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(11, interactions intact the fully the investigate and are to interactions and models CaM–peptide interactions these excellent that CaM–target suggest studies explore several to Alternatively, used experiments. method, often domains are the CaM-binding crystallization to in corresponding the sequences resolution and peptide short spatial NMR the by to study due to target difficult with CaM are of complexes However, (10). resolve structure to performed been have crystallography understanding experiments X-ray for crucial recognition. is multispecific targets protein–protein different of the mechanism to binding molecular CaM the Exploring (7–9). targets com- 300 binary The (7–9). motility plex in involved and protein pumps, kinase, including dinucleotide synthase, adenine proteins oxide nicotinamide kinases, of nitric protein kinase, kinds triphosphate many inositol of phosphatase, activity the cellular incorporating ulates of With range pathways. wide ing a in involved is (2–6). recognition roles protein critical in the flexibility “induced addressing of as selection,” referred “conformational are and considering and fit” the scenarios, emerged two describe binding, (1), during to catalysis flexibility Fischer enzyme in by binding proposed rigid “lock-and- bio- the mechanism, to in addition binding In role key” binding. critical of also viewpoint a dynamics new a conformational have provide or to flexibility This recognized function. been logical long have teins M recognition multispecific model structure-based CaM. of provide binding can multispecific skMLCK into and insights CaM asso- for These proposed targets. characteristics its ciation and CaM discussions between our binding extend multispecific we on Finally, the binding. selection,” and of folding of “conformational synchro- mixture nization the atypical on a depending binding–folding,” the with “simultaneously and landscape fit,” a “induced found atypical We skMLCK the peptide. of changes binding conformational global and CaM of conformational asso- change local this both transi- Furthermore, simultaneously the specificity. involves for high process crucial ciation to are affinity anchors CaM high hydrophobic of from conserved patches tion its binding the and and that skMLCK CaM found of between also recognition We nonspecific target. in an role plays electro- interaction important nonnative hydrophobic nonnative between binding and cooperation interaction skMLCK static the and found CaM We between to peptide. process model binding structure-based the a developed binding explore we protein–protein Here, to understanding recognition. CaM for and of crucial are mechanism targets association multiple its the bind to on capability Investigations the have targets. to found is (CaM) Calmodulin Jos by Edited and 43403; 130022; OH China 11794-3400 Green, of NY Bowling Republic Brook, University, People’s Stony State Jilin, York, Green Changchun, Bowling Sciences, Chemistry, of of Academy Department Chinese Chemistry, Applied of Institute a olg fPyis ii nvriy hncu,Jln epesRpbi fCia130012; China of Republic People’s Jilin, Changchun, University, Jilin Physics, of College amdln(a)i nubiquitous an is (CaM) Calmodulin Ca idn.Telresaedmi eragmnsi pro- protein–protein in rearrangements by domain driven large-scale are The binding. processes biological any a,b 2+ iknChu Xiakun , CMi on ohv h aaiiyt idover bind to capability the have to found is -CaM .Ouhc ieUiest,Hutn X n prvdMrh3,21 rcie o eiwOtbr3 2016) 3, October review for (received 2017 30, March approved and TX, Houston, University, Rice Onuchic, N. e ´ | Calmodulin a,b .PtrLu Peter H. , | itr idn mechanism binding mixture Ca Ca 2+ c Ca n i Wang Jin and , 2+ ions, 2+ idn rti that protein binding Ca dpnetsignal- -dependent Ca 2+ 2+ lae CaM -loaded CMreg- -CaM a,b,d,1 | Ca Ca 2+ 2+ - 1073/pnas.1615949114/-/DCSupplemental at online information supporting contains article This Submission. 1 Direct PNAS a is article This interest. of conflict paper. no the declare wrote authors F.L. J.W. The and tools; X.C., reagents/analytic F.L., and new data; contributed analyzed J.W. J.W. and and H.P.L., X.C., F.L., research; formed the the that select target we the as Here, peptide information (19–24). binding skMLCK quantitative mechanism and underlying insights the more toward gain chal- tool, to powerful the able a meet is as which To serve resolution. simulations dynamics temporal molecular or lenges, spatial of limits to due their methods, single-molecular and biochemical by understood of binding coupled and of process folding the mechanism for the adopt perspective However, thermodynamic and 18). global (17, and state CaM with free complex the the in tures in are analysis peptides coil the CaM-binding random hand, the mostly that other indicated the structures On static (16). on binding its C-terminal bound during only domain with state intermediate an possesses CaM per- we exper- on (FRET) Recently, transfer iment (10–16). energy peptide resonance S1) fluorescence target Fig. a the formed Appendix, C around the (SI wrapping with binding domain structure after N globular the compact and final domain the form