Inactive Conformation Enhances Binding Function in Physiological Conditions

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 adhesive 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 regulatory region remains compressed (27). FimH forms catch bonds because mechanical force induces the elongated state of the regulatory 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. This demonstrates that the inability of the conformation-sensitive antibody. We used atomic force micros- activated FocH variant to mediate binding at moderate to high copy (AFM) and surface plasmon resonance (SPR) to show that flow does not reflect an inability to remain attached. FocH and K12 have similar mechanical strengths, but that acti- Because binding of bacteria from solution requires the ability vated FocH has a much slower bond association rate. Together, to initiate attachment as well as to remain attached, we next these results demonstrate that the inactive state of FimH is nec- measured initial attachment rates from the original binding essary to initiate bacterial adhesion at high flow because it forms videos by counting each bacterium when it first appeared on the bonds quickly. This suggests that FimH binds through a novel surface, whether it then remained bound for less than a second, kinetic-selection model in which ligand selectively binds to the rolled along the surface, or stuck firmly to the surface. At the two inactive state due to its faster association rate, but then induces lowest shears measured, the bacterial attachment rate for K12 the active state, and that mechanical force creates a non- could not be accurately measured because many bacteria in these equilibrium cycle that combines the rapid binding of the inactive conditions appeared in only one frame, suggesting that many state with the slow unbinding of the active state for a higher ef- more may have appeared for too short a time to detect. How- fective affinity than that of either state at equilibrium. ever, in conditions where initial attachment could be accurately measured (Fig. 1C), K12 mediated much more initial attachment Results then did FocH. Thus, the poor performance of the activated Bacterial Adhesion in Flow. To determine whether the inactive state FocH variant at moderate to high flow can be explained by a of FimH is important for mediating adhesion in flow, we com- deficiency in initiating bacterial attachment. pared two strains of E. coli expressing the activated FocH variant of FimH, versus the wild-type K12 variant that is in the inactive Bond Kinetics and Mechanics. To further understand the difference state before binding. The two strains had an otherwise identical in bacterial binding mediated by the two variants, we investigated genetic background, and expressed similar levels of FimH when the difference in association and dissociation of FimH-mannose analyzed by flow cytometry (Table S1). First we performed binding bonds for the two variants. First, AFM was used to study the assays, in which we infused bacteria at various shear rates over mechanical strength of single bonds between mannose and surfaces coated with man-BSA, and used video microscopy to FimH. An AFM cantilever was incubated with man-BSA. A count the number of bacteria bound to the surface. Man-BSA was tissue culture polystyrene surface was incubated with 0.01 mg/mL used since E. coli adhesion to this glycan is similar to that on fimbrial tips, each of which contain a single FimH subunit (33). mammalian epithelial cells (28). As observed previously (25, 32), The cantilever was pressed to the surface for 10 s to allow bonds bacteria expressing K12 displayed shear-enhanced adhesion (Fig. to form, and then retracted at a nominal rate of 1,000 pN/s until 1A); they bound poorly at low shear (0.013 Pa), well at moderate a sudden decrease in force indicated bond rupture (Fig. 2A). shear (0.07 Pa), and poorly at high shear (0.14 Pa). In contrast, Bonds were observed in 15–20% of pulls, so 8% were multiple bacteria expressing FocH bound much better than K12 at low bonds if bond formation follows a Poisson distribution (34). We shear, consistent with the idea that the FocH variant is activated. removed 8.6% from analysis because they showed multiple Bacteria expressing FocH displayed shear-inhibited binding, with ruptures, so the final analyzed data should reflect only single decreased numbers binding at higher shear rates. Indeed, at bonds. For negative controls, 1% aMM was included in the moderate to high flow, far more bacteria expressing wild-type K12 buffer to block FimH from binding to man-BSA. The rupture bound relative to those expressing the so-called activated FocH. forces were binned and presented as histograms in Fig. 2B. When This demonstrates that wild-type FimH provides a functional ad- FimH was not inhibited, both K12 and FocH formed bonds with vantage for adhesion at moderate to high flow. man-BSA that ruptured primarily between 120 and 180 pN, with To determine whether the poor performance of FocH for a peak at 150 pN. This characteristic rupture force was not ob- binding at moderate to high flow was due to an inability of FocH- served in the negative controls, and is a known signature of single expressing bacteria to remain bound in these conditions, we bound FimH–mannose bonds (19, 35). Thus, single FocH–mannose bonds

