Downloaded by guest on September 27, 2021 nanme fssesfreape -eetnadPSGL-1 and found P- (8), con- example, dimer been this systems—for E-cadherin has of in (7), behavior number unknown bond a at is in catch–slip force which counterintuitive with decreases of A then nature and the text, bond), (catch might bond forces catch-slip 40-pN hydrodynamic to to 30- subject clots at vivo. fibrin in bonds shear nascent tension- knob-hole of Strengthen- formation A:a of interface. favor the capable association of pairs their ing understand protein of to remodeling of framework induced response general a mechanical high-affinity provide the and We (slip) states. low-affinity (catch) the the in between transitions structural pocket of binding terms by in explained formulated theory, quantitatively biphasic a binding is using of rupture of discovery bond increase knob-hole The of the lifetimes. kinetics in bond resulting prolonged and closure, affinity ‘a’ hole triggers the in ‘A’ domain B the from tion complex. knob-hole site (residuescalcium-binding A:a flap structural of movable the modeling The provide structures molecular crystal To performed analyzed bonds). we and behavior, (slip catch–slip-bond of value force basis the critical when force tensile a with with decreases exceeds then increases and first bonds) knob- polymers (catch A:a force fibrin noncovalent stabilizing forced of bond impacts single-molecule strength hole load trap-based The optical mechanical experiments. out unbinding how carried reveal we throm- 2018) To 13, in fibrin, February hemostasis. essential review for are and (received stability 2018 bosis 28, mechanical June and approved and formation MD, Fibrin Bethesda, Health, of Institutes National Szabo, Attila by Edited and www.pnas.org/cgi/doi/10.1073/pnas.1802576115 knob- A:a to of up strength force the tensile that with increases discovered bonds We hole 6). (5, bonds structure the influence thrombi. and and clots fibrin of properties of and formation inter- knob-hole govern A:a (1). actions polymerization holes fibrin to for ‘A’ necessary knobs is of ‘a’ interac- contracting Binding knob-hole cross-linking. and the covalent of flow before strength tions blood the by the determined by is platelets, example, for exerted, forces network space-filling the 1 form (Fig. to branch aggregate that and fibers elongate insoluble which into protofibrils, fibrin ‘a’ 1 form (Fig. to (holes) merizes molecule sites fibrin another complementary of the with regions in (knobs) interact ‘b’ sites that and binding ‘B’ exposed and with con- ‘A’ fibrin enzymatically monomeric is to Fibrinogen verted obstruction (thrombosis). thrombotic vessels essential blood and is of (hemostasis) fibrin of bleeding Formation stopping thrombi. for and clots of scaffold cal F remodeling interface Federation; Russian 141700, Federation; Region Russian Moscow 420008, 01854; Kazan MA University, Lowell, Federal Lowell, Kazan Biology, and Medicine Fundamental a Weisel W. John and bonds Litvinov I. slip Rustem to catch from tension-activated transition triggers fibrin in element Regulatory eateto eladDvlpetlBooy nvriyo enyvnaPrla colo eiie hldlha A19104; PA Philadelphia, Medicine, of School Perelman Pennsylvania of University Biology, Developmental and Cell of Department eepoe igemlcl ocdrpueo : knob-hole A:a of rupture forced single-molecule explored We bi steedpouto lo ltigta osiue a constitutes mechani- that filamentous clotting the blood providing of network product proteinaceous end the is ibrin f eateto hmsr,Uiest fTxsa utn utn X78712 TX Austin, Austin, at Texas of University Chemistry, of Department .Mcaia tblt fbodcos nrsos to response in clots, blood of stability Mechanical A–C). | bi polymerization fibrin γ and - | a,b,1 P computing GPU γ a,3 evsa eso esr lpdissocia- Flap sensor. tension a as serves 2 d β laKononova Olga , aoaoyo optradMteaia oeigo ilgclSses ocwIsiueo hsc n Technology, and Physics of Institute Moscow Systems, Biological of Modeling Mathematical and Computer of Laboratory ndls epciey fteltrlD lateral the of respectively, -nodules, γ γ 9 to 295 ndl n rnlcto oknob to translocation and -nodule | utaigbottleneck fluctuating γ α5β e 0)cnann h weak the containing 305) aoaoyo ahmtclMdln nMdcn,Sceo nvriy ocw199,RsinFederation; Russian 119991, Moscow University, Sechenov Medicine, in Modeling Mathematical of Laboratory f > n boetn(9), fibronectin and 1 c,d,1,2 1.Fbi poly- Fibrin (1). A–C) 5p si bonds). (slip pN 35 re Zhmurov Artem , f ≈ 03 pN 30–35 | d,e 2) nrdcdi h otx fsalmlcl idn to binding small-molecule of experiments. the context