Downloaded by guest on October 1, 2021 www.pnas.org/cgi/doi/10.1073/pnas.1702599114 change conformational upon change conformational PKAc binding. of mechanisms sec- fundamental the provide the of will insights for findings barrier These suc- subprocess. from the ATP binding increase hamper ond significantly will PKI and state bound binding closed the cessful that to fact open PKAc-ATP the from of route through because binding pathway main the the that is intermediate find rate we and simulations analyses, dynamics and constant molecular from By paral- intermediate transition states. the closed two an to during open are intermediate) as an there PKAc-ATP sta- as and PKAc-PKI addition, with pathway PKAc In (pathway in state. site pathways active complex lel the at to loop them distal Gly-rich regions bilize of some movements as fit the mechanism well induced drive as binding the can with mixed ligands selection, The a conformational dominant. and suggest fit results induced Our of with PKI). (PKAc PKAc and ternary and ATP PKI), with ATP, PKAc by with (PKAc binding for PKAc during simulations the PKAc dynamics analyzed molecular of and performing changes regu- model conformational allosteric coarse-grained of this a dynamics of developed mechanism closed we the to lation, explore open from to PKAc Aiming of states. changes conformational effectively bio- the can the substrate/inhibitor induce and during ATP confor- closed) Both major process. and three catalysis intermediate, exhibits (open, (PKAc) states PKA mational of subunit catalytic The Jos by Edited pathways different with PKA Chu of Wen-Ting subunit C of change conformational dynamic and mechanism Binding sfudt epeaetadciia oisrglto function regulation its to critical and prevalent cleft, be site which active to PKAc, the closing found of (residue and is dynamics opening them by intrinsic contributed between The mostly linker is 1). binding (Fig. the (12) (substrate/inhibitor and 120–127) lobe 128–300), residue helical-rich site; large small a a 40–119), of a consists comprises which PKAc N-terminal 40–300), structure, (residue (denoted 3D core Ser338 For kidney-shaped and pSer338). in Thr197 and pThr197 at both as sites conserved residues, phosphorylation 350 highly two have The PKAcs is with the (12). general, (PKAc) In structure. substrates/inhibitors PKA and sequence and of active ATP cAMP, critical subunit for the for catalytic include sites sites which binding binding subunits, and the (C) catalytic provide two which and subunits, (R) tory regime. “structure–function” pro- conventional regulation greatly a biological It through PKA’s (3–11). cess on (PDB) understanding well-solved Bank our been Data deepens has Protein which pigs, into PKA, and deposited of mice, struc- and structures humans, in to of developments belongs amount recent mainly arresting the an to biology, of species Thanks tural one many in places. is spread many PKA widely and eukaryotes, 2). in (1, families differentiation largest the cell and metabolism, including memory, pro- activities, growth, biomolecular cellular important many most in involved the cesses of one is PKA dependent T dynamics molecular and China; a tt e aoaoyo lcraayia hmsr,CaghnIsiueo ple hmsr,CieeAaeyo cecs hncu,Jln130022, Jilin Changchun, Sciences, of Academy Chinese Chemistry, Applied of Institute Changchun Chemistry, Electroanalytical of Laboratory Key State h nciePAi oonyecmoe ftoregula- two of composed holoenzyme a is PKA inactive The c asern fAPtria hshtst agtprotein target cAMP- by to phosphorylation, protein phosphates as to terminal referred substrates, ATP of ransferring eateto hmsr n hsc,SaeUiest fNwYr tSoyBok tn ro,N 11794 NY Brook, Stony Brook, Stony at York New of University State Physics, and Chemistry of Department b nttt Madrile Instituto .Ouhc ieUiest,Hutn X n prvdJl 1 07(eevdfrrve eray2,2017) 22, February review for (received 2017 31, July approved and TX, Houston, University, Rice Onuchic, N. e ´ β setdmntdlb APbnigst;residue site; binding (ATP lobe dominated -sheet a,1 iknChu Xiakun , | od suisAazdse aoini IDANnceca,Cmu nvriai eCnolno 84 ard Spain; Madrid, 28049 Cantoblanco, de Universitario Campus Nanociencia), (IMDEA Nanociencia en Avanzados Estudios de no ˜ PKA | binding b,1 n i Wang Jin and , | nrylandscape energy apo a,c,2 | Kc binary PKAc, h ag oe,adtedsac ewe fGu7 n OH and Glu170 of O between distance the to and relative lobe), helix large C Gly-rich of the oxygen position the phosphate the of and (measuring His87 phospho-Thr197 closing of of NE2 and between distance opening the the loop), (measuring Gly186 such of PKAc, between of distance states dis- different the critical the some as classify cat- to out criteria the pointed the have as por- between people tances C-terminal loop, practice, the In Gly-rich in (18). and tion the loops, in peptide-positioning and occur alytic mostly changes PKAc conformational the of process, binding committed) the (dynamically During states 15). (14, closed or to (nucleotide intermediate equilibrium ligand the conformational of favor this presence alters the substrate/inhibitor) while popu- or states, minor other with at catalysis), lations to uncommitted (dynamically state 1073/pnas.1702599114/-/DCSupplemental at online information supporting contains article This 2 Submission. 