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1503160112 Yakovenko et al. Downloaded by guest on September 24, 2021 from solution to immobilized man-BSA in SPR experiments. Fimbriae contain just one FimH molecule per fimbria, so binding reflects monomeric interactions. When identical concentrations of the two variants were injected, similar results were observed as in the AFM experiments. The response for FocH increased 17.4 ± 2.4 fold more slowly than did the response for K12 (Fig. 3B), but the K12 response plateaued so quickly that this ratio again provides only a lower bound. Because fimbriae of both variants are of similar size and were injected at the same concentrations, this shows that the FocH variant has a slower molecular association rate than does K12 when the fimbriae are in solution. To obtain a more quantitative comparison of bond association rates, we injected multiple concentrations of FocH (Fig. 4A) and K12 (Fig. 4B) fimbriae, and fit models to the data to obtain as- Fig. 2. FimH-mannose bond mechanics. (A) A typical AFM curve. (B) Rupture sociation and dissociation constants. FocH fit a one-state binding force histograms for binding of the indicated FimH in fimbrial tips to man- BSA, without and with aMM, which blocks specific adhesion to provide a model and K12 required a two-state model, consistent with the negative control. Data shows mean and SD of two experiments. concept that FocH is locked in the activated state, while K12 can bind in both the inactive and active states. The binding constants for these fits are given in Table 1. Based on the dissociation and K12–mannose bonds break at the same force, so bond strength constants (KD), FocH had a 21-fold higher affinity for man-BSA cannot explain why bacteria expressing FocH fail to initiate at- than did K12, consistent with its much higher affinity for a dif- tachment at moderate to high flow. ferent mannoside, aMM, in a previous study (25). The dissoci- kp We next asked whether the two FimH variants have different ation rate of FocH ( off) was 600-fold slower than the dissociation bond association rates. First we measured the fraction of touches rate of the inactive state of K12 (koff), again consistent with pre- that resulted in specific bond formation in AFM experiments vious measurements (20). Most important to our current question, identical to those described above, except that the contact time the association rate for the inactive state of K12 was 23-fold higher was varied. K12 formed specific bonds so rapidly (Fig. 3A), that

than the association rate for FocH, consistent with the raw AFM BIOCHEMISTRY we could only obtain a lower bound for the rate of bond for- and SPR data. −1 mation, at 0.12 ± 0.03 s . FocH formed specific bonds more These AFM and SPR bond association rate measurements −1 slowly, at a rate of 0.024 ± 0.004 s , but formed more bonds at demonstrate that FocH forms bonds more slowly than does K12. long contact times, consistent with its higher affinity (Fig. 3A). This in turn suggests that the active state has a slower association Both types of fimbrial tips were immobilized using the same rate that reduces initial attachment in high flow conditions. That concentration, which results in equal density and uniform dis- is, attachment at high flow may benefit from the inactive state. tribution of fimbrial tips on the surface (Fig. S1). The difference in the rate of bond formation thus demonstrates that the FocH Role of Conformational State in Bacterial Binding. To verify the variant has at least a 5.2 ± 1.7 fold lower molecular association importance of the inactive state in initiating adhesion at high flow, rate than does K12 when both adhesin and mannose are tethered we expressed nine FimH variants sequenced from clinical isolates to a larger object. of E. coli and eight engineered FimH variants, all in isogenic Differences in association rates of receptors and ligands tethered E. coli strains. We characterized FimH expression using flow to a surface do not always reflect differences in solution association cytometry. Although there was some variation between strains rates (36), so we also compared solution association rates. Fim- (Table S1), these small differences were not well correlated with E. coli briaewereisolatedfromisogenic and tested for binding any of the other measurements we describe below (R2 < 0.41). We chose a selection of variants that cover a wide range of static binding strengths, as indicated by the ratio of the number of bacteria binding to monomannose vs. trimannose ligands (Table S1). This ratio, hereafter called the 1M/3M ratio, provides a normalized measurement of bacterial binding to man-BSA (27).

Fig. 3. Relative association rates of FimH-mannose bonds. (A) Effect of contact time on the frequency of specific bond formation in the AFM experiments for FocH and K12 fimbrial tips binding to man-BSA. Specific bonds are defined as all ruptures between 120 and 180 pN, with aMM controls subtracted. Data shows mean and SD of two experiments. Dashed lines show the initial slopes, which provide an estimate of the rates of bond formation. (B) Binding of FocH and K12 fimbriae injected at 180 nM to man-BSA in BiaCore SPR. Data for 2 d is shown in open and closed symbols, and initial Fig. 4. Quantitative models for FimH bonds. Binding of FocH (A) and K12 slopes for the two dates are shown as dashed and solid lines, providing an (B) fimbriae injected at the indicated concentrations to man-BSA in BiaCore, estimate of the relative rates of bond formation. and fit (black lines) with one-state (FocH) and two-state (K12) models.