conformational to to a adopts undergoes change CaM binding and peptide conformation, The dumbbell target obtained. a without be structure can CaM peptides loaded target binding and the (13–15). of CaM features (CaMKII) between structural II many experiments, kinase these CaM-dependent From and kinase skele- (CaMKI), CaM-dependent (smMLCK), (skMLCK), I kinase kinase chain chain light myosin light tal myosin muscle such smooth complexes, peptide–CaM used as of been structures numerous have resolve techniques to experimental crystallography X-ray uhrcnrbtos ...... n ..dsge eerh ..adJW per- J.W. and F.L. research; designed J.W. and H.P.L., X.C., F.L., contributions: Author owo orsodnesol eadesd mi:[email protected]. Email: addressed. be should correspondence whom To nihstwr utseicbnigo a ihistarget. its with CaM of provide binding can associa- multispecific skMLCK toward and The insights CaM specificity. for and proposed affinity characteristic tion recognition high the to during leading process, changes conformational fine-tune to multiple found the are lead process, association which complicated interactions, this CaM–peptide to underlying the “simulta- The “induced for process. and uncovered recognition atypical selection,” is the binding–folding” “conformational of neously atypical mixture the of a fit,” changes with conformational An landscape simulations. global energy molecular with the skMLCK conformational and peptide CaM-binding local CaM the of both change involves process simultaneously association the that studied we recognition, multispeci- mech- CaM molecular for the the anism solving address To for recognition. molecular crucial in is more ficity targets recruit to binding able 300 is than (CaM) Calmodulin how Understanding Significance b tt e aoaoyo lcraayia hmsr,Changchun Chemistry, Electroanalytical of Laboratory Key State d eateto hmsr n hsc,SaeUiest fNew of University State Physics, and Chemistry of Department Ca 2+ CMbnigt etd 2W hwn that showing C28W, peptide to binding -CaM PNAS Ca | 2+ ulse nieMy1 2017 1, May online Published CMt t agt antb fully be cannot targets its to -CaM c . etrfrPoohmclSciences, Photochemical for Center www.pnas.org/lookup/suppl/doi:10. 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BIOPHYSICS AND BIOCHEMISTRY PNAS PLUS COMPUTATIONAL BIOLOGY to. The binary binding complex skMLCK-CaM has been resolved and skMLCK occurs in these two transitions, leading to the fact by NMR techniques [Protein Data Bank (PDB) ID: 2BBM] SI that skMLCK increases its helicity only by binding to Ca2+-CaM. Appendix, Fig. S1B) and provides the structural basis for our sim- From the free energy landscape, we are able to address a ulation (13). The skMLCK peptide, with 26 residues in length, is mixture mechanism of coupled folding and binding of Ca2+- classified into the typical “1-5-8-14” scenario, which is named by CaM to skMLCK (Fig. 1D). In detail, the pathways O-I1-LB and the number of spacings of hydrophobic anchor residues (25). O-I3-LB correspond to the partial binding between Ca2+-CaM By developing a coarse-grained structure-based model, we and skMLCK happening before and after the partial closing of investigated the binding process of CaM to the skMLCK binding Ca2+-CaM, respectively. Additionally, in the pathway O-I2-LB, peptide. By explicitly taking into account the electrostatic and the partial closeness of Ca2+-CaM is accompanied by the partial hydrophobic interactions, we addressed the critical roles of the binding between Ca2+-CaM and skMLCK, following the simul- residues in CaM participating in tuning the binding from high taneous binding–closing (folding) mechanism. When the par- affinity to high specificity. The underlying binding mechanism tial binding and partial closeness intermediate LB is formed, all obtained from the quantified free energy landscape indicated the three pathways merge together to form one pathway. LB adopts association process is quite complex with the mixture of induced the simultaneous binding–closing mechanism only to form the fit, conformational selection, and simultaneous binding–folding. completely binding and completely folding state C. At the same This unique binding behavior, tuned by multiple conformational time, the skMLCK increases its helicity only when it interacts changes, is further suggested as the source of multispecificity with CaM. in CaM recognition. Our results provide a unique way to gain insights into the promiscuity involved in CaM recognition. Nonnative Electrostatic Interactions Act as a “Steering Force” to Facilitate the Binding Preference of CaM–skMLCK Recognition. Pre- Results vious structural investigations indicated that most of the Ca2+- Affinity and Flexibility Determine the Landscape of Coupled Fold- CaM–associated peptides have the propensity to form hydropho- ing and Binding of Ca2+-CaM to skMLCK. We explored the binding bic and electrostatic interactions at the binding interfaces (13, process with replica exchange molecular dynamics (26), which 15, 29–31). Taking these factors into consideration, the residues generated the free energy landscape along folding and binding of both CaM and skMLCK are divided into hydrophobic, elec- dimensions (Fig. 1A). The reaction