explain model quantitatively the- the to (FB) myoglobin, a in bottleneck developed introduced fluctuating We Zwanzig’s (21), 1). extending (Fig. by for fibrin dynamics ory in catch–slip provided interactions the We of important 17). interpretation (16, with structure-based application bond a states force the upon bound for interface increase of requirement lifetime association a formation affinity, in A:a of binding results tension-dependent mechanism the which (Fig. structural of tension revealed, to the was restructuring polymers resolve fibrin Dynamic of to 1G). response silico catch–slip in dual the and vitro in eluded has duality characterization. catch–slip detailed the of basis (20). structural models the other However, and model (19), sliding–rebinding model network 17), theoretically, hydrogen-bond (16, studied (18), model been two-state have the bonds including catch–slip link- The vinculin–F-actin (15). (13), and age attachments (14), kinetochore–microtubule recep- complexes and 12), microtubule–dynein factor (11, Willebrand GP1b von tor (10), FimH adhesin bacterial ulse nieAgs ,2018. 7, August online Published 1073/pnas.1802576115/-/DCSupplemental. at online information supporting contains article This 3 2 1 the under Published Submission. Direct PNAS a is article This interest. of conflict J.W.W. no and declare D.T., authors V.B., The K.A.M., A.Z., O.K., R.I.L., paper. and O.K., the data; R.I.L., wrote analyzed model; V.B. the and developed D.T. K.A.M., and A.Z., V.B., exper- J.W.W. O.K., the simulations; and performed performed R.I.L. R.I.L. O.K. simulations; research; iment; designed V.B. designed and J.W.W. O.K. experiment; and the V.B., designed O.K., R.I.L., contributions: Author ent .Marx A. Kenneth , owo orsodnemyb drse.Eal [email protected] of Email: addressed. valeri University or be Engineering, may correspondence and whom To Science Materials 94720.y CA of Berkeley, California, Department address: Present ...adOKcnrbtdeulyt hswork. this to equally contributed O.K and R.I.L. lt ntecirculation. blood the nascent in of breakup clots polymerization prevent might fibrin and flow favor blood under non- might bonds of knob-hole strengthening bio- covalent structural shear-enhanced fibrin response, The of quanti- force aspect nanomechanics. important catch–slip theory an biphasic Our comprises the which bond. for knob-hole accounts tatively the of tension-activated strengthening behav- of mechanism with catch–slip structural dual the bond) resolved rare We (slip ior. a decreased displaying thereby then of force, and tensile properties bond) mechanical (catch first bonds increased the knob-hole of “knob- strength determine the noncovalent Counterintuitively, clots. that by prevent driven bonds that is clots hole” blood formation of Fibrin scaffold the bleeding. is network fibrin The Significance ee ecmie igemlcl ocdubnigassays unbinding forced single-molecule combined we Here, [email protected]. PNAS NSlicense. PNAS c eateto hmsr,Uiest fMassachusetts of University Chemistry, of Department | c aeiBarsegov Valeri , uut2,2018 21, August y y | o.115 vol. www.pnas.org/lookup/suppl/doi:10. c,d,3 .Thirumalai D. , b nttt of Institute | o 34 no. | 8575–8580 f ,

CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY To distinguish the A:a binding from other interactions, we performed control experiments. Nonspecific surface-to-surface adhesion showed signals with τ < 0.03 s. Specificity was tested by coating the surfaces with proteins lacking knobs and holes [bovine serum albumin (BSA)] or lacking knobs ‘A’ and ‘B’ (Fg). The control interactions (Fg/BSA, Fn/BSA, and Fg/Fg inter- faces) revealed mostly (93–97%) short-lived signals with τ < 0.03 s and a small fraction (0.6–2.5%) of more stable inter- actions lasting 0.04–0.5 s (SI Appendix, Table S1). The bond lifetimes of control background interactions did not show depen- dence on force (Fig. 2A). To distinguish the A:a binding from other associations, we measured the Fg–Fn interaction in the absence/presence of the GPRPam peptide (knob ‘A’ inhibitor). GPRPam suppressed specific A:a knob-hole interactions lasting >0.5 s (SI Appendix, Fig. S2A). The binding probability for these interactions was several fold higher in the Fg–Fn system than in control systems, including Fg–Fn interactions inhibited by GPRP (SI Appendix, Fig. S2A and Table S1). To test whether the intermediately strong interactions with 0.03 s < τ < 0.5 s contribute to the observed dependence of hτi on f , we also compared the bond lifetimes obtained either by including or excluding these intermediate-strength