Yakovenko et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 Table 1. Rate constants and other parameters discussed in the bacterial attachment can be explained by a difference in bond paper association rates, as measured in both AFM and SPR, with K12 Parameter Value Source forming bonds 23-fold more quickly. For 17 FimH variants, the number of bacteria binding at high flow was inversely correlated p × 4 −1 −1 kon 5.6 10 M s SPR for FocH with the fraction of FimH in the active state. Together, these p × −3 −1 −1 koff 1.3 10 M s SPR for FocH data demonstrate that the inactive state of FimH forms bonds 6 −1 −1 kon 1.3 × 10 M s SPR for K12 more quickly and is needed for bacteria to initiate binding at −1 koff 0.80 s SPR for K12 high flow. −3 −1 kact 3.7 × 10 s SPR for K12 Cells can benefit from any mechanism that helps initiate −2 −1 kin 1.5 × 10 s SPR for K12 binding in moderate to high flow. FimH allows E. coli to colonize KK12 490 nM koff kin D kon kin + kact the mouth where shear stress due to salivary flow is estimated at FocH p = p KD 23 nM koff kon 0.08 Pa (38), and the intestines where shear stress due to peri- force p = KD 1.0 nM koff kon stalsis is estimated at 1 Pa (39, 40). Our data showing the su- perior binding of wild-type K12 over activated FocH in this flow range (Fig. 1) can therefore explain the evolutionary dominance To characterize the conformational state of each variant, we of variants like K12 that are in the inactive state. The need to E. coli characterized purified fimbriae in an ELISA with the Mab21 bind in this shear regime is not unique to FimH and . Many monoclonal antibody, which only recognizes the active state of pathogens as well as blood cells must bind in these and other FimH (37). Mab21 recognized the activated FocH variant rap- niches that involve moving fluid, such as venous and arterial – idly, reaching half-maximal binding in 30 s (Fig. S2A). In con- blood vessels, which experience a wall shear stress of 0.1 5Pa trast, Mab21 recognized a small fraction of the K12 variant at (41). Although flow increases the effective attachment rate of 30 s, and then recognition increased slowly over 120 min (Fig. macromolecules by replenishing the depleted layer near the S2B). This demonstrates that a small fraction of K12 is initially in binding surfaces (42, 43), high flow inhibits adhesion of cells the active state, and the rest activates slowly over time either (44), even when moderate flow enhances adhesion (32, 45). Drag spontaneously or due to the presence of Mab21. Either way, the force is often assumed to explain the reduced adhesion at high binding to Mab21 at 30 s reflects the amount of a variant that is flow, but although 0.1-Pa shear stress may create enough drag force to shorten significantly bond lifetimes on larger eukaryotic initially in the active state without mannose. Because FocH is E. coli primarily in the active state (25), the ratio of Mab21 binding at cells (46), drag force on at 0.1 Pa is only 1.6 pN (47), which is much too low to break FimH bonds (20). A more likely reason 30 s for each strain relative to FocH (hereafter called the Mab21 for reduced cell adhesion at moderate to high flow is that hy- ratio) thus indicates the fraction of FimH that is in the active drodynamic lift reduces the concentration of microparticles near state (Table S1). By this measurement, 6 ± 6% of K12 was in the the surface as flow increases (48, 49). Hydrodynamic lift should active state. For the full set of variants, the ability of bacteria to affect both bacteria and eukaryotic cells, which are microscale bind man-BSA in static conditions, as detected by 1M/3M, particles, but not nanoscale macromolecules. Although in vitro roughly correlated with the fraction of FimH in the active state, devices are usually oriented to take advantage of gravity to force as detected by the Mab21 ratio (R2 = 0.89; Fig. S3). the cells toward the surface, which can reduce or even eliminate We then measured the number of bacteria that bind to a man- the effect of hydrodynamic lift, this is not the case in vivo. It BSA surface after 3 min at low (0.013 Pa) and at high (0.14 Pa) therefore makes sense that cells that must adhere in flow in vivo flow, for each of the 17 strains (Table S1). Although the re- have evolved mechanisms to increase bond association rates. lationship was weak, the two measurements were inversely cor- R2 = These same fast rates will also enable cells to roll along a related ( 0.63; Fig. S3), demonstrating that there is a general surface instead of detaching when high drag force shortens bonds trade-off between low- and high-shear adhesion, but neither pro- lifetimes. Many adhesive receptors that mediate rolling adhesion vides a clear prediction of the other. We also attached each strain in flow have also been shown to have inactive states (46), in- at 0.035 Pa, then washed at 2 Pa for 2 min and 5 Pa for 1 min. cluding von Willebrand factor (45), selectins (50), and integrins None detached, and the fraction that remained stationary at 5 Pa (21). Rolling cells expressing activated high affinity variants of R2 = was not correlated to the number binding at 0.14 Pa ( 0.2; Fig. P-selectin detached as much (51) or more (50) as cells expressing S3). However, binding at low shear was fairly well correlated to the wild-type P-selectin. SPR data on both activated variants appear R2 = A Mab21 ratio ( 0.85; Fig. 5 ), demonstrating that the active to show lower association rates than data on wild-type P-selectin, state is important for binding in low shear conditions. Binding at but this was only confirmed quantitatively when the data were fit high-shear shear showed a biphasic relationship to the Mab21 with a two-state model (51), not with the one-state model (50), ratio (Fig. 5B). Variants with extremely low ratios had slightly likely because the latter did not kinetically fit the data. Our lower binding, likely because they activate poorly even under conclusions demonstrate the need to accurately determine ki- force. For the rest, the number bound at high shear was strongly netic as well as thermodynamic parameters to understand the 2 inversely correlated with the Mab21 ratio (R = 0.96; Fig. 5B), and importance of the inactive state in cell rolling. thus the fraction of FimH in the active state. This demonstrates We propose a novel kinetic-selection model, in which the in- that E. coli binding to man-BSA via FimH at high shear stress active state is important to initiate binding. We hypothesize that (e.g., >0.1 Pa) requires the inactive state of FimH. this model may apply to all bonds with gates (5–7) or lids (8–11) that close over the ligand in the active state, as observed for Discussion FimH (Fig. 6A). As illustrated in Fig. 6B, the kinetic-selection Here we showed that a FimH variant called FocH that is in the model hypothesizes that the inactive (R) and active (R*) state active state bound more poorly to a surface at high flow, relative may coexist before binding, but that ligand selects for the in- k kp to a wild-type FimH variant called K12 that is primarily in the active state due to a faster association rate ( on on), and then inactive state. The difference in high flow binding did not reflect induces the active state (R → RL → RL*). This model is distinct a difference in the mechanical strength of the two variants be- from the common conformational selection model (Fig. 6C), cause single bonds formed by both variants withstood the same which also assumes that both states coexist before binding, but force, and bacteria expressing both variants withstood similar requires that ligand selects the active state, rather than the in- flows. Instead, the difference reflected lower attachment rates by active state (R → R* → RL*). The kinetic-selection model is bacteria expressing FocH versus K12. The difference in initial distinct from the common induced-fit model (Fig. 6D), which

