The Unique Disulfide Bond-stabilized W1 β4-β1 Loop in the α4 β-Propeller Domain Regulates Integrin α4β7 Affinity and Signaling

Abstract

Background: Integrin ␣ 4 ␤ 7 is unique for mediating rolling and firm adhesion of lymphocytes pre-and post-activation. Results: Disrupting the disulfide bond-stabilized W1 ␤4-␤1 loop in the ␣ 4 ␤-propeller domain impaired ␣ 4 ␤ 7 -mediated, low-affinity ligand binding and bidirectional signaling. Conclusion: The W1 ␤4-␤1 loop regulates integrin ligand binding and signaling. Significance: Our findings reveal a particular molecular basis for ␣ 4 ␤ 7 -mediated rolling cell adhesion.

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

“…The C-terminal domain (residue 334-596) has a canonical α/β hydrolase fold with a central eightstranded, mixed β-sheet flanked by α-helices on both sides. A short α-helix (helix α1, residues [5][6][7][8][9][10][11][12][13][14] in the N-terminal region protrudes from the β-propeller domain and connects it to the hydrolase domain.…”

“…On the contrary, the ring closures of the non-Velcro type are mediated by hydrophobic interactions between continuous β-sheets [12]. Disulfide bonds can also be found between the first and last blades [13,14]. The non-Velcro structure allows flexibility in the ring and even a widening of a central cavity.…”

Kindlin-3 Is Essential for the Resting α4β1 Integrin-mediated Firm Cell Adhesion under Shear Flow Conditions

“…The cells remaining bound to VCAM-1 substrates (5 g/ml) at each wall shear stress were determined as a percentage of initial adherent cells at 1 dyne/cm 2 . Fluorescence Resonance Energy Transfer (FRET) Assay-FRET was measured as described (33,39). For detecting the orientation of integrin ectodomain relative to cell membrane, cells were seeded on a poly-L-lysine (100 g/ml)-coated surface in serum-free DMEM with the indicated divalent cation and incubated for 30 min at 37°C.…”

“…mAb to CD45 was from Sino Biological (10086-H02H). mAb Act-1 against human ␤7 integrin was as described previously (33). Fab fragments were produced as described (34), and direct labeling of antibodies with Alexa Fluor 488 was performed using a protein labeling kit according to the manufacturer's instructions (Invitrogen).…”

“…We next used a FRET assay to study the effect of kindlin-3 knockdown on integrin conformation. To assess the orientation of integrin ␣4␤1 ectodomain relative to the plasma membrane, ␣4␤1 was labeled with Alexa Fluor 488-conjugated AIIB2 Fab fragment, which binds to the top of ␤1 I domain, as donor (44), and the plasma membrane was labeled with a lipophilic probe, FM4-64 FX, as acceptor (33,39). In 1 mM Ca 2ϩ /Mg 2ϩ , kindlin-3 knockdown cells showed higher FRET efficiency than the control and luciferase shRNAtreated cells, suggesting a more bent conformation of the resting ␣4␤1 when kindlin-3 was knocked down (Fig.…”