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

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Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rate measurements are similar for integrins on cell surfaces and ectodomain fragments. Although the extended-open conformation’s on-rate is ∼20-fold slower, its off-rate is ∼25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. These kinetic measurements, together with previous equilibrium measurements of integrin conformational state affinity and relative free energy on intact cells, are key to a definitive understanding of the mechanism of integrin activation.

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  1. Evaluation Summary:

    Li, Yan and Springer report ligand binding on- and off-rates for three different conformations of α4β1 as well as α5β1 integrin. This is the first report that provides these numbers, which are important to understand the 'mode of integrin activation'. The study is - from a technical stand point - flawlessly performed and the calculated data is in perfect agreement with the previously published data.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Public Review (Reviewer #1):

    This study reports measurements of ligand binding on- and off-rates for three different conformations of α4β1 as well as α5β1 integrin. These measurements were carried out with a well characterized set of Fabs that arrest the integrin in different conformational states. In line with their previously published work (Li et al. EMBO J, (2017); Li & Springer PNAS, (2017); Li & Springer, J Cell Biol, (2018), the measured binding kinetics appear to follow a conformational selection model of integrin-ligand binding, whereas the on- and off-rates for the active (EO) integrin binding for ligand is surprisingly slow, thereby potentially explaining the integrin activation trajectory on a cell encountering the ECM. These findings are novel and important. The drawback of the study is that the three different conformational states were stabilized with Fabs, which, although well characterised, may interfere with ligand binding and not necessarily work as expected.

  3. Public Review (Reviewer #2):
    This manuscript describes a detailed measurement and calculation of integrin ligand-binding kinetics, which are very important for the understanding of integrin activation. The data clearly indicated that low-affinity binding states of closed conformation of integrin bind ligand with the mode of "fast on fast off", while the high-affinity binding of the open conformation results from the much slower of the off-rate. The kinetics measurements were well designed and a lot of work was done in this study.

  4. Public Review (Reviewer #3):

    The manuscript by Li et al. entitled "Low affinity integrin states have faster binding kinetics than the high affinity state" addresses the fundamental question of how the different conformational states of an adhesion receptor control its adhesive properties and thus cell adhesion. In particular, the authors studied the binding kinetics of distinct conformational states of integrins, both on and off rate, and the relative abundance of these different conformational states at the cell surface.

    The manuscript by Li et al. builds on previous articles by the same group, which essentially (among other groundbreaking studies on cell adhesion and in particular integrins) established the structural knowledge on integrins, in particular the fact that integrins can exist in 3 conformational states: the low-affinity bent-closed (BC) and extended-closed (EC) conformations and the high-affinity extended-open (EO) conformation.

    The main messages of the manuscript are: 1. low affinity states of integrins (BC, EC) bind faster than the high-affinity state (EO); 2. the higher affinity of the EO state results from a slower off-rate compare to low affinity BC and EC states; 3. Low affinity integrin states are denser compared to the high affinity integrin state at the cell surface. These results could shed new light on the sequence of molecular events leading to integrin activation/adhesion in the cellular context. In particular, on the relative contribution of the outside-in versus inside-out mechanisms to activate integrins.

    The authors used well-characterized conformation-specific Fab combinations to stabilize integrins (α4β1 and α5β1) in different conformational states and measured ligand binding/unbinding kinetics (on rate and off-rate). They used two experimental configurations: 1. Flow cytometry on intact suspended cells (Jurkat cells), to study α4β1 and α5β1 integrins in their cellular environment; 2. bio-layer interferometry to study an ectodomain fragment of α5β1.

    Using the first experimental configuration, to stabilize the EC and EO conformations of α4β1 integrin they used 9EG7 and to stabilize the EO conformation of β1 integrins they used a combination of 9EG7 and HUTS4Fab. Under basal conditions (BC, EC, EO) association to and dissociation from a ligand (FITC-LDVP) were the fastest, association and dissociation were slower for extended conformations (EC, EO) and even slower when only the EO conformation was present. Then found the same results, association and dissociation were slower when only the EO conformation was present, with VCAM D1D2 binding to integrin α4β1, although they could not measure these parameters for the basal condition since the affinity of α4β1 for VCAM D1D2 is too low.

    Then, they studied the binding of Fn39-10 to α5β1 integrin on K562 cells and found the same results, association and dissociation were slower when only the EO conformation was present compared to when the EC and EO were present. Then they quantified the apparent Kon and Koff assuming a 1 vs. 1 Langmuir binding model. Thus, overall, their results show that the ligand associates and dissociates more slowly to/from the EO conformation than to/from the BC and EC conformations.

    Then using bio-layer interferometry they performed the same analysis on soluble α5β1 ectodomain binding to Fn39-10. In that configuration the authors could also quantify the basal ensemble Fn39-10 binding kinetics, by raising the population of the EO state by truncation and favoring high mannose glycoforms posttranslational modification. The results confirmed that the open EO state associates and dissociates more slowly compared to mixture of the close BC and EC states.

    To measure the off-rate of the closed states, the authors first enabled ligand binding to integrins to reach steady state, and then added closure-stabilizing Fab to measure the dissociation kinetics. By using this strategy, they could measure the koff for the mixture of closed conformations (BC + EC) and also for the extended closed conformation only. In these conditions, the dissociation from the basal and extended α4β1 ensembles on Jurkat cells were similar. They found the similar results with Fn39-10 dissociation from basal or extended ensembles of the α5β1 ectodomain. These results confirmed that the Koff for the closed states (BC + EC) are much faster than for the open state EO.

    Finally, assuming that integrin conformational transition kinetics are sufficiently fast and therefore do not influence the measured kinetics, since integrins and ligand-bound integrins can be considered to equilibrate between their conformational states, the authors used a 1 vs. 1 Langmuir binding model to calculate the ligand binding kinetics from the ensemble measurements.

    Overall, the authors found that integrins α4β1 and α5β1 closed states (BC and EC) have a lower affinity compared to their open states, but closed states have higher on-rates than their EO open states. The higher affinities of the open states compared to the closed conformations for α4β1 and α5β1 integrins lie in a much slower off-rate. These results are supported by published structural data showing that the open conformation of the integrin has a tighter ligand binding pocket than the closed conformations, which creates a steric barrier to ligand binding but also stabilises ligand binding (dissociation barrier).

    The fact that the closed conformations (BC and EC) have a faster Kon compared to the open conformation (EO), could explain how integrins can probe the extracellular matrix (ECM) without the need for the EO state which could be stabilized by interaction with intracellular regulators and the actin cytoskeleton (inside-out). In that case integrin binding to its ligand (outside-in) could initiate an adhesive structure mainly in regions where the actin flow generates enough force to stabilize the transition to the EO state (e.g. lamellipodium, focal adhesions). The fact that BC and EC closed conformations possess faster Koff compared to the EO state is also very interesting. In this scenario, there could be a transient period where integrins have the possibility to unbind their ligand by transition to the EC and BC, before full activation by force generated by the actin cytoskeleton. This will also enable reversible binding of integrins once connection with the actin cytoskeleton is lost, which is consistent with integrin turnover or diffusion-immobilization cycles found in adhesive structures.

    The conclusions of the manuscript are convincingly supported by the results. The authors have performed a very comprehensive characterization of the kinetic parameters of α4β1 and α5β1 integrins association to and dissociation from their ligands. These results could provide a better understanding of the mechanisms that control the binding of integrins to their ligands in adherent cells. In particular, the results could shed light on the sequence of molecular events leading to integrins that are simultaneously bound to their extracellular ligand and connected to the intracellular actin cytoskeleton.