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1503160112 Yakovenko et al. Downloaded by guest on September 24, 2021 dissociation from the active state (RL*→R*), followed by inactivation (*→R) now that ligand and force are gone. This nonequilibrium cycle creates a higher effective affinity than is possible for either state without mechanical force, by combining the fast association rate kon of the inactive states with the slow dissociation k* K rate off of the active state. Although the D (inverse affinity) of the K12 and FocH variants for man-BSA in the SPR fits is 490 and 23 nM respectively, the effective KD of the K12 variant under kforce = k* k = force is D off / on 1.0 nM. This raises the question as to whether the effective affinity of other receptors is increased through nonequlibrium cycles involving kinetic selection. Although the cycle can be driven by mechanical force for adhesive receptors, it might also be driven by the chemical energy of downstream Fig. 5. Importance of FimH conformation to function. The number of signaling events such as autophosphorylation for signaling re- bacteria bound at (A) low 0.013-Pa shear stress and (B) high 0.14-Pa shear ceptors, so this concept should be considered for all receptors with stress were normalized to the highest binding strain at each shear, and conformational dynamics in the pocket, not just adhesive proteins plotted against the Mab21 ratio (the fraction of FimH in the active state) for – exposed to mechanical force. 17 FimH variants. Data shows mean and SD of 2 3 experiments. The K12 It should be noted that the kinetic-selection model does not variant is indicated by a gray triangle and FocH by a black square. The cor- relation between binding and Mab21 ratio is shown with the R2 statistic for make any assumptions about the internal structural dynamics of a linear fit. In B, the variants with lowest Mab21 ratio (black X’s) were ex- the receptor. The model should be equally valid whether there is cluded from the fit because the relationship was biphasic. an allosteric or localized conformational change in the receptor, and whether the change occurs in a single concerted step, or in sequential steps with one or more intermediates, as suggested for also requires that binding occurs through the inactive state (R → FimH (27, 52). For the kinetic-selection model, like the induced- RL → RL*), but assumes that this occurs because the active state fit model, the only requirement about the internal conforma- does not exist before binding, not because of a difference in rate tional dynamics of the receptor is that it be able to bind the constants. The kinetic-selection model may be considered a ligand, at least transiently, in some form of the inactive state. mixture or variation of these common models, and like these and BIOCHEMISTRY other models for conformational dynamics predicts virtually by Materials and Methods definition that stabilizing the active state will increase affinity. Reagents. Man-BSA was obtained from V-LABs, Inc. All other chemicals were However, the kinetic-selection model predicts a functional ad- obtained from Sigma. Recombinant strains were constructed using a fim null K12 derivative, AAEC191A as described previously (53). Fimbraie were pu- vantage not predicted by the other models in nonequilibrium rified as described previously (37). conditions such as those found during cell adhesion. The kinetic- Flow cytometry analysis was performed to determine FimH expression selection model alone predicts that chemical or mutational sta- levels for the different strains, using Mab21 in the presence of 1% aMM and bilization of the active state will dramatically reduce binding Alexa Fluor 488-labeled goat anti-mouse IgG (Molecular Probes). Because before reaching equilibrium, thus explaining the counterintuitive aMM stabilizes the active state of FimH, Mab21 probes the total amount of results of Fig. 3. Moreover, because mechanical stabilization of the FimH rather than its state of activation in these conditions (37). active state can only occur after the complex forms, this disad- vantage of the active state is completely bypassed during the 1M/3M Ratio. Static assays of bacterial adhesion to immobilized 1M-ligands (1M-BSA or yeast mannan) and 3M-ligand (bovine RNAseB) were carried out nonequilibrium cycle that is driven by force, as illustrated in Fig. in 96-well plates as described previously (54). E R→ RL 6 . That is, ligand binds quickly to the inactive state ( ), ELISA with Mab21 were performed for immobilized fimbriae as described allowing mechanical force to induce the active state (RL→RL*) previously (37) except that Mab21 was incubated for the time indicated in and prevent inactivation, so unbinding must occur through slow each experiment. To calculate the Mab21 ratio, the optical density after 30-s