coordinates “rmsd-1CLL” trostatic, and plain ones in our work (SI Appendix, Materials and “Qbinding ” were respectively used to monitor the structural and Methods). Both native interactions and nonnative interac- change of Ca2+–CaM and the binding degree between Ca2+– tions are supposed to play important roles in the protein–protein CaM and skMLCK. To have a clear description of how recogni- binding. The native interactions contribute to the binding affin- tion occurs, we identified three stable states (the O, C, and LB ity and specificity whereas nonnative interactions act on the ini- states) and three unstable regions (I1, I2, and I3) on the land- tial recognition before forming native interactions (32–35). The scape. The O and C states are open and closed states, corre- interchain nonnative electrostatic interactions are sometimes sponding to the target free state of Ca2+-CaM before binding regarded as the steering force to facilitate the protein recog- with both the N and C domains of the CaM open and bound nition (32–36). To see the role of native and nonnative inter- state of Ca2+-CaM with N and C domains wrapping around actions in Ca2+-CaM–skMLCK, we calculated the interaction skMLCK, respectively, whereas the LB state is an on-pathway energies and contact map at each state (Table 1 and SI Appendix, binding intermediate state (Fig. 1). We found that there are three Fig. S6). It is worth noting that the nonnative plain Lennard– parallel pathways going through the intermediate “LB” state Jones interactions are represented only by an exclusive volume from the transition between “O” and “C” states. Each pathway repulsive term. passes through the unstable regions “I1,” “I2,” and “I3,” respec- By exploring interaction energies during binding (Table 1), we tively, before reaching the LB state. We can see the LB state is found part of nonnative electrostatic interaction is formed and partly binding with Qbinding around 0.4 and partly closed with no nonnative hydrophobic interaction is formed in the state O. rmsd-1CLL at 1.15 nm (Fig. 1A). Quantitatively, the degree of This implies that the formation of nonnative electrostatic inter- closeness of the LB is about 3/4 relative to the final completely actions is before the formation of nonnative hydrophobic inter- compact globular state (Fig. 1C). Using the specific native con- actions and native interactions, illustrating again the role of elec- tact probability map to explore the structural features of the LB trostatic interactions as a steering force in Ca2+-CaM–skMLCK (SI Appendix, Structural Characteristic for Each State), we found recognition. Regarding the native interactions, we found that that the long helical structure of the central linker is broken they are first formed between the C domain of CaM and the and the bending of the linker contributes to the closeness of N terminus of the skMLCK and then between the N domain of Ca2+-CaM in the LB state. The N terminus of skMLCK mainly CaM and the skMLCK. The nonnative electrostatic interactions binds to the C domain and the linker close to the C domain of will contribute to the binding preference between C-domain– CaM (Fig. 1D) (SI Appendix, Structural Characteristic for Each CaM and skMLCK from the O to the LB state. This result is con- State). Our results provide a dynamical basis for understand- sistent with the fact that binding preference is strongly dependent ing the previous findings that the C-terminal domain of CaM on and adapted through the electrostatic interactions between has a higher affinity to target than the N-terminal domain (14, Ca2+-CaM and the targets (37). Most nonnative electrostatic 27, 28) and the high plasticity of the linker is determined by its interactions are formed in the transition from the O to the LB intrinsic flexibility (13–16). In addition, we used the distributions state (Table 1). E119, E120, and E123 in the C domain of CaM of rmsd-skMLCK for each state along the pathway to monitor begin to form nonnative electrostatic interactions with K1, R2, this dynamical process (Fig. 1B). We found that the distribu- R3, and K5 in the N terminus of the skMLCK in the state O. tions of rmsd-skMLCK vary in different states and regions. The E80 of the linker forms the nonnative electrostatic interaction distribution of rmsd-skMLCK for the C state is narrower and with R16 of the skMLCK and E47, D50, and E54 of N-domain smaller than for the O state and the I3 state. The distributions CaM begin to form nonnative electrostatic interactions with R3 for the I1 state and the I2 state are in the middle range (Fig. 1B). and K5 of skMLCK in the state LB (SI Appendix, Fig. S6A). This indicates that the skMLCK-binding peptide gradually forms The native interaction is hardly formed in the N domain of CaM α-helical structures from a random coil along the binding process in the LB state (SI Appendix, Fig. S6B). The nonnative electro- to CaM. The distributions of rmsd-skMLCK are almost the same static interactions contribute to the binding preference between in I1 and LB. The distributions of rmsd-skMLCK for O and I3 N-domain–CaM and skMLCK in the next dynamical binding step are also almost the same. No binding between the Ca2+-CaM from LB to C.

