Inactive conformation enhances binding function in physiological conditions Olga Yakovenkoa, Veronika Tchesnokovab, Evgeni V. Sokurenkob, and Wendy E. Thomasa,1 aDepartment of Bioengineering, University of Washington, Seattle, WA 98195; and bDepartment of Microbiology, University of Washington, Seattle, WA 98195 Edited by Timothy A. Springer, Harvard Medical School, Boston, MA, and approved July 8, 2015 (received for review March 10, 2015) Many receptors display conformational flexibility, in which the processes, where the kinetics, or rates, of individual steps are more binding pocket has an open inactive conformation in the absence important than overall affinity because the process does not reach of ligand and a tight active conformation when bound to ligand. equilibrium. An example of a nonequilibrium process is cell ad- Here we study the bacterial adhesin FimH to address the role of hesion. Because adhesive receptors bind to ligands that are an- the inactive conformation of the pocket for initiating binding by chored to other cells or surfaces, the adhesive bonds are subjected comparing two variants: a wild-type FimH variant that is in the to tensile mechanical force due to cytoskeletal contraction, fluid– inactive state when not bound to its target mannose, and an flow-induced drag forces, or other stresses. This tensile force can engineered activated variant that is always in the active state. Not only be applied between receptor and ligand after the bond forms, surprisingly, activated FimH has a longer lifetime and higher so binding and unbinding occur in different conditions, and are affinity, and bacteria expressing activated FimH bound better in thus not occurring in a reversible manner as required to reach static conditions. However, bacteria expressing wild-type FimH equilibrium. Many adhesive receptors form catch bonds (15–19), bound better in flow. Wild-type and activated FimH demonstrated which are bonds that are activated by tensile force to become similar mechanical strength, likely because mechanical force in- longer lived, so the influence of force on this binding process is duces the active state in wild-type FimH. However, wild-type FimH nontrivial. The functional advantage of the inactive state in this displayed a faster bond association rate than activated FimH. nonequilibrium process is not known. Moreover, the ability of different FimH variants to mediate The Escherichia coli adhesin FimH is a model for addressing the BIOCHEMISTRY adhesion in flow reflected the fraction of FimH in the inactive role of the inactive state in the nonequilibrium process of cell state. These results demonstrate a new model for ligand-associ- adhesion, because FimH forms catch bonds that involve allosteric ated conformational changes that we call the kinetic-selection regulation (19, 20), like many other adhesive proteins (21–24) and model, in which ligand-binding selects the faster-binding inactive the structural basis of allostery in FimH is well understood (20). state and then induces the active state. This model predicts that in The isolated mannose-binding lectin domain has an elongated physiological conditions for cell adhesion, mechanical force will regulatory region and a tightly closed binding site that binds drive a nonequilibrium cycle that uses the fast binding rate of the α-methyl-mannose (aMM) with an affinity of 1–2 μM(25,26)and inactive state and slow unbinding rate of the active state, for a mannosylated BSA (man-BSA) with a lifetime of many minutes higher effective affinity than is possible at equilibrium. (20). However, FimH is normally incorporated into fimbrial ad- hesive organelles, and in this state, the naturally predominant conformational dynamics | receptor | ligand | adhesion | mechanical force variants such as K12 have a compressed regulatory region and a mannose-binding pocket that is 3 Å wider because the gatelike t is now known that few proteins recognize ligands through a lock clamp loop is open (20). We refer to this as the inactive state Iand key mechanism, in which the binding pocket is in essentially the same conformation whether or not ligand is bound. Instead, the Significance conformation of the binding pocket is usually dynamic. Confor- mational dynamics have many functions for receptors as well as enzymes, so for simplicity, we will use the term ligand to describe The binding pocket of a receptor often switches between two “ ” substrates and products as well as molecules that are not changed conformations, with the tight active conformation binding ligand “ ” by binding. For allosteric proteins, an obvious function of confor- with higher affinity than the loose inactive one. We demonstrate mational changes in the pocket is to allow regulation of ligand here that in physiologically relevant nonequilibrium conditions, binding by an allosteric effector. For other proteins, flexibility in stabilizing the active conformation actually reduces