Jizhong Lou

Flow-enhanced adhesion regulated by a selectin interdomain hinge.

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin–ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.

Forcing switch from short- to intermediate- and long-lived states of the αA domain generates LFA-1/ICAM-1 catch bonds.

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 α7-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.

Mechanisms for Flow-Enhanced Cell Adhesion

Cell adhesion is mediated by specific receptor–ligand bonds. In several biological systems, increasing flow has been observed to enhance cell adhesion despite the increasing dislodging fluid shear forces. Flow-enhanced cell adhesion includes several aspects: flow augments the initial tethering of flowing cells to a stationary surface, slows the velocity and increases the regularity of rolling cells, and increases the number of rollingly adherent cells. Mechanisms for this intriguing phenomenon may include transport-dependent acceleration of bond formation and force-dependent deceleration of bond dissociation. The former includes three distinct transport modes: sliding of cell bottom on the surface, Brownian motion of the cell, and rotational diffusion of the interacting molecules. The latter involves a recently demonstrated counterintuitive behavior called catch bonds where force prolongs rather than shortens the lifetimes of receptor–ligand bonds. In this article, we summarize our recently published data that used dimensional analysis and mutational analysis to elucidate the above mechanisms for flow-enhanced leukocyte adhesion mediated by L-selectin-ligand interactions.

Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells

A biomembrane force probe visualizes force-regulated reversible switches between bent and extended conformations of αLβ2 integrin on the surface of a living cell.

Dynamic phosphorylation of CENP-A at Ser68 orchestrates its cell-cycle-dependent deposition at centromeres.

The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.

Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds

As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catch-slip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.

Molecular Dynamics Simulations of Forced Unbending of Integrin αVβ3

Integrins may undergo large conformational changes during activation, but the dynamic processes and pathways remain poorly understood. We used molecular dynamics to simulate forced unbending of a complete integrin αVβ3 ectodomain in both unliganded and liganded forms. Pulling the head of the integrin readily induced changes in the integrin from a bent to an extended conformation. Pulling at a cyclic RGD ligand bound to the integrin head also extended the integrin, suggesting that force can activate integrins. Interactions at the interfaces between the hybrid and β tail domains and between the hybrid and epidermal growth factor 4 domains formed the major energy barrier along the unbending pathway, which could be overcome spontaneously in ∼1 µs to yield a partially-extended conformation that tended to rebend. By comparison, a fully-extended conformation was stable. A newly-formed coordination between the αV Asp457 and the α-genu metal ion might contribute to the stability of the fully-extended conformation. These results reveal the dynamic processes and pathways of integrin conformational changes with atomic details and provide new insights into the structural mechanisms of integrin activation.

Flow-Induced Structural Transition in the β-Switch Region of Glycoprotein Ib

The impact of fluid flow on structure and dynamics of biomolecules has recently gained much attention. In this article, we present a molecular-dynamics algorithm that serves to generate stable water flow under constant temperature, for the study of flow-induced protein behavior. Flow simulations were performed on the 16-residue beta-switch region of platelet glycoprotein Ibalpha, for which crystal structures of its N-terminal domain alone and in complex with the A1 domain of von Willebrand factor have been solved. Comparison of the two structures reveals a conformational change in this region, which, upon complex formation, switches from an unstructured loop to a beta-hairpin. Interaction between glycoprotein Ibalpha and von Willebrand factor initiates platelet adhesion to injured vessel walls, and the adhesion is enhanced by blood flow. It has been hypothesized that the loop to beta-hairpin transition in glycoprotein Ib alpha is induced by flow before binding to von Willebrand factor. The simulations revealed clearly a flow-induced loop-->beta-hairpin transition. The transition is dominated by the entropy of the protein, and is seen to occur in two steps, namely a dihedral rotation step followed by a side-group packing step.

The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold

B lymphocytes use B cell receptors (BCRs) to sense the physical features of the antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here, we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN. We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect. In contrast, the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold. These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs. DOI: http://dx.doi.org/10.7554/eLife.06925.001

Dimeric Ube2g2 simultaneously engages donor and acceptor ubiquitins to form Lys48‐linked ubiquitin chains

Cellular adaptation to proteotoxic stress at the endoplasmic reticulum (ER) depends on Lys48‐linked polyubiquitination by ER‐associated ubiquitin ligases (E3s) and subsequent elimination of ubiquitinated retrotranslocation products by the proteasome. The ER‐associated E3 gp78 ubiquitinates misfolded proteins by transferring preformed Lys48‐linked ubiquitin chains from the cognate E2 Ube2g2 to substrates. Here we demonstrate that Ube2g2 synthesizes linkage specific ubiquitin chains by forming an unprecedented homodimer: The dimerization of Ube2g2, mediated primarily by electrostatic interactions between two Ube2g2s, is also facilitated by the charged ubiquitin molecules. Mutagenesis studies show that Ube2g2 dimerization is required for ER‐associated degradation (ERAD). In addition to E2 dimerization, we show that a highly conserved arginine residue in the donor Ube2g2 senses the presence of an aspartate in the acceptor ubiquitin to position only Lys48 of ubiquitin in proximity to the donor E2 active site. These results reveal an unanticipated mode of E2 self‐association that allows the E2 to effectively engage two ubiquitins to specifically synthesize Lys48‐linked ubiquitin chains.