E3928 | www.pnas.org/cgi/doi/10.1073/pnas.1615949114 Liu et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 i tal. et Liu of change structural the monitor to 1CLL structure the to 1. Fig. nemdaeL.I stecnomto xrce rmteI ein h kLKde o idt h a n h nraigdge ftecoigof the closing is the C state. of O degree free increasing completely the the and to than CaM compared I2 less closed region. the are partly this closing to and in and bind binding binding state not partly both O does is of the degrees LB skMLCK with the intermediate the compared but The change region; CaM binding. not I3 of skMLCK does domain the further CaM Ca C for from of the door extracted to conformation the only conformation the opens binds the and CaM domain skMLCK CaM is N the of and I3 region; linker, domain therefore I2 LB. domain, C We the C intermediate respectively. the from the states, to extracted respectively C conformation only are The and the binds regions state. LB, is green skMLCK lines, O, free and the the black target blue, region; in and red, the I1 distance The green, is the simulation. C–N red, O the of The The from distribution colors. extracted CaM. the different landscape of for by the ((4.36 stand marked on LB nm, regions are in 2.60 unstable respectively CaM pathway and and regions. of pathway the nm, and closeness Each along 3.07 of states state states. degree different nm, the each at closed 4.36 intermediate estimated skMLCK for at to stable of distance” located open a distributions are “C–N from Conformational is peaks of (B) transition there whose distributions state. the states, The LB in C the closeness. state to and of LB getting O degree before intermediate the I3 the Besides and through landscape. I2, go I1, the (C regions pathways in unstable three regions the all unstable through that three passes found and We states state. stable LB three identified We binding. monitor fbt –2L n BCdces stennaiehy- nonnative the as nonnative decrease of increase LB–C the With increase. and interactions each drophobic O–I2–LB in O–I2–LB heights both for barrier are that of The O–I3–LB than strength. interaction higher and hydrophobic are O–I1–LB nonnative and for same heights the barrier 2A almost Fig. the 2. and that Fig. electrostatic in shows parameters different interaction in hydrophobic respectively, nonnative pathway, each non- along the in of interactions roles electrostatic Ca different and the LB–C interactions the explore hydrophobic during native To also signifi- but 1). change transition (Table O–LB interaction transition the in native only not each cantly from and Different hydrophobic energy S6A). nonnative Fig. interaction the Appendix, interactions, (SI native electrostatic CaM the nonnative in forming wide inter- is site hydrophobic nonnative actions the of distribution around the inter- and formed interactions hydrophobic mostly electro- nonnative the are the by actions found driven hydrophobic We stage initial that interactions. the fact static after the formed inter- to and electrostatic due actions, with compared is state short-ranged, LB It are the S6A). interactions in CaM Fig. hydrophobic of Appendix , domain nonnative C (SI of the on amount formed significant are interactions a found we state, Interactions. Hydrophobic Nonnative ofrainldsrbtoso a ndfeetsae.W sdtecnri itnebtenteCdmi n oano a omntrits monitor to CaM of domain N and domain C the between distance centroid the used We states. different in CaM of distributions Conformational ) 2+ 2+ CMsMC ope;CMaot h opc ofraino ohN n -emnldmisbnigt skMLCK. to binding domains C-terminal and N- both of conformation compact the adopts CaM complex; -CaM–skMLCK CMsMC eonto,w hwtebrirheights barrier the show we recognition, -CaM–skMLCK h w-iesoa reeeg adcp ln md1L and rmsd-1CLL along landscape energy free two-dimensional The (A) hncmaigt h O the to comparing When Ca 2+ CMaot ubelcnomto eoebnigo kLK 1i h ofrainetatdfrom extracted conformation the is I1 skMLCK. of binding before conformation dumbbell a adopts -CaM − 3.07)/(436 Ca 2+ CMaduetefato fntv otcsbetween contacts native of fraction the use and -CaM − 2.60) = tutrlilsrtosfrtesal states stable the for illustrations Structural (D) 3/4. about is C to 0.733)relative li aieLJ native Plain electrostatic Native hydrophobic Native hydrophobic Nonnative electrostatic Nonnative type Interaction al .CMsMC neato nryi ahcmlxstate complex each in energy interaction CaM–skMLCK for interaction 1. Table electrostatic height the barrier inter- as the electrostatic change not However, the and does 2B). as almost strength (Fig. decrease LB–C interaction heights increase electrostatic height heights barrier actions barrier each barrier three the in than The all higher O–I2–LB O–LB. are O–I3–LB transition for tran- and the the O–I1–LB accelerating hydropho- than for on nonnative LB–C influence the bar- more sition of Therefore, have decrease interactions O–I2–LB. the bic of than height (4×) reduction rier larger undergoes LB–C significantly of a height barrier the interactions, hydrophobic Q binding h nto nry kJ energy: of unit The eaotdtero ensur eito rs-CL relative (rmsd-1CLL) deviation square mean root the adopted We . · mol PNAS −1 −0.01 −0.00 −0.01 −0.04 −7.82 J Lennard–Jones. LJ, . C LB−C LB O | ulse nieMy1 2017 1, May online Published h nryo ahstate each of energy The Ca −11.98 −13.65 −37.03 2+ −3.97 −1.90 CMadskMLCK(Q and -CaM −15.33 −21.02 −38.85 −6.17 −3.61 binding | −30.85 −38.29 −21.00 −7.63 −4.56 E3929 to )

BIOPHYSICS AND BIOCHEMISTRY PNAS PLUS COMPUTATIONAL BIOLOGY CaM are W4, F8, F17, R2, R3, K5, and V11 and M144, E84, M145, M124, L112, and M109, respectively (SI Appendix, Fig. S6). To investigate the evolution of the native contacts along the routes from LB to C, we show the distribution of native contact for the barrier region (marked by LB–C) between the LB and C states. In the LB–C region, we found that the residues ranking top seven in average contact number for skMLCK and CaM are W4, F8, F17, V11, I9, N7, and R3 and E84, E11, F92, M144, M124, L112, M109, F68, and F19, respectively (SI Appendix, Fig. S6). These results show that the native hydrophobic interac- tions and native electrostatic interactions play important roles in contributing to the affinity and specificity of the CaM–skMLCK binding. During the recognition, the conserved hydrophobic residues with regular spacing serve as the anchors to form hydrophobic interactions with residues in Ca2+-CaM. Based on the conserved position of hydrophobic residues, we term the skMLCK corresponding locations in 1-5-8-14 (25, 38). Our sim- ulation shows W4, F8, and F17 of skMLCK rank top three in average contact number in the LB state and W4, F8, F17, and V11 of skMLCK rank top four in average contact number in the LB–C state. It shows the important role of hydrophobic anchors in specific CaM–target binding. Among these four hydropho- bic anchors, the W4 anchor contributes the most. Because three electrostatic anchors (R2, R3, and K5) are near W4, the synergis- tic effect is significant. The number of the hydrophobic residues in MET is high in the skMLCK (SI Appendix, Fig. S6). It again supports that the interactions between hydrophobic anchors in peptides and Met-rich hydrophobic binding patches in Ca2+- CaM are important for the high-affinity bound complex (38, 39).