binding. We the binding pocket allows proteins to bind structurally distinct li- furthermore show that ligand binds most rapidly to the inactive conformation, but detaches most slowly from the active con- gands (1), which may in turn facilitate evolution of new structures formation, so that each conformation provides an advantage and functions (2, 3). In many cases, the inactive conformation of to binding. Mechanical force regulates many adhesive re- the pocket is relatively loose, and the active conformation tightens ceptors in a way that creates a nonequilibrium cycle that would around the ligand, often due to the closing of a hinge (4) between take advantage of the best property of each conformation, two domains, or loops that are described as a gate (e.g., refs. 5–7) – creating a higher effective affinity than either state alone. This or a lid (e.g., refs. 8 11) because they close over the ligand in provides new insights into the importance of both the inactive binding pocket. The ability of the pocket to close around different state and nonequilibrium conditions. ligands can contribute to specificity (5). Switching to the inactive state of the receptor dramatically increases the dissociation rate, so Author contributions: O.Y., V.T., and W.E.T. designed research; O.Y. and V.T. performed pocket dynamics controls the residence time of ligands (4, 12), and research; O.Y., V.T., and W.E.T. analyzed data; and O.Y., E.V.S., and W.E.T. wrote the thus the catalytic rate of most enzymes (3). The importance of the paper. inactive state to association rates is less clear; inactive states have The authors declare no conflict of interest. been shown have lower (4, 12), similar (13), or higher (14) associ- This article is a PNAS Direct Submission. ation rates relative to the active state, and the functional importance Freely available online through the PNAS open access option. of the differences is unclear. 1To whom correspondence should be addressed. Email: [email protected]. We hypothesize that inactive states can have faster ligand as- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sociation rates, and that this is important for nonequilibrium 1073/pnas.1503160112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1503160112 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 because K12 has an affinity of 300 μM for aMM (25). Most bonds between K12 and man-BSA last under 1 s, and a small fraction are much longer lived, demonstrating that K12 can bind to mannose in both the inactive and active states (20). The structure of the short-lived inactive bound state is not known, but mutational studies suggest weak, or sequential, allosteric coupling, in which the clamp loop closes transiently around mannose and the regu- latory region remains compressed (27). FimH forms catch bonds because mechanical force induces the elongated state of the reg- ulatory region, which stabilizes the closed form of the clamp loop (20, 28). Many variants of FimH have been either engineered or discovered in clinical isolates that increase binding to mannose in Fig. 1. FimH-mediated E. coli binding to man-BSA in flow. (A) Number of static conditions (27, 29, 30) by destabilizing the inactive state E. coli bound to a man-BSA surface after 5 min injection of free-floating relative to the active state (27). Evolution selects against these bacteria at the indicated shear stress. (B) Percent of E. coli that remained bound or stationary on a man-BSA surface after 30 s at the indicated shear mutations (30, 31), which strongly suggests that the inactive state stress. (C) Initial attachment rate of E. coli on a man-BSA surface at the in- provides a functional advantage in vivo, but the mechanism for dicated shear stress. In A and C, data were normalized to the number or this advantage remains unknown. attachment rate of K12 at 0.07 Pa. All panels show the mean and SD of two Here we compare FimH variants to determine whether and how experiments on different days. the inactive state provides a functional advantage to cell adhesion in nonequilibrium conditions. We refer to K12 as wild-type FimH. The FocH variant has the same affinity for aMM as the isolated both strains of bacteria to the surface at 0.035 Pa and then in- lectin domain (25), so we refer to FocH as activated FimH. Al- creased flow stepwise while tracking bound bacteria to measure though activated FocH mediated more bacterial attachment than movement or detachment (Fig. 1B). Even at 1 Pa, the highest did wild-type K12 at low flow rates, it mediated less attachment at shear stress studied in Fig. 1A, both strains remained bound high flow rates. Indeed, for over a dozen FimH variants, the without even moving. Indeed, all bacteria remained bound through number of bacteria binding at high flow was inversely proportional 10 Pa, and the fraction that moved at 3 and 10 Pa was similar for to the amount of FimH in the active state, as determined by a the two FimH variants.