Receptor–Ligand Binding: 'Catch' Bonds Finally Caught Dispatch

Receptor–Ligand Binding: ‘Catch’ Dispatch Bonds Finally Caught

Konstantinos Konstantopoulos, William D. Hanley existence of catch bonds as well as their transition to and Denis Wirtz slip bonds upon exposure to stronger disjoining forces. It is noteworthy that the observation of catch–slip transitional bonds was also evident in flow chamber assays Leukocyte recruitment to sites of inflammation is ini- in the low/intermediate force regime (<15–30 pN) [10]. tiated by the selectin family of adhesion receptors. The apparent discrepancy of these data [10] when Recent research reveals that P-selectin binding to compared with those of previous studies [7,8] is attrib- its ligand exhibits ‘catch’ to ‘slip’ bond transition that uted to the fact that the tether forces examined in may help explain the shear threshold phenomenon. earlier investigations were higher than the range required to observe catch–slip transition. The biphasic modulation of P-selectin–PSGL-1 During inflammation, leukocytes first tether and roll on binding affinity by increasing tensile forces may vessel walls where they become activated, due to provide a rational basis at the single-molecule level to exposure to locally produced chemokines, before they account for the shear threshold effect. The demon- firmly adhere and ultimately extravasate into the tissue stration of this phenomenon for another member of space [1]. These distinct types of adhesive events are the selectin family (L-selectin) in a cell-free system [11] mediated by highly specific receptors that display supports the concept that the origin of the shear unique kinetic and mechanical properties [1]. The threshold effect is predominantly molecular, and inti- selectin family, comprising E-, L- and P-selectin, is pri- mately associated with the intrinsic kinetic and marily responsible for the tethering and rolling of leuko- mechanical features of receptor–ligand bonds. There- cytes on inflamed endothelium under shear stress fore, cellular features such as deformation by increas- [1,2]. Both in vivo [3] and in vitro [3,4] studies have doc- ing shear forces which increase the cell–substrate umented the shear threshold phenomenon in which the contact area and the probability of bond formation number of rolling leukocytes first increases and then [12] may play a role, but do not control the effect. decreases while monotonically increasing wall shear Moreover, higher wall shear stresses, which favour stress. How cell–substrate adhesive contacts can be molecular contacts at the expense of encounter time amplified by the application of progressively stronger [13], may effectively increase selectin–ligand bond for- dispersive hydrodynamic forces is counterintuitive. mation [12,13], and thus support more leukocyte teth- Force is traditionally thought to shorten the lifetimes of ering/rolling interactions, contributing to the shear receptor–ligand bonds, referred to as ‘slip’ bonds, as it threshold effect. lowers the energy barrier between bound and free Shear-threshold phenomena have also been noted states [5]. However, a model that describes the in biological systems other than selectin-mediated physics of receptor–ligand binding has predicted the leukocyte rolling, including bacterial cell attachment to occurrence of adhesive links — termed ‘catch’ bonds host cells relevant to the process of microbial colo- — whose lifetime is actually prolonged by the applied nization via the lectin-like adhesin FimH [14], and force [6]. This model was previously discarded as a platelet adhesion to sites of vascular injury via binding potential explanation for the shear threshold effect of of von Willebrand factor to glycoprotein Ibα [15]. While selectin-mediated events, on the basis of extensive catch bonds had not been previously resolved at the data showing that the lifetimes of transient leukocyte single-molecule level, they had been implicated in the tethers on selectins shorten exponentially with increas- shear-enhanced Escherichia coli cell attachment to ing shear [7,8], as quantified by flow chamber assays. guinea pig red blood cells mediated via FimH [14,16]. The advent of ultrasensitive force devices has Using steered molecular dynamics simulations, enabled researchers to probe the kinetic and mechan- Thomas et al. [14] provided a mechanistic interpreta- ical properties of receptor–ligand bonds with unprece- tion for FimH affinity modulation by rapid force- dented resolution at the single-molecule level [5,9,10]. induced conformational changes in the FimH tertiary Work recently published in Nature using atomic force structure. This methodology revealed that application microscopy now reveals that bonds between P- of an external force induces extension of a FimH linker selectin and P-selectin glycoprotein ligand-1 (PSGL-1) chain connecting the lectin and pilin domains of FimH, display a biphasic relationship between lifetime and due to destabilization of hydrogen bonding between force, whereby increasing tensile forces initially prolong the linker region and the lectin domain. These struc- and subsequently diminish the lifetimes of these bonds tural changes could propagate from the carboxy-ter- [10]. This work provides the first direct experimental minal region to the amino-terminal region of the lectin observation at single-molecule resolution showing the domain and cause a conformational switch of the receptor binding site from ‘low affinity’ to ‘high affin- Department of Chemical and Biomolecular Engineering, The ity’ or alternatively expose an additional ‘cryptic’ Johns Hopkins University, 3400 N. Charles Street, Baltimore, binding site [14,16]. Along these lines, a readily flexi- Maryland 21218, USA. ble linker chain has the innate ability to promote adhe- E-mail: [email protected]; E-mail: [email protected] sion in the absence of shear force input, whereas Dispatch R612