1 Direct PNAS a is article This interest. of conflict no declare authors wrote The J.W. and X.C., W.-T.C., and data; paper. analyzed the J.W. and X.C., W.-T.C., research; formed of conformation The the open, 18). 15, on into 14, (12, based classified cleft site states be active intermediate can and which closed, conformations, main three an as serving 17). (16, PKI PKAc block of for will evidence inhibitor PKAc-ATP the and each supporting PKI by binding, between enhanced substrate cooperatively affinity binding be high the will The that PKI other. accessi- and revealed PKAc ATP experiments of of Additional affinity structure 15). the binding (14, high-affinity making ble the PKI, PKAc by quenched from The pseu- be (13). interactions high-affinity to signal found export a are nuclear including dynamics a 20-aa protein, and a region stable (PKI), dosubstarte heat inhibitor of binding cocrys- via protein peptide (7) high-affinity al. et a Knighton structure by talizing crystallographic solved successfully first was The PKAc elusive. of is the PKAc characteristics, of dynamic structure highly the of because However, (12). uhrcnrbtos .TC,XC,adJW eindrsac;W-..adXC per- X.C. and W.-T.C. research; designed J.W. and X.C., W.-T.C., contributions: Author owo orsodnesol eadesd mi:[email protected]. Email: addressed. be should correspondence whom To work. this to equally contributed X.C. and W.-T.C. omtoa hnepoesfo pnt lsdstates. closed con- to the open from during process binding change of formational stages different the an at indicate as studies residues PKAc-ATP our critical Also, with first). pathway binding conforma- (ATP the intermediate and favor fit and induced selection sug- of tional results mechanism coarse-grained binding Our weighted mixed simulations. the gest dynamics using molecular by and ligands, PKAc, different model to two PKI of and and ATP mechanism dynamics binding PKAc of the We landscapes activity. uncovered energy catalytic is free and the (PKAc) The recognition quantified PKA substrate eukaryotes. of the in to subunit families linked catalytic kinase of largest change the conformational of one is PKA Significance tutrlivsiain eeldta Kcppltsat populates PKAc that revealed investigations Structural PNAS | ulse nieAgs 0 2017 30, August online Published α abnao C)o e5 n CA and Ser53 of (CA) atom carbon . apo www.pnas.org/lookup/suppl/doi:10. Kcdmntsa open at dominates PKAc | E7959–E7968

PHYSICS BIOPHYSICS AND PNAS PLUS COMPUTATIONAL BIOLOGY 26 apo AB 24 22 20 Glycine-rich loop 18 Tyr330 Ser53 α Mg-positioning C 16 ATP Gly186 loop Glu127 His87 14 Catalytic loop Glu170 Asp166 pThr197 12 PKI 10 8 6 8 10 12 14 16 18 20 22

open-PNAS closed-PNAS intermediate-PNAS

CDE 26 26 26 binary-ATP binary-PKI ternary 24 24 24 22 22 22 20 20 20 18 18 18 16 16 16 14 14 14 12 12 12 10 10 10 8 8 8 6 8 10 12 14 16 18 20 22 6 8 10 12 14 16 18 20 22 6 8 10 12 14 16 18 20 22

0 1 2 3 4 5 (kT)

Fig. 1. (A) PKAc structure and (B–E) the free energy landscapes of PKAc dynamics along critical distances between Ser53 and Gly186 (x axis) and Ser53 and Asp166 (y axis) in different systems [(B) apo PKAc (apo), (C) PKAc-ATP (binary-ATP), (D) PKAc-PKI (binary-PKI), and (E) PKAc-ATP-PKI (ternary)]. PDB ID code 1ATP is illustrated as the PKAc-ATP-PKI ternary complex. Ligand ATP is shown as spheres (between small and large lobes of PKAc), and ligand PKI is shown as purple cartoons. For PKAc, small lobe (40–119), large lobe (128–300), and linker (120–127) are shown as red, light orange, and blue cartoons, respectively. Glycine-rich loop (46–57; colored in yellow) belongs to small lobe, and catalytic loop (116–171) and Mg-positioning loop (both colored in green) belong to large lobe. Three main distances of six residue pairs (Ser53-Gly186, His87-Thr197, and Glu170-Tyr330), which are used to distinguish different states of PKAc, are labeled in A. The classification of open, closed, and intermediate states is referred to the data in the work of Masterson et al. (15).

