arterial blood flow enhances glycoprotein Ibα (GPIbα) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbα/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbα dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbα-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flowenhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbα on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.

Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short-to intermediate-and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the ␣ 7 -helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the ␣A and ␤A domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.Integrins are membrane molecules broadly expressed on a wide variety of cells as ␣␤ heterodimers that bind ligands on another cell or the extracellular matrix (1, 2). Integrin/ligand interactions are thought to be capable of not only transmitting forces but also transducing signals bi-directionally across the cell membrane, thereby playing a key role in mechanosensing and mechanotransduction (3, 4).Integrins can assume distinct conformations with different ligand binding affinities (1, 2). Several types of conformational changes have been described based on structural (5-8) and functional (9 -14) studies (see below Integrin/ligand bonds are often subjected to forces externally applied to the cell, e.g. during leukocyte adhesion to vascular surfaces, or internally generated by the cell, e.g. during migration. Mechanical forces have been suggested to regulate integrin binding affinity by inducing conformational changes. For example, applying a shear flow to cells has been shown to enhance integrin/ligand binding (12,17,18). Atomic force microscopy single-bond experiments have demonstrated that ␣ 5 ␤ 1 , an ␣A domain-lacking integrin, forms catch bonds with fibronectin (FN) in which force prolongs bond lifetimes in the 10 -30 pN range (19). Steered molecular dynamics simulations have suggested how force might activate integrin ␣A domains (20) and the headpiece of integrin ␣ V ␤ 3 (21-24). However, many mechanistic details about the integrin mechanochemistry are still missing.Using force clamp (25) and thermal fluctuation (26) experiments to measure single bond interactions by a biomembrane force probe (BFP), here we show that lymphocyte functionassociated antigen-1 (LFA-1), an ␣A domain-...

In Gram-negative bacteria, the assembly of β-barrel outer-membrane proteins (OMPs) requires the β-barrel-assembly machinery (BAM) complex. We determined the crystal structure of the 200-kDa BAM complex from Escherichia coli at 3.55-Å resolution. The structure revealed that the BAM complex assembles into a hat-like shape, in which the BamA β-barrel domain forms the hat's crown embedded in the outer membrane, and its five polypeptide transport-associated (POTRA) domains interact with the four lipoproteins BamB, BamC, BamD and BamE, thus forming the hat's brim in the periplasm. The assembly of the BAM complex creates a ring-like apparatus beneath the BamA β-barrel in the periplasm and a potential substrate-exit pore located at the outer membrane-periplasm interface. The complex structure suggests that the chaperone-bound OMP substrates may feed into the chamber of the ring-like apparatus and insert into the outer membrane via the potential substrate-exit pore in an energy-independent manner.