interac- tions to or from the datasets. The profiles of hτi (SI Appendix, Fig. S2C) and the cumulative binding probability vs. f (SI Appendix, Fig. S2D) were similar with a corresponding small shift to shorter bond lifetimes (SI Appendix, Fig. S2D) and to a higher binding probability (SI Appendix, Fig. S2C). We con- cluded that the bond lifetimes τ > 0.5 s represented specific Fig. 1. Major steps and main structural determinants in fibrin polymer- Fg–Fn or A:a knob-hole interactions. We included these data ization. ( – ) Cleavage by thrombin of the fibrinopeptides A and B (FpA A C in subsequent analyses and modeling and discarded the bond and FpB) in fibrinogen (A) converts it into fibrin with exposed knobs ‘A’ and ‘B’ (B), which bind to hole ‘a’ in the γ-nodules (blue) and ‘b’ in the β- lifetimes τ < 0.5 s. nodule (green), facilitating fibrin oligomerization (C). (D) Fibrin fragments Bond lifetimes of the D:E complex as a function of tensile force. used in dynamic force assays in vitro and in silico. (E) Atomic model of To minimize the role of nonspecific interactions due to the a fibrin oligomer fragment (2) with the α-, β-, and γ-chains colored in large size of full-length Fn/Fg molecules, we replaced Fg with its orange, green, and red, respectively. The β- and γ-nodules are in light blue smaller proteolytic fragment D, containing the globular portion and light gray, respectively. (F) A:a knob-hole complex containing the γ- (with hole ‘a’) and Fn with fragment E comprising the central nodule with hole ‘a’ and the N-terminal part of the α-chain with knob ‘A’ globule. Fragment E exists in three variants: (i) bearing both (orange); hole ‘a’ is formed by domains A, B, and P (3); the three-stranded knobs ‘A’ and ‘B’ upon cleavage of fibrinopeptides A and B with β -sheets stack in B domain is shown in purple. Loop I (blue), interior region ii (green), and movable flap (red) interact with knob ‘A’ (4). In simulations, thrombin (fragment desAB-E); ( ) bearing only knobs ‘A’ upon the tensile force was applied to αCys36, and γLys159 was constrained. (G) cleavage of fibrinopeptide A with batroxobin (fragment desA- Schematic of the energy landscape for A:a knob-hole interactions. Tension E); and (iii) having no knobs if uncleaved (fragment E) (Fig. perturbs the equilibrium LR1 ↔ LR2 between the low-affinity bound state 1D). Specific interactions between fragments D and desAB-E LR1 and force-stabilized high-affinity bound state LR2 changing the kinetics also displayed the catch–slip character with hτi increasing with f of forced rupture, LR1,2 → L + R. up to f = 30–40 pN and then decreasing for f > 40 pN (Fig. 2B). The bell-like curve of hτi was quite similar to that for the Fg:Fn interactions (Fig. 2B), but with shorter average bond-lifetimes. Results The dual catch–slip behavior was largely suppressed with the A:a Knob-Hole Bond Dissociation Under Tensile Force. addition of GPRP (SI Appendix, Fig. S2E), and it disappeared Fibrin–fibrinogen bond lifetime as a function of constant ten- sile force. The A:a knob-hole interactions were probed at an interface during repeated touching of two microbeads coated covalently with fibrinogen (Fg), a source of holes ‘a,’ or monomeric fibrin (Fn), a source of knobs ‘A’ (SI Appendix, Fig. S1). By touching the Fn-coated surface with the Fg- coated surface, we allowed these molecules to form the A:a knob-hole complex (binding phase). Next, the surfaces were retracted by applying a constant tensile force to dissociate the A:a knob-hole bond (unbinding phase). Repeating the binding– unbinding cycles, we measured the lifetimes of Fg:Fn bonds, from which the force (f )-dependence of A:a knob-hole bond lifetimes was obtained (SI Appendix, Fig. S2A). The distribu- tions of bond lifetimes P(t; f ) were collected for f =10–60 pN (SI Appendix, Fig. S3). These were used to calculate the aver- Fig. 2. Kinetics of rupture of A:a knob-hole bonds from single-molecule age bond lifetimes hτi, which first increased with f up to f = experiments. (A) Average bond lifetime hτi vs. force f for Fg:Fn interactions without and with Ca2+ (3 mM) and for control protein pairs both lacking 30–35 pN and then decreased for f > 40 pN (Fig. 2A). Impor- knobs ‘A’ (Fg:Fg) or one of interacting proteins (BSA) or lacking knobs ‘A’ tantly, the addition of 3 mM CaCl2 weakened the A:a bond, and holes ‘a’ (Fg:BSA and Fn:BSA). (B) hτi (>0.5 s) vs. f for fragment D (holes and the nonmonotonic dependence of hτi on f disappeared ‘a’ and ‘b’) coupled to fragments desAB-E (knobs ‘A’ and ‘B’) or desA-E (knob (Fig. 2A). ‘A’), or E (no knobs).