of Tyr330 (measuring the distance of the C-terminal tail to the Results and Discussion active site) (12, 19). Dynamic Conformational Equilibrium in apo PKAc. The structure- Based on the abundant static structures (bound states) of based model (SBM) (29–31), which generally has only one basin PKAc, additional insights are moving to understand the rela- at the bottom of the energy landscape representing the native tionship between conformation and function by uncovering the structure, has successfully described the protein folding and conformational dynamics of PKAc during binding. Recently, binding processes. However, such a simplified model may fail NMR-based investigations by Masterson et al. (14, 15) por- to describe the particular case where protein has multiple con- trayed an energy landscape of PKAc binding with substrate formations, each corresponding to a specific function within the phospholamban (PLN) and inhibitor PKI. Different binding well-folded native basin. To deal with the conformational transi- partners have different effects on the conformational dynamics tion within the bottom of the energy landscape, great efforts have of PKAc, manipulating the regulation process through a del- been put forth to improve the SBM with multiple basin potential icate circulation of dynamically committed, uncommitted, and (32–37). One key to intuitively construct a model with multiple quenched states. In addition, Hyeon et al. (20) described the basins is to build a mixed contact map, which integrates multi- process of binding-induced conformational changes of PKAc by ple structural information together (38–40). Following this strat- ATP through a coarse-grained molecular simulations. Because egy, we construct the weighted contact map based on all possible of the high flexibility and associated underlying free energy native interactions in all well-known PKAc structures deposited landscape, it is valuable to investigate the PKAc systems with in the PDB (SI Appendix, Fig. S5). computational methods (15, 20–28). However, fully understand- In this study, we have searched about 180 structures of PKAc ing the process of intrinsic dynamics and binding-induced con- in the PDB. By monitoring five critical residue–residue distances formational change of PKAc, in particular accomplished by a of all of the PKAc structures (SI Appendix, Fig. S4), it shows that, complete biomolecular process with binding of two ligands, is from open to closed states, these distances are getting decreased still challenging because of the complexity of underlying free (15). Furthermore, it seems that the distance between Ser53 energy landscapes and large numbers of elements of the sys- and Asp166 is more appropriate to classify these structures than tem. Here, we developed a coarse-grained model for PKAc sys- other criteria. As a result, these PKAc structures can be classified tems (apo, PKAc-ATP, and PKAc-PKI binary complexes and into open (D2 > 17.00), intermediate (17.00 > D2 > 12.80), PKAc-ATP-PKI ternary complex). With quantified thermody- and closed (D2 < 12.80) states, shown in SI Appendix, Fig. S4A. namic binding energy landscape and kinetics, we show dynamic Except for some structures with no phosphorylated residues and conformational changes occurring in PKAc and address the some with incomplete essential residues, all of the other 165 critical residues during the binding process in different PKAc structures were selected to build the model of PKAc. The native systems. Our results provide a comprehensive way to under- contact map was individually calculated by Shadow algorithm stand the molecular mechanism of the PKAc family by using a (41) for each structure, and the strength of each native contact “structure–dynamics–function” framework, which makes impor- was reweighted by its probability appearing in all 165 structures. tant contributions to understanding the biological mechanism of The overall map of the native contact in our model is shown in PKAc family. SI Appendix, Fig. S5, showing that most native contacts appear