The Effects of Flexibility and Ca2+ on the Mixture Binding Mechanism. To explore how the linker flexibility influences the mixture mech- anism of the Ca2+-CaM binding to the skMLCK, we show the barrier heights along each pathway, respectively, in different strengths of the linker flexibility in Fig. 3. The site-specific con- stant Hinge is the parameter determining local strain energies (SI Appendix, Materials and Methods), which is used to control Fig. 2. The barrier heights along each pathway in different electrostatic the strength of the linker flexibility in our simulation. High Hinge and nonnative hydrophobic interaction strengths. Fi(O–LB) (i = 1,2,3) shows corresponds to small flexibility of the linker. We found that the the barrier heights for the pathway O–Ii–LB. F(LB–C) is the barrier height increase in the linker flexibility decreases all four barrier heights for LB–C. (A) Barrier height changes with different strength of nonnative (Fig. 3A). In addition, we found that the barrier height for hydrophobic interactions. Nonnative−hydrophobic is the parameter represent- ing the strength of the LJ potential of the nonnative hydrophobic contacts O–I3–LB is the lowest in high linker flexibility (Hinge < 6) among in the Hamiltonian energy (SI Appendix, Materials and Methods), altering all of the barrier heights of the O to LB pathway (Fi(O–LB), i = the strength of the nonnative hydrophobic interaction. (B) Barrier height 1,2,3). The barrier height for O–I3–LB also has the biggest vari- changes with different salt concentrations. CSalt is the salt concentration. ation from low to high linker flexibility. The barrier height for O–I2–LB becomes the lowest for low linker flexibility. This indi- cates that the pathway O–I3–LB is the easiest to occur in the increases, implying the electrostatic interactions play little role high linker flexibility. At the same time, we note that the path- at the last stage of the binding. In addition, we found elec- way O–I1–LB is the hardest to occur in the high linker flexibility. trostatic interactions have less significant influence on driv- O–I2–LB becomes the easiest to occur at low linker flexibility. ing conformational change of the skMLCK compared with the We also see that the barrier height (F(LB–C)) from LB to C is nonnative hydrophobic interactions (SI Appendix, The Nonna- always lower than or comparable to the barrier heights from O tive Interactions Drive Conformational Change of the Target). to LB in each strength of the linker flexibility. Based on the above discussions, the nonnative hydrophobic Comparing the landscapes (SI Appendix, Fig. S11) of the interactions have a different role compared to nonnative electro- Ca2+-CaM binding to skMLCK with and without Ca2+, we static interactions in Ca2+-CaM–skMLCK recognition: At the found that C and LB are more stable in the presence of Ca2+, beginning, nonnative electrostatic interactions steer the two units which is consistent with previous experiments (40). In fact, closer in space; then, nonnative hydrophobic interactions are structural analysis revealed that these hydrophobic residues are formed transiently to drive and adapt the interface close to the buried inside of apo-CaM. Upon Ca2+ binding, the structure native bound state. of CaM adopts a more extended dumbbell conformation with the hydrophobic interior exposed to solvent to facilitate the The Native Interaction Contributes to the Affinity and Specificity subsequent binding with targets (41–43). In addition, although of CaM–skMLCK Binding. Each native interaction changes signifi- Ca2+ does not interact with the central linker directly, it can cantly not only the O–LB transition but also the LB–C transition control the plasticity of the linker (10). Ca2+ influences both in our simulation (Table 1). The native interactions contribute the inherent flexibility and the conformation of each domain to the affinity and specificity of the protein–protein binding (32– of CaM (SI Appendix, Fig. S9) (43–46). In our simulations with 34, 36, 37). In the LB state, we found that the residues ranking Ca2+, all barrier heights (F1(O–LB), F2(O–LB), F3(O–LB), and in the top seven in average contact number for skMLCK and F(LB–C)) are decreased (Fig. 3B). The conformation fluctuation

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CaM How LB between a close- peptide. binding determines 3/4 skMLCK the which a the work, for our addition, mechanism in In binding found 1D). mixture is (Fig. LB change intermediate cen- not ness the does to of conformation only linker the bind and tral targets CaM of the strong domain very provides that C-terminal linker, states not the central intermediate although the the region, of of I1 flexibility evidence the the to in due CaM stable on proposed based results, analysis was modeling structural linker From the central (47). study significant the experimental without in an CaM targets by conformation of the the domain that of C-terminal intermediate change the An of to (27). domain only target C-terminal bind its the N-terminal to only bound may the of CaM formation state than complex intermediate CaM–target higher) an final involve higher The times much 27). 