Figure 1. A model of force-enhanced ABCinteractions between P-selectin and Amino PSGL-1 at the single-molecule level. terminus (A) No force (static conditions); (B) low of PSGL-1 force; (C) intermediate force. Hemody- namic forces due to blood flow induce a large conformational change in the EGF domain of P-selectin, which propagates along the lectin domain to the putative P-selectin PSGL-1 binding pocket. Similarly, mechanical forces in atomic force microscopy can C type lectin induce these conformational changes domain during the compression–tension cycle. Small distortions of the binding pocket would properly align critical binding determinants by exposing buried residues, EGF domain which progressively enhance P- selectin–PSGL-1 bond lifetime from low to intermediate forces (B->C; catch bond behavior). Intermediate and large disjoining forces ultimately dominate these attractive Increasing force forces and shorten the bond lifetime (slip Current Biology bond behavior; not shown). Red lines (in the binding pocket) represent hydrogen bonds, whereas high red dot density rep- resents a positive electrostatic potential. structural mutations predicted to stabilize the linker the largest change in the P-selectin conformation chain against extension require a shear threshold for occurs at the interface of the lectin and EGF domains, mediating adhesion of bacterial cells to host cells [14]. whereas smaller structural changes are evident in the Although the crystal structure of P-selectin in a putative PSGL-1-binding site [17]. complex with a PSGL-1-derived glycosulfopeptide has We cannot exclude the possibility that mechanical been resolved at equilibrium [17], a mechanistic inter- forces could also elongate the highly flexible pretation of how force elicits the catch bond behav- PSGL-1 molecule, and thus better expose its critical iour in P-selectin–PSGL-1 pairs is still lacking. determinants for recognition by P-selectin. Weak High-affinity binding of the lectin domain of P-selectin P-selectin–PSGL-1 interactions induced by low hemo- to PSGL-1 requires three clustered tyrosine sulfate dynamic forces (Figure 1) may be mediated by either residues, adjacent peptide components, and fucose the sialyl Lewis x present on a short core-2 O-glycan or and sialic acid residues on an optimally positioned any of the sulfotyrosine residues [17,18]. Increasing short core-2 O-glycan within the anionic amino-termi- hemodynamic forces may strengthen binding, that is, nal region of PSGL-1 [18]. All of these moieties have catch bond behaviour (Figure 1), by inducing confor- distinct contributions to the binding affinity of PSGL-1 mational changes in the binding pocket that ultimately for P-selectin, and must be presented in a stereo- present the critical binding determinants on both P- chemically precise configuration for optimal binding selectin and PSGL-1 in a proper configuration for which involves a combination of electrostatic and cooperative binding [17,18]. These structural changes hydrophobic contacts [17,18]. Somers et al. [17] may result from progressive exposure of buried showed that the sulfotyrosine-containing region of residues that initiate new attractive interactions, PSGL-1 binds P-selectin in an area of positive elec- thereby prolonging the lifetime of the bond. Alterna- trostatic potential, whereas its sialic acid/fucose com- tively, if the pathway to debonding involves the defor- ponent interacts with P-selectin in a region of neutral mation of compliant domains — distal from the binding and negative electrostatic potential. pocket — of the protein complex, then application of a Based on the structural information of P-selectin force may retard the rate of deformation, therefore bound with a PSGL-1-derived glycosulfopeptide and enhancing the stability of the P-selectin–PSGL-1 bond in light of reports suggesting that the epidermal [6]. Past a threshold force, increasing disjoining forces growth factor-like (EGF) domain of P-selectin may reg- will gradually disrupt the electrostatic and hydrophobic ulate its function [17,19], a model that can explain the interactions between PSGL-1 and P-selectin, thereby catch and catch–slip transitional bond behaviour of decreasing the lifetime of the bond, leading to the slip the P-selectin–PSGL-1 complex in vivo is presented in bond regime (Figure 1). Figure 1. Mechanical forces induce a conformational Although nuclear magnetic resonance and X-ray change at the interface of the lectin and EGF domains crystallography provide high-resolution structural of P-selectin that propagates along the lectin domain information about receptor–ligand pairs at equilib- ultimately triggering the PSGL-1 binding site to rium, they cannot determine the crystal structure of change from a low-affinity to a high-affinity conforma- receptor–ligand pairs exposed to non-equilibrium tional state (Figure 1). Indeed, comparison of the forces. Micromanipulation methods suffer from the crystal structures of uncomplexed P-selectin and opposite shortcoming. Optical tweezers, atomic force P-selectin–ligand complex at equilibrium reveals that microscopy, and biomembrane force probes measure Current Biology R613