E7960 | www.pnas.org/cgi/doi/10.1073/pnas.1702599114 Chu et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 hs nFg )a ela h te uvst asindsrbto.Frdsrbtos h range the distributions, For distribution. Gaussian to curves fitted the as well the as and 1) Fig. in those 2. Fig. much have the for PKAc-ATP-PKI (except and distances lower five PKAc-ATP the 87–197), (14, of all distance states almost consis- quenched for However, are dynamically 15). results and uncommitted, These committed, dynamically of values. dynamically findings further experimental lower can the even with complex) tent (ternary to ligand them that second decease and the distances of five binding the the decrease can complexes) of (binary PKI results The (∼ width. distributions Gaussian to here, five results the of the in all distances fitted 2, of Fig. ranges larger in much shown have As distances systems. different in distances PKAc-ATP. in than PKAc-ATP-PKI PKAc- intermediate in in and higher open much than is between PKAc-ATP-PKI states difference in energy and should the lower intermediate and It much between ATP, PKAc-ATP. is difference in states energy the closed that that with toward noted moving compared be is PKAc- state distribution in the closed than complex, the PKAc-PKI ternary in the In expanded ATP. more narrow much becoming dis- is is this Interestingly, tribution distances state. intermediate PKAc- two the In at the concentrated small. and of rather distribution is the states ATP, closed 1 and Fig. intermediate, in open, shown in and that, quantified obvious is are PKAc in of Asp166 complexes and Ser53 and Gly186 and the between and terminus. termi- C lobe, C the large the and the within lobe and small lobe, lobe small large the the between within nus, lobe, small the within h tal. et Chu efrhraayetedsrbtoso l ftefiecritical five the of all of distributions the analyze further We Ser53 between distances the along landscapes energy free The µ σ y h itiuin ffieciia itne btenrsdepiso 316 316 717 7–3,ad1730 nfu ytm sm as (same systems four in 127–330) and 170–330, 87–197, 53–166, 53–186, of pairs residue (between distances critical five of distributions The (A) wdh hnPA-K,wihmasta h idn of binding the that means which PKAc-PKI, than (width) stelcto ftecne ftepa,and peak, the of center the of location the is h oiinadwdhrfrto refer width and position The (B) (probability). states of number of fraction the represents axis apo Kc h reeeg ifrnebetween difference energy free the PKAc, µ B Probabilty A ugs httebnigo T or ATP of binding the that suggest 0.06 0.08 0.12 0.02 0.04 0.1 0 0.6 0.8 Probabilty Position (nm) 1 0.02 0.04 0.06 0.08 0.12 0.14 0.16 Distance (nm) 0.1 1.2 1.4 1.6 1.8 0 1.2 1 2 apo apo 1 1.2 1.4 binary-ATP 1.4 Kca ela other as well as PKAc 1.6 1.6 53-186 1.8 1.8 Distance (nm) System 2 2 2.2 binary-PKI e apo 2.2 −( 2.4 170-330 2.6 x Kc We PKAc. −µ) 2.8 0.02 0.04 0.06 0.08 0.12 0.1 σ 0 It B–E. 3 ternary 2 0.8 /2σ 3.2 sthe is 1 2 1.2 Width (nm) ); 1.4 Distance (nm) 0.08 0.12 0.14 0.16 0.18 0.22 0.24 0.26 0.02 0.04 0.06 0.08 0.12 0.14 0.16 0.1 0.2 0.1 1.6 0 apo 1.2 the device quenching dynamic highest, the the as 15). PKI is (14, of PKAc-ATP subpro- role different and the the confirms PKI of which subpro- between all affinity of binding the addition, second