8576 | www.pnas.org/cgi/doi/10.1073/pnas.1802576115 Litvinov et al. Downloaded by guest on September 27, 2021 Downloaded by guest on September 27, 2021 hrb omn diinlbnigcnat ewe residues between contacts , the binding γ toward additional translocated forming and thereby extended flap mov- the movable in between the domain density B contact and in flap increase able an also see 3); Fig. n otcsbtenko A n h niebniginter- binding entire the and ‘A’ knob con- face, with between simulations contacts performed ing we nanoswitch. flap, force mechanical movable stant the a of is role flap Movable residues Appendix, forma- between to contacts leading binding γ straightening, additional I loop of triggered tion This 3). state Fig. bound in high-affinity the stabilized which state bound (low-affinity open was interface LR binding the S5C), neg- When charged positively 3). γ with interacted Fig. flap in movable tension- the 2b a and charged as 2a atively serves (snapshots which interface the of flap, binding opening/closing the movable triggering element the regulatory sensitive by controlled from dissociation is states along bound accessed of complex the profiles knob-hole the A:a the shows of 3 prolonged Fig. of tension. origin the to linked state is bond). (catch which bound lifetimes force, high-affinity by the stabilized from dissociation bound the low-affinity the from rupture state bond the unlike that, found o of ior flap, movable the time contacts knob-hole the of of in maps function the (residues changes analyzed a instantaneous also We reflecting as strength. bond affinity ‘A’ binding of knob sure and ‘a’ contacts, binding hole persistent of ber bond. catch the for basis Structural .Hne oc tblzsa lentv on tt not state Appendix, bound (SI alternative an at respectively stabilizes populated trajectories, force 10 Hence, the S4B). Fig. of 30% and 70% speed at pulling tured the For B). ivnve al. et Litvinov trajecto- of 20% remaining the in For while at dissociated runs, bonds of the ries, 30% in pN 90 higher vs. scenarios lower unbinding at force dissociation Two molecular slower 1E). vs. faster (Fig. by complex characterized knob-hole A:a force of ramped using simulations f performed we silico. findings, in mental measurements force Interactions. Dynamic Knob-Hole A:a of Modeling Molecular A:a was interactions. the behavior knob-hole A:a reflected catch–slip specific they observed to due the and Hence, specific, fragments bonds. are and knob-hole D desA-E evi- fragment and provides between desAB-E This interactions 2B). the (Fig. that D dence fragment and with A nonreactive fibrinopeptides (Fig. was uncleaved its fashion B with catch–slip preserved E fragment the ‘B’) intact in The D knob 2B). of fragment (without with interact Exposure desA-E to fragment ability bonds. in knob-hole ‘A’ knob the frag- reflect they of of that and Appendix, formation–dissociation density specific are interactions surface (SI D/desAB-E the that in D reduction ment substantial a following (t Asp297–γ l33i opIadresidues and I loop in Glu323 the of Arg275 ecaatrzdcnomtoa rniin nhl a under ‘a’ hole in transitions conformational characterized We ) 1 n iscaino nb‘’wsrpd(npht ain 2a (snapshots rapid was ‘A’ knob of dissociation and ) ν n h tmcsrcua model structural atomic the and Methods) and (Materials LR f Q 10 = Q ihtednmc fcnat ewe nb‘’and ‘A’ knob between contacts of dynamics the with , 1 F γ .A eut h oe‘’closed. ‘a’ hole the result, a As S5D). Fig. and ligand; (L, Ser240–γ h34i h a and flap the in Phe304 4 ≈ f f /,tebnsyeddat yielded bonds the µm/s, = 0p n5%o h 0smlto usadat and runs simulation 10 the of 50% in pN 60 0 = Q β 0p.W orltdtednmc fbind- of dynamics the correlated We pN. 30 γ F MF IAppendix, (SI domain B the in stack -sheet Asp291, . LR Q IAppendix, (SI observed were Lys380; a u otetligbc n ot of forth and back tilting the to due was R MF 1 eetr,tesrcue cesdalong accessed structures the receptor), , h bevdnnoooi behav- nonmonotonic observed The . and IAppendix, SI γ i.S2F Fig. ν Asp294, IAppendix, SI F f LR 10 = ≈ i2 nko A (SI ‘A’ knob in αGlu22–His24 2 IAppendix, (SI pN 70 The . emntrdtettlnum- total the monitored We l2 nko ‘A’, knob in αGly17–Glu22 3 Q .Teerslsindicate results These ). γ /,teAabnsrup- bonds A:a the µm/s, ewe h eiusin residues the between , s27 and Asp297, F ne tension, Under S5C. Fig. nprdb u experi- our by Inspired F LR and i.S5 Fig. ≈ 1 → oeuiaethe elucidate To LR Q 0ad10p in pN 130 and 90 t LR . 2 n structures and i.S4 Fig. C Q γ saso 2b (snapshot 2 and γ r26and Arg256 s31in Asp301 LR samea- a is i.S4A). Fig. transition γ Lys321– .We D). 