100 a (16, have domain (about to shown affinity previously been target State. has Folded CaM of Partially main a as Intermediate The Conclusion and Discussion skMLCK. the to binding CaM of hs eut hwthat show results These of binding the by caused has system the that ( have condition not flexibility. the does under linker system the the that condition smaller the under the (small) linker, constant Large of site-specific simulation. flexibility our The in LB–C. flexibility for linker height barrier the  is F(LB–C) O–Ii–LB. 3. Fig. Hinge steprmtrdtriiglclsri nrisadmodulates and energies strain local determining parameter the is iOL)i=123 hw h are egt o h pathway the for heights barrier the shows 1,2,3) = Fi(O–LB)(i (A) Ca Ca 2+ 2+ ly oiierl nteprocess the in role positive a plays a aiiaetebnigprocess. binding the facilitate can Ca 2+ .  Hinge Ca h -emnldo- C-terminal The 2+ h e asare bars red The B) h re asare bars green The . ed olw(high) low to leads tpclidcdfi n h tpclcnomtoa selection. conformational the atypical to the according and place take fit O–I3–LB induced from (Fig. and state atypical mechanism O–I1–LB different LB fact, a the shows In to pathway 5). each pathways and main state O three the are This skMLCK. there to because binds fit it is when induced mechanism binding of complex and model (2–6), peptide, folding–binding selection the conformational simple of or changes the conformational to change global compared conformational the local and both CaM in of involved as peptide process target binding and CaM a between con- recognition Taking the change. coopera- of conformational and sideration local consistency than better binding with have conformational tion peptide global target The of binding. changes the by accompanied always peptide? and of changes receptor conformational of simultaneously global changes process the conformational binding local the this both for Accordingly, involving mechanism (54). the IDP is of been binding–folding What has the folding for induced proposed of also by mechanism A followed 53). selection (52, conformational folding” before before “binding “folding and “cou- noncooperative binding” cooperative as well into as classified binding−folding” pled be conventional the can folding, mechanism and investigat- association binding By of changes (49–51). synchronization binding the conformational dis- their ing by global “intrinsically accompanied as the always are known (IDPs), coils, proteins” plastic- random by ordered configurational driven intrinsically local processes For the bind- biological ity. with the during for binding flexibility proposed protein–protein for conforma- been and have (2–6) induced-fit ing lock-and- mechanisms rigid the the the selection (1), solve to catalysis tional to enzyme addition Fischer in by In binding proposed 48). and mechanism, folding 20, binding biomolecular key Many (19, study con- to peptide. transitions out global binding skMLCK-binding carried the been the and have works of CaM both of changes involves change process formational binding conformational The local itself. the wrapping to CaM coil the random by a from sition idn rcs novsBt h oa n lblConformational Ca Global and Local the Changes. Both Involves Process Binding linker, led central state the LB CaM. the of of in closeness region skMLCK partial the and breaking to site in E84 the LB the F8 4C). near between around (Fig. is binding the site site site LB LB The the the site. W4 with in near I1 binds site the and is the by approaches LB, site skMLCK to bound I1 I1 is the note From skMLCK implying state. We in LB LB, state. the in LB to top number important among the are also contact in skMLCK average in anchor W4 in and binding CaM seven and in a site) M124 as and (LB M109 region act that M144, binding-patches to E84, a reason, tends form same F8 the to of rather For tend anchor 4A). I1 an M145 (Fig. in as and state patches act LB binding to the in the tendency contacts than higher with average a interacts more has which has and skMLCK, W4 state the addition, I1 act in the In than to rather in 4B). tendency state higher (Fig. I1 the a state in has LB skMLCK which for can site,” patches residues binding “I1 three as and an These have F8, 4A). as F92 (Fig. regarded and N7, I1 be M124, W4, in M109, and contacts 4A). F92, average CaM (Fig. E84, more peptide in are skMLCK M145 state in and LB V11 M144, the M124, with compared M109, contacts native age 4. and Fig. I1 in for it contacts show and calcu- native this (4ANC) We average used LB LB. between also and difference I1 We the between linker. lated difference the the C analyze in the to of E84 method residues electrostatic such Met and contacts, rich native skMLCK, domain, averaging of of anchors method hydrophobic the as using by LB state nfc,w on httegoa ofrainlcagsare changes conformational global the that found we fact, In nteI tt,tersde hthv ag ifrnei aver- in difference large a have that residues the state, I1 the In 2+ CMt kLKppie h etd nege tran- undergoes peptide the peptide, skMLCK to -CaM uigtepoeso ope odn n idn of binding and folding coupled of process the During PNAS hlclsrcue accompanied structures α-helical | ulse nieMy1 2017 1, May online Published Ca 2+ CMaot mixture a adopts -CaM | E3931

BIOPHYSICS AND BIOCHEMISTRY PNAS PLUS COMPUTATIONAL BIOLOGY Fig. 4. (A) The difference between the I1 and LB states in average of native contacts. 4ANC are the differences between native contact number in the I1 state and the LB state and the values that are larger than 1.0 are marked correspondingly. (B) Key residues in PDB structure. The 1CLL and 2BBM are reference structures for the O and C states, respectively. (C) The structures of I1 and LB states that are extracted from our simulation.