In cesses, pathway. the each increases on of ligand cess of especially binding thermostability, that found the mea- We experimental 1). with (Table consistent surements are and The events respectively. binding (∆ ferent stage, illustrated pathways energies P binding As free and parallel (B). binding stage two bound A are intermediate PKI there possessing and 3A, ATP Fig. both in and PKI (P), extracted intermediate (A), be bound bound energy can ATP free intermediate (basins) the (U), unbound stages From as binding PKAc. stable to binding four PKI landscapes, and land- ATP energy Stability. free of the the scapes quantified Enhance we and 43), (42, Energy metadynamics the Using Decrease Can ATP/PKI Ligand these pocket. that binding fact ATP the the of of vicinity because in mainly are 1D), regions Fig. PKAc of in regions that C-terminal to and (similar loop Gly-rich stabilize will ATP nebtentetasto tt n h non tt,adi is it and state, unbound the and state transition the between ence the calculated (∆G we barrier addition, In binding complex. binary in the barrier that in from limited binding ferent ATP and of second subprocess the a in is there Interestingly, PKAc-ATP. the in binding ATP the in 0.42 binding about ATP at of state 0.28 transition about the at located are states 1.8 1.4 uigtePIbnigsbrcse via subprocesses binding PKI the during 3C, Fig. in shown As iayAP bnr-K ternary binary-PKI binary-ATP I 2 1.6 A 53-166 2.2 Distance (nm) 1.8 aha n the and pathway 2.4 System 2 2.6 2.2 2.4 Q 127-330 x 0.04 0.06 0.08 0.12 0.02 inter 0.1 2.6 xssostemnmmadmxmmo h distance, the of maximum and minimum the shows axis 0 0.6 2.8 I 0.8 ‡ fPA-T,wietetasto tt of state transition the while PKAc-ATP, of P 3 ,wihi enda h reeeg differ- energy free the as defined is which ), PNAS aha slctda bu 0.56 about at located is pathway 1 µ Distance (nm) I 1.2 and binary-ATP eecluae eaaeyfrdif- for separately calculated were G) binary-PKI P 127-330 170-330 53-186 53-166 | 87-197 ternary 1.4 aha,bt ftetotransition two the of both pathway, σ ulse nieAgs 0 2017 30, August online Published apo epciey fGusa distribution. Gaussian of respectively, , 1.6 Q 87-197 1.8 inter 2 2.2 fPA-K.However, PKAc-PKI. of I P I aha,wihi dif- is which pathway, A aha slocated is pathway I A Q and inter | E7961 I of P

PHYSICS BIOPHYSICS AND PNAS PLUS COMPUTATIONAL BIOLOGY A B

C

Fig. 3. (A) The free energy landscape projected onto two reaction coordinates (the fraction of native contacts numbers between PKAc and ATP, Qinter of PKAc-ATP; the fraction of native contacts number between PKAc and PKI, Qinter of PKAc-PKI). The four minima correspond to U stage (both unbound, both Qinter = 0), the A stage (only ATP bound, Qinter of PKAc-ATP ∼ 0.93, Qinter of PKAc-PKI = 0), the P stage (only PKI bound, Qinter of PKAc-PKI ∼ 0.93, Qinter of PKAc-ATP = 0), and the C stage (both bound, both Qinter of PKAc-ATP and Qinter of PKAc-PKI ∼ 0.93). For forward binding (from U stage to B stage), there are two pathways with different intermediates, IA (ATP binds first to reach A stage) and IP (PKI binds fist to reach P stage). (B) Schematic diagram of two pathways (IA pathway and IP pathway), four subprocesses (UA, AB, UP, and PB), and four stages (U, A, P, and B) during forward binding process. The IA pathway is colored blue, and the IP pathway is colored red. (C) The free energy landscapes along four different directions. The four different directions correspond to four subprocesses of ATP binding to apo PKAc (UA), PKI binding to apo PKAc (UP), ATP binding to PKAc-PKI (PB), and PKI binding to PKAc-ATP (AB). The unit of free energy is in kT (k is the Boltzmann constant, T is the temperature).

