High integrin α _V β ₆ affinity reached by hybrid domain deletion slows ligand-binding on-rate

Abstract

The role of the hybrid domain in integrin affinity regulation is unknown, as is whether the kinetics of ligand binding is modulated by integrin affinity state. Here, we compare cell surface and soluble integrin αβ truncation mutants for ligand-binding affinity, kinetics, and thermodynamics. Removal of the integrin transmembrane/cytoplasmic domains or lower legs has little effect on αβ affinity, in contrast to β integrins. In integrin opening, rearrangement at the interface between the βI and hybrid domains is linked to remodeling at the ligand-binding site at the opposite end of the βI domain, which greatly increases in affinity in the open conformation. The larger size of the βI-hybrid interface in the closed state suggests that the hybrid domain stabilizes closing. In agreement, deletion of the hybrid domain raised affinity by 50-fold. Surface plasmon resonance and isothermal titration calorimetry gave similar results and the latter revealed tradeoffs between enthalpy and entropy not apparent from affinity. At extremely high affinity reached in Mn with hybrid domain truncation, αβ on-rate for both pro-TGF-β1 and fibronectin declined. The results suggest that the open conformation of αβ has lower on-rate than the closed conformation, correlate with constriction of the ligand-binding pocket in open αβ structures, and suggest that the extended-closed conformation is kinetically selected for ligand binding. Subsequent transition to the extended-open conformation is stabilized by its much higher affinity for ligand and would also be stabilized by force exerted across ligand-bound integrins by the actin cytoskeleton.

General structural features that regulate integrin affinity revealed by atypical αVβ8

“…Mn 2+ increases affinity of αVβ6 in part by stabilizing headpiece opening 12 . Mn 2+ boosted affinity of native αVβ6 by 12-fold and of αVβ8 by 2-fold, correlating with the lack of hybrid domain swing-out in αVβ8.…”

“…Higher affinity results from tightening of the ligand-binding site in the integrin β-subunit βI domain at the β1-α1 loop and α1-helix around the metal ion-dependent adhesion site (MIDAS) (Fig. 1b, c) 11,12 .

Fig. 1Overall integrin conformational states.

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Mechanical stress determines the configuration of TGFβ activation in articular cartilage

“…The exact mechanism for TGFβ activation in chondrocytes, particularly in an active mechanical environment, has not been studied. It has been shown that the arginylglycylaspartic acid (RGD) motif in LAP is the natural ligand for the αV subunit containing integrins 43 , 44 . Cell contractile forces may induce a conformational change of LAP and consequent TGFβ activation through the bond between αV integrins and the RGD sequence in LAP 45 , 46 .…”

Low affinity integrin states have faster binding kinetics than the high affinity state

“…Many previous studies have emphasized the importance of force in regulating integrin adhesiveness (Alon & Dustin, 2007;Astrof et al, 2006;Li & Springer, 2017;Nordenfelt et al, 2016;Nordenfelt et al, 2017;Sun et al, 2019;Zhu et al, 2008). Recent measurements of the intrinsic ligand-binding affinity of each conformational state and the equilibria linking them enabled a thermodynamic comparison of integrin activation models (Li & Springer, 2017, 2018Li et al, 2017). Remarkably, only the combination of adaptor binding and cytoskeletal force can activate integrins in an ultra-sensitive manner, with the switch between on and off occurring over a narrow range of signal input {Kuriyan, 2012 #24442}; the large increase in length between the bent and extended conformations (Fig.…”

“…If subsequent conformational change to the high affinity state is rapid, fast ligand binding kinetics to the low-affinity state efficiently couples ligand binding to stabilization by applied force of the high-affinity state, which has a long lifetime (Yakovenko, 2015). Work from our group on integrin αVβ6 showed that removal of the hybrid domain in the αVβ6 head resulted in a 50-fold increase in affinity for ligand yet decreased the apparent on-rate of ligand binding (Dong et al, 2018) suggesting that the open conformation has a lower on-rate than the BC and EC states. However, the intrinsic ligand-binding kinetics for each state of integrin αVβ6 could not be determined due to the lack of tools to stabilize specific conformational states.…”