changes in bond energy and lifetime of receptor/ligand 15. Doggett, T.A., Girdhar, G., Lawshe, A., Schmidtke, D.W., Laurenzi, under force with picoNewton and millisecond resolu- I.J., Diamond, S.L. and Diacovo, T.G. (2002). Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the tions, but provide no direct structural information. GPIb(alpha)-vWF tether bond. Biophys. J. 83, 194–205. Recent advances in molecular dynamics simulations 16. Isberg, R.R. and Barnes, P. (2002). Dancing with the host: flow- [20] are partially filling the gap by calculating non-equi- sependent bacterial adhesion. Cell 110, 1–4. librium conformational changes that occur when 17. Somers, W.S., Tang, J., Shaw, G.D. and Camphausen, R.T. (2000). Insights into the molecular basis of leukocyte tethering and rolling receptor–ligand pairs are subject to external forces revealed by structures of P- and E-selectin bound to SLe(X) and from known crystallographic data at equilibrium. It PSGL-1. Cell 103, 467–479. would therefore be highly informative to conduct mol- 18. Leppanen, A., White, S.P., Helin, J., McEver, R.P. and Cummings, R.D. (2000). Binding of glycosulfopeptides to P-selectin requires ecular dynamics simulations of the P-selectin–PSGL-1 stereospecific contributions of individual tyrosine sulfate and sugar pair and determine structural and interaction-force residues. J. Biol. Chem. 275, 39569–39578. field changes within, near or distal from the putative 19. Kansas, G.S. (1996). Selectins and their ligands: current concepts and controversies. Blood 88, 3259–3287. binding pocket upon application of forces that mimic 20. Lu, H. and Schulten, K. (1999). Steered molecular dynamics simula- hemodynamic shear. These simulations focusing on tions of force-induced protein domain unfolding. Proteins 35, the entire receptor–ligand complex as an integrated 453–463. mechanical unit will be challenging. Direct evidence for force-induced conformational changes may ultimately come from advances in fluorescence spectroscopy (such as fluorescence resonance energy transfer) at the single-molecule level. Coupling single-molecule fluorescence spectroscopy to micromanipulation tools as well as point mutations within the binding pocket, and in distal regions such as in the EGF domain, will help identify the mechanosen- sitive residue(s) and elucidate the molecular mecha- nism by which the lifetime of P-selectin–PSGL-1 binding is initially prolonged and then decreased for increasing applied force.

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