closely related to the binding rate. Among the four subprocesses by αD, and the loop between αF and αG (P–11 site), the coiled of binding (two subprocesses in each pathway), the subprocess tail of which is surrounded by Gly-rich loop, the loop between of ATP binding in the IP pathway has the highest binding barrier αB and αC, and peptide positioning loop (P+1 site). These find- (9.75 kT), which implies that the kinetic rate of this subprocess ings are consistent with the previous experimental and theoret- is the slowest one. Therefore, it suggests that the whole binding ical studies (12, 15). Intriguingly, there is no direct interaction rate of the process with ATP binding first (IA pathway) will be between the C terminus of PKAc and PKI, but PKI can increase much higher than that of the process with PKI binding first (IP the stability of C terminus of PKAc. Moreover, the RMSF of Gly- pathway). rich loop in PKAc-ATP is lower than that in PKAc-PKI, which is The 100-ns molecular dynamics (MD) simulation without bias consistent with the results in Fig. 2. was performed on each system of apo PKAc, PKAc-ATP com- plex, PKAc-PKI complex, and PKAc-ATP-PKI ternary complex Binding-Induced “Open” to “Closed” Conformation Transition. To individually. Then, the last 90 ns of each trajectory was analyzed monitor the conformational changes of PKAc, we calculated the for the rms fluctuations (RMSFs), which are the time averages of rmsds for each residue (with reference to the average struc- ture) during the simulation (15). As illustrated in Fig. 4, of all of Table 1. Experimental Kd (12, 16, 17) and simulated data of the four systems (at the four stages), the region of some loops different binding subprocesses of PKAc (binding free energy: ‡ (Gly-rich loop, loop between αB and αC, loop between αF and ∆G = Gbound − Gunbound; binding barrier: ∆G = GTS − Gunbound) α G) and N and C termini show high RMSF values. Catalytic Experimental Modeled Simulated Simulated loop (166–171) and Mg positioning loop (184–187) are relatively Subprocess K ∆G ∆G ∆G‡ stable. The binding of ligand can decrease the RMSF of some d regions, indicating that the existence of ligand can increase the UA 10,000 −2.88 −4.24 7.96 stability of PKAc. It is worth noting that Gly-rich loop and C ter- PB 60 −5.44 −6.27 9.75 minus (colored gray in Fig. 4A) of PKAc-ATP have lower RMSF UP 230 −4.76 −5.63 5.20 than those of apo PKAc. In contrast, the loop between αB and AB 0.2 −8.29 −7.19 6.37 αC (76–85), αD (129–137), peptide positioning loop (198–205), The definitions of UA, PB, UP, and AB are stated in Fig. 3. The units of Kd and the loop between αF and αG (240–248) of apo PKAc have and ∆G/∆G‡ are nanomolar and kT (k is the Boltzmann constant, T is the similar RMSFs as that of PKAc-ATP, which is higher than those temperature), respectively. Modeled ∆G is the experimental measured bind- of PKAc-PKI and PKAc-ATP-PKI. These regions consist of the ing free energy based on the simulation environment, and the calculation binding pocket of PKI, the helical head of which is surrounded details of modeled ∆G are listed in SI Appendix.

E7962 | www.pnas.org/cgi/doi/10.1073/pnas.1702599114 Chu et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 PA-T,PA-K,adPA-T-K minus PKAc-ATP-PKI and PKAc-PKI, (PKAc-ATP, 4. Fig. h tal. et Chu binding ATP similar the is of It unfolding”) PKAc-PKI. “partial (or binary 29.3% “cracking” the the the to by with binding obvious more ATP to even for binding be increase ATP will phenomenon For the processes. while binding PKI and apo ATP in ently effect. while similar binding, have of not subprocess does first next the binding the decrease PKI can during first PKAc binding of ATP lowest flexibility that (the suggest stable most results of subpro- the The all are the SD). PKAc-ATP Of in to P). distances binding and the PKI of of (A cess all complex subprocesses, binary binding bound four of the that (B) complex than ternary lower bound sim- is our of in “intermediate” SD stages The A/P and (12). and respectively B, ulations, closed, U, in open, PKAc to into corresponding states, PKAc the well-classify of to able that conformation are loop implying distances Gly-rich these others, the Furthermore, at than region. occurs PKAc significant in change more conformational is most 53–166) and 186 bind- ( the during PKAc in of in shown distances As critical process. ing five the of changes figure. this in labeled are systems four the among RMSF Q nrgigy efudta l-ihlo eae ut differ- quite behaves loop Gly-rich that found we Intriguingly, inter apo Kc hr sa1.