2 A were Fig. and lce h oal a oin hc ih upesthe any suppress of absence might the with which well motion, agreed This flap formation. catch-bond movable the blocked Ca binding: calcium upon and changes structural significant Ca without and with ‘a’ hole γ Ca structures, seven site In binding 2). ref. in S1 the of site structures Ca-binding crystal weak and flap Movable Structures. Crystal of Analysis 3). (Fig. correlated (state positively increase were affinity translocation flap binding Appendix, the (SI the back in Hence, tilting decrease S5D). flap the the Fig. to with corresponded which correlated nm, was 1.3 17 to Appendix, periphery 25 (SI the ‘A’ from knob translocation the flap to movable the to responded a by in D γ and flap movable the and Appendix, flap We 3). the (Fig. elongation between forces higher flap at the bond formed quantified A:a the ‘A’ be- of strength knob enhanced contacts and binding γ flap Additional movable the (γ αPro18, domain. the with tension in B interactions under residues off the and tween of on Appendix, switching stack (SI flap flap the movable the bound High-affinity 2a). structure in 2b). structure arrow (structure (red inter- state domain dissociated through state B to of 1) and stack (structure state sheet 2b) state bound and Low-affinity native 2a 3). the (structures state from mediate rupture of of progress evolution tonic/nonmonotonic state ity (Upper complex. 3. Fig. s28 and Asp298, and Gly283, and Phe303–αTrp33, MF Q 2+ Q γ h nraein increase The . LR ec,teXrydt niae htC-idn to Ca-binding that indicated data X-ray the Hence, . .-odices in increase ∼1.5-fold LR gen s time vs. (green) safnto ffreadcreae hne in changes correlated and force of function a as s31rttdaa rmko A,frigcnatwith contact forming ‘A’, knob from away rotated Asp301 2 1 nefc eoeigadfre utr fteAaknob-hole A:a the of rupture forced and remodeling Interface h oal a oe oadcthsko A rdarwin arrow (red ‘A’ knob catches and to moves flap movable The : /LR γ Asp301–αArg19, .W eetdtrepiso eiusi the in residues of pairs three selected We S5B). Fig. 2 γ scaatrzdb oe/ihrvle of values lower/higher by characterized is 2)codntdb residues by coordinated (22) 2 γ rfie frpueforce rupture of Profiles ) γ Asp298–γ s31i h oal a.Tesrcue of structures The flap. movable the in Asp301 PNAS Pro299–αVal20, β t setstack: -sheet rmtefrerm iuain.Rpuefrom Rupture simulations. force-ramp the from Q γ | a2)wr seta o the for essential were Phe304–αVal20) γ rm1–0t 53 a accompanied was 25–30 to 15–20 from LR uut2,2018 21, August l24 o ahpi,w calculated we pair, each For Gly284. oueo ua bi(gn (table fibrin(ogen) human of nodule D 1 2+ β γ h oal a neat ihthe with interacts flap movable The : MF Lys302–αGlu22, setsako h oan(SI domain B the of stack -sheet .Tedces in decrease The S5B). Fig. in (Lower Q. D γ rm15t . m hc cor- which nm, 2.3 to 1.5 from MF revealed 4E, Fig. in overlaid 2, γ .Ti eutdin resulted This S5B). Fig. Gly296–γ γ 2+ Ser300–αGly17, ymauigtedistance the measuring by si h o-fnt Ca- low-affinity the in is npht – hwthe show 1–3 Snapshots ) | F γ 2. o.115 vol. rd n idn affin- binding and (red) eaaye l 27 all analyzed We γ Ala282, γ Asp294, D Phe303–αPro18, MF | o 34 no. F rm23to 2.3 from γ n mono- and LR γ D γ γ Asp297– Ser300– Gly296, MF Q β Asp297 2 -sheet | and ) from 8577 and γ β 2 -

CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY Hole ‘a’ is a fluctuating bottleneck. Force affects the dynam- ics of hole ‘a’ closure and the kinetics of knob-A escape. The bond lifetime t and bottleneck width X are coupled random variables (Fig. 4E), described by the Smoluchowski equation dP(X ,t) for the joint distribution, dt = [L(X , t) − K (X )]P(X , t) (SI Appendix, Eq. S7), where the rate K (X ) = kX 2 depend- ing on rate constant k(f ) = k0 exp[σy f /kB T ] (26) (k0-attempt frequency and σy -transition distance for rupture) represents the kinetics of knob ‘A’ escape from the bottleneck, and in h i ∂ κ kB T ∂ the operator L(X ; t) = ∂X ζ (X − hX i) + ζ ∂X the first and second terms describe tension dependence and fluctuations of X , respectively (ζ-friction coefficient). This equation can be solved as described in SI Appendix to obtain the Green’s function solu- tion G(X , X0; t). The distribution of bond lifetimes is P(t) = R ∞ R ∞ 0 dX 0 dX0G(X , X0; t)P(X0), and the average bond life- R ∞ time is hτi = 0 dtP(t)t. However, the millisecond timescale of bottleneck fluctuations (η = ζ/κ; κ-interface stiffness) is much shorter than the bond lifetimes, and the initial distribu- tion P(X0) = δ(X0 − x0) is peaked around a constant value x0. 