Different from the classic induced fit and conformational selec- ity of first binding and then folding for IDP recognition (55, 56). tion, which do not involve the global conformational change A recent study addresses how linker flexibility affects the bind- of the ligand, during the binding process of both the atypical ing mechanism of IDPs (57). The initial binding of an IDP with induced fit and the atypical conformational selection, the ligand its target mostly occurs in just a segment instead of the entire skMLCK changes its helicity only when it binds to CaM (Fig. IDP and the long-range electrostatic interactions have impor- 5). Apart from the atypical induced fit and atypical conforma- tant biasing effects (55–57). In the present work, we have found tional selection, the pathway O–I2–LB takes place according to a that skMLCK and CaM have similar properties in their mixture- “simultaneous” binding–folding mechanism due to the synchro- binding pathways. In addition, we found the skMLCK binding nization of folding (both local conformational change of CaM peptide gradually forms α-helical structures binding to CaM, and the global conformational changes of the skMLCK) and the which is in accordance with a “divide-and-conquer” mechanism binding (between the Ca2+-CaM and skMLCK peptide). The proposed for the IDP binding–folding (54). In a word, we find simultaneous binding–folding mechanism is a unique mechanism many interesting intrinsically disordered properties of skMLCK for the transition from the LB to the C state because of the syn- during its binding process with CaM in our work. chronization of binding and folding (Fig. 5). Multispecificity. Ca2+-CaM is found to have the capability to bind over 300 targets (7–9). However, the underlying factors to The Intrinsically Disordered Properties of Target Peptide. The distri- control such multispecific binding remain unclear. Based on our bution of rmsd-skMLCK for the C state is narrower and smaller studies, we are able to get some insights on this issue. than that for the O state and the I3 state, and the widths of distributions for the I1 state and the I2 state are in the mid- dle range (Fig. 1B and SI Appendix, Fig. S15). The distributions i) At the beginning, nonnative electrostatic interactions steer of rmsd-skMLCK for the states also show the similarity in the the two units to be closer in space. The nonspecific nonna- conformation of the skMLCK relative to the reference structure tive electrostatic interactions are responsible for the binding 2BBM. In the reference structure 2BBM, the conformation of preference between CaM and many kinds of targets due skMLCK adopts the α-helical structures. It indicated that the to the dipolar charged distribution of N- and C-terminal tightly binding states have higher helicities than the loosely bind- domains in CaM. Then nonnative hydrophobic interactions ing states (Fig. 1B). These results indicate that skMLCK has a are formed transiently to drive and adapt the interface close more disordered conformational ensemble of the loosely bind- to the native bound state, which is beneficial to different ing state than of the tightly binding state, which show the binding targets forming the specific native interaction with CaM. nature of the IDP (49–51). Previous work has shown the possibil- The cooperation between two kinds of nonnative interactions

E3932 | www.pnas.org/cgi/doi/10.1073/pnas.1615949114 Liu et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 i tal. et Liu iii) transition the for mechanism C. unique to the LB from also The mechanism. is O–I2–LB binding–folding mechanism and simultaneously simultaneous selection, a according to conformational according place atypical place take and takes of fit –LB change bind- induced O–I3 conformational atypical the and global to CaM, O–I1–LB the respectively. of and peptide, change skMLCK, skMLCK conformational and local CaM the between ing for stand lines green 5. Fig. ii) hsmliihafiiycnb eadda h oreo mul- of source the as regarded tispecificity. be complexes. can high-affinity affinity to lead multihigh to This able interactions are hydrophobic CaM with resulting formed the biologi- 38), their (25, on differ- functions based cal have anchors hydrophobic peptides of locations bound binding ent high-affinity different the bind- to Although important hydrophobic complex. are Met-rich CaM and in peptide patches ing the hydropho- in between anchors interactions bic hydrophobic native again It the CaM. conserved that to binding underscores of to contributing importance skMLCK in anchors the addressed than We targets different interaction. to electrostatic bind nonnative to mere easier much CaM the makes ifrn agt r bet eettems utbeways suitable most the select to that expected able is are It targets CaM. form with different to interactions supposed of are kinds which different targets, different the may on mechanism depend mixture-binding inter- the hydrophobic that modu- implies and strongly this electrostatic actions, of the are strengths that pathways the found binding by we lated the Because of target. its heights processes the to barrier from binding vary CaM may the folding synchro- of and the binding that of indicated skMLCK nization mech- the mixture with a adopts binding CaM anism global that of fact plasticity The and sites. the binding CaM provide their for skMLCK of of search change change conformational sites conformational conformational by binding Local accompanied other their change. each that target to bind its process then and and a CaM