%wdroeiga h l-ihloop, Gly-rich the at opening wider 15.6% a is there PKAc, MF(m uvsof curves (nm) RMSF (A) Kc( PKAc ∼ .Tecag ftedsacso eiu 3(53– 53 residue of distances the of change The 0.9). Q inter r ihrta hto on PKAc bound of that than higher are 0) = 0.15 0.05 0.1 0.2 0.4 0.6 l v distances five all S6, Fig. Appendix, SI B A 0 0 apo C-terminus 0 0 0 10 0 20 0 350 300 250 200 150 100 50 0 Kc KcAPcmlx KcPIcmlx n KcAPPItraycmlxa ela MFdfeec n)curves (nm) difference RMSF as well as complex ternary PKAc-ATP-PKI and complex, PKAc-PKI complex, PKAc-ATP PKAc, α Gly-rich loop D p iayAPbnr-K ternary binary-PKI binary-ATP apo apo α B- h Kcsrcueclrdwt Do MF(m.Tergoswihhv ag ifrneof difference large have which regions The (nm). RMSF of SD with colored structure PKAc The (B) PKAc). α C loop α F α catalytic loop D Mg positioning Residue number α loop G α C peptide positioning fPA ob lsrt h tutr fcoe tt PBID (PDB state closed of structure the to closer be to PKAc of of rmsds Over- the S8 ). loop, decrease and can S7 binding Figs. Ser53- ligand Appendix, all, of (SI curves Ser53-Gly186 distance and the Asp166 as Also, behaviors states. similar have closed terminus and of open curves the rmsd the distinguish Ser53-Gly186 (Fig. and to Ser53-Asp166 PKAc convenient of of are distances stages conforma- the binding largest Therefore, activation the different 4). site, with between region active changes the the is tional of loop region Gly-rich proximal The one loop. as distal well the as site, including nus, active complexed, the or of isolated regions is PKAc when subprocess. this ation in larger of is extent loop the Gly-rich because the PKAc-PKI, of to opening binding cleft the ATP the for energy close more cost then that be implies and will it entrance, Moreover, the interactions. of stabilizing can gate by which the loop, Gly-rich as the regarded PKAc to be to opening the binding induce ATP initially that to bind- has present PKI we the in Therefore, found this processes. be ing However, cannot transition (20). open” study “more theoretical to open previous a in found pocket Gly-rich loop eas oio h ein ihlrecnomtoa fluctu- conformational large with regions the monitor also We α B loop op n emnsadfreteconformation the force and terminus C and loop, αF–G peptide positioning α F- loop α G loop standard deviation ei,atvto loop, activation helix, αC of RMSF(nm) PNAS 0.06 helix, αC 0 C-terminus | ulse nieAgs 0 2017 30, August online Published op n termi- C and loop, αF–G op n C and loop, αF–G ei,activation helix, αC | E7963

PHYSICS BIOPHYSICS AND PNAS PLUS COMPUTATIONAL BIOLOGY code 1ATP). Moreover, the extents of rmsd changes of the αF– αG loop [about 0.1 nanometer (nm) from unbound to bound state] and C terminus (about 0.1 nm) are higher than those of αC helix (about 0.05 nm) and activation loop (about 0.03 nm). The binding of ATP does not decrease the rmsd of the αF–αG loop significantly (about 0.01 nm from apo PKAc to PKAc-ATP), probably because ATP does not interact with this region directly. However, the rmsd curve of the αF–αG loop undergoes simi- lar significant fluctuations (peaks at transition states) during the binding process from unbound to bound state as those of the other regions. The C terminus has a dramatic effect on captur- ing the ATP and PKI at the first stage through the “fly-casting” conformation 1 of PKAc conformation 5 of PKAc mechanism (44, 45). The following coordination of ATP to the binary complex binary complex active sites and PKI to the substrate binding site seems to be unrelated to the C terminus, since no apparent direct interac- ATP to PKAc PKI to PKAc 1CMK 1ATP tions are observed (shown below). However, as PKAc possesses a delicately complicated interaction network inside, the effects of ATP/PKI binding on the C terminus at the late stage may be indirectly triggered by other regions of PKAc, as binding is sup- posed to tune the PKAc’s conformational distribution and sta- bilize the PKAc in a large scale. The rmsd of the C terminus along the binding process additionally shows a sharp decrease at the late stages. We addressed the roles of C terminus partic- ipating into the whole stage of the binding process. As a result, ATP binding or PKI binding can influence the conformation of both proximal and distal regions of binding site, including Gly- rich loop, activation loop, αC helix, αF–αG loop, and C termi- nus, because of the delicate interactions networks within PKAc. Although ATP directly binds to the Gly-rich loop, while PKI does conformation 1 of PKAc conformation 5 of PKAc not, the dynamic conformational changes of this region as well as ternary complex ternary complex other regions are obviously revealed by the simulations. ATP to PKAc-PKI PKI to PKAc-ATP 1BX6 1ATP To see the motion of the small and large lobes during ligand binding, principal component analysis (PCA) was performed on Fig. 5. Comparison between conformation 1/conformation 5 of PCA and each trajectory of metadynamics run. Building up PCA here is some representative crystal structures of open (1CMK), intermediate (1BX6), similar with that used by Masterson et al. (15). The proportion and closed (1ATP) states. of each principal component (PC) is illustrated in SI Appendix, Fig. S9. As shown in SI Appendix, Figs. S10 and S11, the first PC corresponds to the open-to-closed