2 Under these conditions, P(t) = khX i2e−khX i t , where the aver- −t/η −t)/η age width is hX (t)i = [x0e + (x0 − f /κ)(1 − e )]Θ(fc − f ) + x1Θ(f − fc ) [Heaviside step function Θ(x) accounts for the excluded volume interactions at a critical force fc ]. Theory quantitatively explains force dependence of A:a bond life- times. A fit of the theoretical predictions of hτi vs. f calculated with the FB model to experimental data (Fig. 2B) showed very good agreement (Fig. 5). The model has five parameters [fric- tion was set to ζ = 10−3 pN nm−1s (diffusion of amino acids in water)]: interface stiffness κ=16.7 pN/nm, force-free inter- face width x0 = 2.8 nm (in state LR1), minimal interface width 2 −1 (in LR2) x1 = 0.6 nm, escape rate constant k0 = 0.11 nm s , and transition distance σy = 0.25 nm. Of these, κ, x0, and x1 can be directly accessed in the simulations, which reveal: κ = 10 − 30 pN/nm, x0 = 2.0 − 2.8 nm, and x1 = 0.5 − 0.9 nm. The critical force fc ≈ 33 pN agrees well with the experimental 30- to 35-pN Fig. 4. Structural basis of the FB model of A:a bond rupture. (A) Snapshots range (Fig. 5), thus justifying the proposed mechanisms for the of the A:a knob-hole complex with hole ‘a’ open (low-affinity state LR1) catch bond. With these parameters, the distributions of bond and closed (high-affinity state LR2). (B and C) The interface width X from two representative simulation runs, which resulted in bond rupture from state LR1 (B) and LR2 (C). B and C, Insets show probability distribution P(X), sampled over a 0.1-µs time window. The shift of P(X) over time (shown by arrows) reveals the continuous evolution of X.(D) Hole ‘a’ modeled as a FB of width X defined to be the radius of a circle circumscribing the trian- gle formed by residues γAsp297, γGlu323, and γAsn361. Under tension, X decreases from x0 (LR1) to x1 (LR2), which makes the rupture rate K depend on X.(E) Crystal structures of hole ‘a’ in the γ-nodule with (gray-blue) and without Ca2+ (gray) in the γ2 binding site in the movable flap (boxed) and the GPRP-terminal part of knob ‘A’ (orange). Upon binding of Ca2+ (cyan ball), γAsp297 in the movable flap rotates by 70◦ and shifts by 5 A˚ away from the GPRP-peptide (terminal part of knob ‘A’; red arrow). Also shown is γAsp301 mutated to His in recombinant Fg Finland P21.

catch-bond regime in the Fg:Fn bond rupture kinetics in 3 mM CaCl2 solution (Fig. 2).

Fluctuating Bottleneck Theory. A:a interface remodeling. We analyzed the evolution in interface width X , defined as the radius of cross-sectional area formed by residues γAsp297, γGlu323, and γAsn361 in the movable flap, loop I, and interior region, respectively (Fig. 4). In state LR1, X fluctuated between 1.3 and 2.5 nm (open hole ‘a’; Fig. 4B); in state LR2, X decreased from 1.3 to 0.8 nm (Fig. 4C), implying Fig. 5. Catch–slip force response of A:a knob-hole bond. The nonmono- that the bottleneck is formed when hole ‘a’ closes. The profiles of tonic behavior of the average bond lifetime hτi as a function of tensile distribution P(X , t) (Fig. 4 B and C), corresponding to the evo- force f (Fig. 2) is modeled by using FB theory. Snapshots show evolution of lutions of X , shifted to larger X (rupture from LR1) or smaller the A:a knob-hole complex from the low-affinity bound state LR1 populated X (from LR2). Hence, hole ‘a’ was a bottleneck (21, 23–25) with at low forces (f <10 pN) to the high-affinity bound state LR2 stabilized at tension-dependent width X . higher forces (f ≈30–35 pN).

8578 | www.pnas.org/cgi/doi/10.1073/pnas.1802576115 Litvinov et al. Downloaded by guest on September 27, 2021 Downloaded by guest on September 27, 2021 .-clmlvlefrstate for value −7.1-kcal/mol con- binding disrupted of number total tacts the counting by mated the of S3 curves theoretical and experi- ‘a’ data between mental of agreement hole good multitude math- Very a and a states. for bound into translocation high-affinity evidence mapped provides flap were which the framework, observations ematical capture These model 4). to FB (Fig. 29) the closure 24, extended We 23, remodeling. (21, interface binding the S6 bond state Fig. bound to low-affinity correspond the which from observed, dissociation were to forces 60- rupture Low 90-pN (27). thrombosis with associated hypofibrinogenemia P21; (Finland flap con- able mutation with with variants and not flap fibrinogen movable is silico strained assays in single-molecule reconstituted catch-regime We for a feasible. necessary of amounts CaCl in absence mutants mM the explains 3 which Ca in blocked, coordinate is flap tion movable the upon in flap mecha- movable the this Ca in for showed alterations which evidence fibrin(ogen), structure of Additional large-amplitude structures crystal 4). from A:a (Fig. came nism the and closing translocation, of flap ‘a’ movable remodeling hole the to dynamic due from interface, association results A:a of the slip-regime bond of the strengthening knob-hole Tension-induced to 3). catch-regime (Fig. rupture the bond from cross-over the gers Ca mM suppressed. was 3 behavior of were catch-bond ‘B’ presence the and and the catch– weakened, ‘A’ In The knobs 2). 