The unusual strength of the FimH catch bond under force

Abstract

Many receptor ligand interactions have developed catch bond properties to overcome and even utilize forces that may oppose the attachment. The lifetime of a catch bond increases under force. FimH is an E. coli adhesive protein that forms a catch bond with mannose via a force-induced transition from a low-ligand-affinity state to a high-affinity state. The high-affinity state possesses incredibly strong binding properties that have never been fully characterized. In this work we sought to measure the lifetime of FimH in the high-affinity state using a constant force assay on a multiplexed magnetic tweezers. The magnetic tweezer applies a constant force to the bond between an anchored receptor and a ligand-coated bead. The applied force can vary between beads and is estimated from the velocity of the untethered bead along the focal axis. Movement in this direction requires analysis of diffraction rings whose pattern depends on the z-position of the bead. These are compared to diffraction ring images of beads taken at known z-locations. However, we often encountered errors in bead positions that could not be attributed to anything other than the tracking. By repeatedly manipulating a magnetic bead between a chamber floor and ceiling we found our bead tracking method resulted in unexpectedly high variability in the estimated bead positions and velocity. We found that excluding the center-most diffraction rings significantly improved the precision of bead tracking. With this new tracking method, we then measured the lifetime of the high-affinity state of FimH under force. We found the lifetime to be incredibly long, yet the rates of unbinding could not described by the expected single exponential decay model, but rather a double exponential decay model – suggesting multi-state unbinding kinetics. While this behavior could be the result of the magnetic beads forming multiple fimbria attachments, this explanation is highly unlikely as the effect of force on the rate of unbinding is not what we would expect if multiple bonds were forming. Rather it appears the FimH-mannose bond has at least two different states upon force activation. These results are the first functional evidence demonstrating this heterogeneous behavior.