Fig. 6. Models for conformational dynamics. (A) The inactive state of FimH with mannose (orange) docked into the pocket (20), and the active state coc- rystallized with mannose (26). A gate consisting of residues 1, 140 and 142 (cyan), closes around ligand in the active state. (B–E) Models are compared; the transitions and states that control model behavior are shown in black and those that are assumed to occur too slowly or rarely to affect model behavior are shown in light gray. In these models, a short wide and a long thin shape are used to illustrate the inactive and active states, respectively, independentof ligand binding. This is intended only to help the reader distinguish the inactive from the active states, rather than to reflect any assumption about the conformation of the various low-energy or transient states. (B) The kinetic selection model predicts that all four states coexist, but the active state binds and unbinds very slowly. (C) The conformational selection model assumes that ligand binds selectively to the active conformation. (D) The induced fit model assumes that the active unbound conformation does not exist. (E) In the kinetic-selection model with mechanical force, the active complex inactivates much more slowly than it unbinds, switching which rate constant determines model behavior.

Yakovenko et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 incubation with Mab21 for each strain was divided by that for FocH, to es- suspected to be locked in the active state. A two-state binding model timate the fraction of each strain in the active state. was used for K12 because this variant is known to switch states (19). For the K12 fit, the Rmax was restricted to be similar to that for FocH be- Dynamic Adhesion. Binding, initial attachment, and detachment of E. coli cause the low affinity of K12 meant the model would be under- were measured in flow. All bacteria were tested with surfaces prepared in an determined without this restriction, and the restriction is justified identical manner; each was incubated with 0.2 mg/mL man-BSA as described because the same amount of man-BSA was immobilized on the chip in before (32). The shear stress used is noted in each case. both sets of injections, the average mass of the two types of pili is SPR was performed as described previously with a BiaCore 2000, using man-BSA the same. as the immobilized ligand and fimbriae as the analyte (25). Molar concentrations were calculated from total protein concentrations assuming an AFM. Measurement of rupture forces was performed as described previously average molecular mass of 15,000 kDa, which assumes average 1,000 (19), except that 0.01 mg/mL fimbrial tips were incubated with the surface FimA subunits per fimbriae. Model fits were performed using BiaEvaluation for both strains, and loading rate and contact time were varied as indicated software, with RI set to zero because any change in refractive index was in the figures. We use a small compressive force of 50 pN (Fig. 2A) during the subtracted out using a control channel that lacked mannose on the BSA. A contact, which is not expected to denature FimH because FimH functions one-state binding model was used for FocH because this variant was well for 20 s at 50 pN of tensile force (20).

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