2). both (Fig. due when (Fig. exposed enhanced was bonds was knob-hole Control behavior transition single slip catch–slip 1). of the (Fig. dissociation to that polymerization of nonco- confirmed fibrin dynamics strongest experiments in catch–slip bonds—the knob-hole interactions of A:a valent basis of structural rupture forced the we revealed theory, and have simulations, experiments, single-molecule Using Discussion ec,tebnigfe-nrydfeec ewe h catch- the between is difference slip-bond free-energy and binding the Hence, For simulations. from range For bonds). hydrogen and N contacts, electrostatic polar, the over integrating by estimated near-equilibrium, bond, A:a for force(F the energy rupture binding to the assays, solution is in populated not for the energies theory, (κ/2)(x FB binding From in distance). state difference transition equation; and rate differential (escape stochastic dependence (through temperature Appendix, and rupture friction bond binding describe of cou- the to of of (ii) interface fluctuations pocket); the (conformational at biomolecules changes pled structural dynamic quantify to experimental of lifetimes ivnve al. et Litvinov Ca lower at time clotting Ca the of effects the with sistent the eea oeso ac-odeit(8,btnn con for account none but (28), exist catch-bond of models Several trig- which sensor, force molecular a is flap movable The u eut nteefcso Ca of effects the on results Our bond, A:a the For and ) 15 = 2+ ∆G . clmlfo Btheory. FB from kcal/mol −5.9 N idn Fg 4E (Fig. binding ). = × 0 )-distance(X and hτ − P . clml rmpligsmltos h work the simulations, pulling From kcal/mol. −7.1 .5ka/o eeg/otc vrgdoe nonpolar, over averaged (energy/contact kcal/mol 0.75 omptefe nrylandscape energy free the map to iii) ( and S7); Eq. (t i x 1 Fg )vldtdtemdl Bter ep:(i) helps: theory FB model. the validated 5) (Fig. ) ∆ ) 2 opr elwt oprmti est profiles density nonparametric with well compare G = 2 P ∆∆G = (t . kcal/mol. −5.9 ouin bann eobnn fibrinogen recombinant Obtaining solution. ∆ ) 12ka/o.Hne h experimental the Hence, kcal/mol. −11.2 G uv,is curve, ) for = and = IAppendix, SI K f . kcal/mol. −5.1 7375 clml hc scoeto close is which kcal/mol, −(7.3–7.5) d = = IAppendix, SI 2+ LR IAppendix, (SI pN 10–40 LR 2+ 5.8 ncotn.Climin shorten ions Calcium clotting. on 2 1 ees(0,bta ihrCa higher at but (30), levels , . clml n assuming and kcal/mol, −5.1 .Asmn that Assuming µM. ∆G si the in is 2+ LR 2+ ,wihlasto leads which S6), section nteAabn r con- are bond A:a the on n h a transloca- flap the and , 1 = γ and P 1.–87 kcal/mol. −(12.4–18.7) ∆ s31i ntemov- the in Asp301His .ApadGly and Asp S7). Fig. (t 511.)kcal/mol −(5.1–11.2) G ) LR LR IAppendix, (SI a lob esti- be also can 2+ 2 1 h : bond A:a the , IAppendix, (SI is i.S3 Fig. ∆∆G LR LR LR = ). 2 Fig. 2+ SI − 1 is 1 , h edwste eue ni hytuhdec te eetdywt a with repeatedly other each touched they and until pedestal reduced the the then of and was separation bead the trapped, the oscillation, was bead the 1–2 bead starting within pedestal After single a bead. a bring chamber to the stage, manually moved After microscope was surface. stage the their on on placed E fragment was or fibrin immobilized with (10 suspension same 50 bead the D-coated CaCl in fragment mM 3 out or of addition carried the with were reduce but to experiments buffer added X-100 Some containing Triton 7.4) (vol/vol) interactions. 0.1% (pH and nonspecific Hepes BSA, mg/mL mM 2 20 NaCl, in mM 150 temperature room at performed in were described are Interactions. preparation Protein–Protein protein of Measurements analyze and stage, piezoelectric the move off-line. deflection, data used was beam software laser LabVIEW record method. to force Stokes’ the with confirmed were (bond proteins of stiffness surface-attached trap separate to required lifetime) time The stiffness). compres- keep of force surfaces to duration interacting the loop bead of between feedback control contact the electronic enabled sive on system an This trap with constant. force corrected the the was by position exerted trap force the amount The an position. by trap displaced used the was control deflector acousto-optical to 2D computer-operated (λ A an mode. laser was TEM-00 Nd:YAG system FCBar trapping optical a 60× the with a of and core microscope inverted subject used The Z1 force. was complexes AxioObserver tensile 38 protein–protein constant ref. of a in strength to described mechanical trap the force optical measure piconewton custom-built to the A polystyrene generate micrometer-size 36–40). as to (5, such beam particles, beads microscopic laser move focused and hold a to uses that S1) Fig. Trap. Optical Methods and Materials an clotting. blood is of fibrin understanding in our structure–function, advances bond fibrin which catch of the aspect mechanochemical of their important discovery is The fibrin since basis. thrombi for structural pathological relevant and is clots mechanism hemostatic ischemic rupture), catch-bond all causing The (clot vessels, example. smaller embolization for in strokes, prevent mech- lodge range thrombi to catch-bond which the help The during in also bonds. is could stress knob-hole anism shear the