essentially between binding is bind- the multispecificity the Furthermore, pep- for ing. its precondition and the CaM of provided sites tide binding the between native) native, Arguments h itr-idn ehns ndti.Terd lc,and black, red, The detail. in mechanism mixture-binding The i and ii hwteseilt fitrcin (non- interactions of speciality the show CLt oio h tutrlcag of change structural structure the to relative monitor rmsd-1CLL to the adopted 1CLL we simulation, our in skMLCK to Coordinate. Reaction double- the of in (Details are model interaction. well hydrophobic and interactions trostatic .W sd25 used We 1A). (Fig. landscape energy thermodynamic (REMD) the dynamics explore molecular to exchange (26) replica used we achieve To sampling, ps. tem- sufficient 1.0 a of a to time coupling coupled a was with simulation dynamics the Langevin via and bath ps perature 0.0005 is step time The (65). 4.0.5 Protocols. Simulation binding peptide. monitor skMLCK skMLCK of to of change 2BBM(rmsd-skMLCK) rmsd structural structure the the reference used the we to hand, relative other peptide the On process. binding between contacts native of by model our into and map Hamiltonian, mixed-contact a integrate term We repulsive ume. the and energy torsion potential local The n yrpoi Arsde.W ul w-eddul-elSMand SBM double-well expression two-bead the by a given nonhydrophobic built is for We Hamiltonian potential the residues. LJ CA 10–12 hydrophobic the to and a compared by potential, represented LJ is potential 6–12 hydrophobic between CB–CB only The exist the residues. interactions hydropho- by hydrophobic hydrophobic represented and The is interactions (64). potential model electrostatic electrostatic The Debye–Huckel the SBM. our the added in (13, and we interactions recognition interactions bic CaM 54), charged in 29–31, in the 25, role Because 15, important 61). an 60, play interactions 32, mixed-contact hydrophobic a 13, by (10, structures the To 2BBM) of model ID: 60–63). information (PDB map (32, integrated closed and skMLCK we 1CLL) basins, target ID: two (PDB its with open to systems to binds SBM CaM a which extend by process the plore Model. Double-Well Methods and Materials iv) tpclidcdfi ahrta h itr ehns for mechanism mixture the than S14 ). skMLCK. rather Fig. the fit following , Appendix pathway induced (SI a atypical select binding to skMLCK tends compared smMLCK stable the Therefore, more in is state state that LB I1 with the the but that of populated found simulations hardly we the is in binding, state smMLCK LB In the form is skMLCK. to which critical S13), be Fig. to Appendix, found (SI “5” hydrophobic conserved the of lacks position smMLCK that is skMLCK and smMLCK smMLCK and CaM Appendix, simula- for (SI same tions skMLCK the for with used CaM procedure to tion binding peptide simulations different performed smMLCK we by of conclusions, To our determined selected. of are is validity the anchor LB test binding and the state and regions conformations I1 binding-patch distinct the in the CaM skMLCK, target. of and for CaM analysis between our mechanism From binding decisive fea- the the structural as for their acts factor binding to the suit- according along most intermediate CaM, the The to tures. select bind can to targets ways different able that expected is It for mechanism mixture skMLCK. our the from binding–folding different simultaneous is a and essentially mechanism peptide is CaMKI which and 59), CaM (58, for and proposed CaM was A between peptide (40). binding CaMKI scenarios for binding scenario conventional induced-fit to two mutually leads the This peptide. of adap- by mixture induced conformational a structure by bound the followed to peptide, tion the structure compact embrace that entirely to than shown rather has partly experiment the The support selects 59). CaM bind- to 58, recently 47, multispecificity (40, evidence the proposal increasing our from been has benefiting There CaM, ing. to bind to + U Attraction U IApni,MtrasadMethods.) and Materials Appendix, SI U electrostatic edvlpdasrcuebsdmdl(B)t ex- to (SBM) model structure-based a developed We Local odsrb h rcs ywihthe which by process the describe To l ftesmltoswr efre ihGromacs with performed were simulations the of All U + sdvddit odsrthn,agebnig and bending, angle stretching, bond into divided is total U Ca Repulsive and PNAS (Γ 2+ open CMadskMLCK(Q and -CaM U hydrophobic , | + Γ .Temi ifrnebetween difference main The ). close ulse nieMy1 2017 1, May online Published U U Electrostatic Repulsive ) Ca = r sdt nrdc h elec- the introduce to used are 2+ U Local CMadue h fraction the used and -CaM rvdsteecue vol- excluded the provides + U hydrophobic binding Ca omntrthe monitor to ) U h Simula- The Attraction 2+ . CMbinds -CaM | nthe in E3933

BIOPHYSICS AND BIOCHEMISTRY PNAS PLUS COMPUTATIONAL BIOLOGY replicas and the neighbor replicas attempted to exchange with each other Technology, China, Grants 2016YFA0203200 and 2013YQ170585. J.W. thanks every 2,000 MD steps. For all replicas, the total simulation time was 1.25 µs. the support in part from National Science Foundation Grant NSF-PHY-76066. H.P.L. acknowledges support from the National Institutes of Health National ACKNOWLEDGMENTS. F.L. and J.W. acknowledge support from National Institute of General Medicine Science and the Ohio Eminent Scholar Science Foundation of China Grant 91430217 and Ministry of Science and Endowment.

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E3934 | www.pnas.org/cgi/doi/10.1073/pnas.1615949114 Liu et al. Downloaded by guest on October 1, 2021