incipient hydrodynamic strengthens the the is that when and fibrin This formation, small favors is fibrin clots. clot during stressed that on of bonds early damage important knob-hole prevents A:a and polymerization the blood to of system strengthening regulatory physiological a of formation. part clot Ca a thermody- Ca low-affinity be a and the that might kinetically In that notion both and flow. the formation namically blood support fibrin data of modulate our forces inter- can context, knob-hole the general A:a under the more catch-bonds enhancing pre- via by clotting clot actions of fibrin incipient site [Ca the low the formation, at fibrin aggregation cedes Ca platelet strong [Ca Ca local Because to of the due (35). clotting, absence of low site the be the in at might that bonds fact knob-hole the rel- by A:a physiological reinforced the The (34). of formation evance fibrin of Ca initiation Ca a the precede that for bonds implying knob-hole fibrin, A:a by initial uptake the of formation Ca and low at stronger [Ca are at abolished [Ca is at tion fibrin inhibits Ca GPRP with (32). interferes of peptide GPRP Binding mimetic (31). knob-A slowed the is polymerization fibrin concentrations, ucinly h ac-odbhvo ed shear-enhanced lends behavior catch-bond the Functionally, ftewrigbfe n oe noacabrcnann pedestals containing chamber a into flowed and buffer working the of µL f c n h antd ftetnieforce tensile the of magnitude the and τ a esrd xeiet eecnutdwt h average the with conducted were Experiments measured. was IAppendix, (SI trap optical an on based is system model Our k opt = PNAS 0.19 ∆ ± 2+ z | 2+ .2p/m oc airto n rpstiffness trap and calibration Force pN/nm. 0.02 a esrdwt udatdtco,and detector, quadrant a with measured was uut2,2018 21, August ees(3.Teepsr fkosA knobs of exposure The (33). levels = 10 = ] ,6 m ih4Wpwri continuous in power 4-W with nm) 1,064 2+ 2+ IAppendix, SI −5 2+ ih rmt omto of formation promote might ] paeb ciae platelets activated by uptake 2 10 = ] 2+ T oa f1 of total A . ,bcueteAabonds A:a the because M, h antd fcompressive of magnitude the , 7 fe eimi favorable is medium -free ed e L a de to added was mL) per beads 2+ f | .-AFurln combined lens Fluor 1.3-NA = bnigstsi fibrin in sites -binding ufc oicto and modification Surface o.115 vol. −3 k Experiments S1. section opt ,btteinhibi- the but M, ∆z fteFg-coated the of µL ftetrapped the of µm (k | opt o 34 no. pia trap optical , 2+ binding | 2+ 8579 2+ 2+ 2+ is ]

CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY compressive force fcomp = 20–30 pN and contact duration T = 0.5 s. The con- mational space (42). In force-ramp simulations, we used time-dependent 3 stant pulling force was varied from f = 5–60 pN. Trap displacement signals pulling force f(t) = kopt (νf t − ∆x), where νf = 10 µm/s is the virtual pulling were recorded at 2,000 scans per second, and a bond lifetime was measured speed, kopt = 100 pN/nm is the trap spring constant, and ∆x is the displace- for each pedestal–bead-touching event. Several tens of pedestal–bead pairs ment of a pulled residue. In force-clamp runs, we used constant tensile force were analyzed for each set of conditions. The binding–unbinding events from f = 30–80 pN (Fig. 1G). We constrained the Cα-atoms of γLys159 and tagged individual files were summarized; the total number of bond lifetime val- the Cα-atom of αCys36 (Fig. 1G). ues recorded for each set of experimental conditions varied from ∼3,000 to ∼4,500. The bond lifetimes <0.5 s represented nonspecific interactions and ACKNOWLEDGMENTS. We thank Dr. Henry Shuman, who built the optical were not susceptible for specific inhibition. These short bond lifetimes were trap instrument with force-clamp modification, for his help with exper- not included into data analysis and modeling. iments, data processing, and analysis; and Andrey Mekler for technical assistance. This work was supported by NSF Grant DMR 1505316 (to J.W.W. Dynamic Force Measurement of the A:a Bond Rupture in Silico. The atomic and V.B.); American Heart Association Grant-in-Aid 13GRNT16960013 (to V.B. and J.W.W.); NIH Grants UO1 HL116330 and RO1 HL135254 (to J.W.W.); structure of the A:a knob-hole complex is described in SI Appendix, sec- Russian Foundation for Basic Research Grants 15-01-06721 (to A.Z.) and 14- tion S3. We used all-atom Molecular Dynamics simulations on a GPU using 04-32066 (to O.K.); Russian Science Foundation Grant 17-71-10202 (to A.Z.); the Solvent Accessible Surface Area model of implicit solvation with the the Program for Competitive Growth at Kazan Federal University; National CHARMM19 force field (41). We used a lower damping coefficient γ = 3.0 Institutes of Health Grant R01 GM089685 (to D.T.); and Collie-Welch Chair ps−1 for faster system equilibration and efficient sampling of the confor- F-0019 (to D.T.).

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