conformational transition at (including the activated state) during the binding. These two most parts of the small lobe and the αF–αG loop of the large scenarios can be distinguished in theoretical framework by the lobe, while the second PC corresponds to a shearing motion conformational transition occurring before or after the bind- ing. However, they also can be found to coexist (conforma- orthogonal to the open-to-closed conformational transition. Of tional selection can be classified as a special case of induced fit; all of the regions, the Gly-rich loop has the largest movement. induced fit can also be regarded as a special case of confor- The distances of the movement of Ser53 (distance between con- mational selection). From a structural perspective, the ligands ˚ formation 1 and conformation 5) in PC1 are 11.438 A (ATP generally trigger the PKAc conformational transition from open to PKAc), 9.433 A˚ (PKI to PKAc), 13.604 A˚ (ATP to PKAc- to closed state; however, the details of the binding mechanism PKI), and 6.560 A˚ (PKI to PKAc-ATP). We then compared the are elusive because of the highly intrinsic dynamic behavior of results of PCA with experimental structures, illustrated in Fig. 5. PKAc. For PKI, the high-affinity binding PKAc inhibitor, the The open extents of Gly-rich loop of ATP binding to PKAc and experimental X-ray crystallography and thermodynamic titration PKI binding to PKAc are similar to that of 1CMK, the structure approach have already underscored its strong inhibition inter- of apo PKAc. The behavior of conformation 1 of Gly-rich loop actions with PKAc and addressed the associated binding as an during PKI binding to PKAc-ATP is similar to that in the crys- enthalpy-driven process in contrast to the general PKAc bind- tal structure of the intermediate state (1BX6). However, open ing substrates, such as PLN, of which the binding to PKAc is extent of the Gly-rich loop of ATP to PKAc-PKI is a bit higher an entropy-driven process preferentially exhibiting a conforma- than that of 1CMK, which is consistent with the open to more tional selection scenario (14, 15). open transition. Conformation 5 of both ATP binding and PKI To clearly address the underlying binding mechanisms of binding processes is similar to 1ATP. These results are consis- PKAc with different binding ligands (ATP/PKI), we addition- tent with the results of distances distributions during binding (SI ally calculated the evolution of populations of open, interme- Appendix, Fig. S6). diate, and closed states of PKAc, individually classified by the critical distance between residues 53 and 166, along the bind- Mixed Binding Mechanism of PKAc. There are two existing mech- ing reaction (Qinter ) during different subprocesses (Fig. 6). As anisms to well-describe the conformational changes of protein Qinter increases with ligand binding proceeding, the populations that occur during ligand binding: induced fit (46, 47) and con- of all of the three conformational states (open, intermediate, and formational selection/population shift (48). “Induced fit” mech- closed) of PKAc change progressively, indicating that the con- anism refers to ligand binding that drives a ligand-free (usu- formational dynamics of PKAc is constantly modulated by the ally open) enzyme toward the activated conformation (usually ligand interactions during binding. Our analyses unambiguously closed). “Conformational selection” refers to protein selecting have pictured that all of the binding processes of PKAc fall into the bound conformational from the multiple functional states the framework of the typical induced fit scenario. However, our

E7964 | www.pnas.org/cgi/doi/10.1073/pnas.1702599114 Chu et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 ahasfo h einn U oteed()bnigsae As stage. binding (B) end the to (U) beginning the from pathways Step. I its of mechanisms dynamics. because conformational binding mainly intrinsic different complex ligands, binding use different simulation to accommodate our capability to and the investigations has previous results, the protein on the addition, based binding In popu- PKAc, dynamically. PKAc the PKAc induces in in selection constantly preexisting role ligands lations conformational of critical binding a and the plays as fit processes, fit induced induced the both processes behaviors, binding have ligands PKAc form the to of Although ATP conformation. to embrace site closed and active accommodate a at then, open more and be ATP PKAc to recruit that induced find be we to results, has with our conformation accompanied In fit (20). induced (cracking) unfolding be local to binding found ATP, theoretically For been scenario. the has fit biasing induced significantly the lures to fly mechanism PKI– strong binding strong and mechanism, radii the fly-casting capture the that big use with find affinity) we theo- (high results, previous interactions our of PKAc In prediction (49). the studies binding as of retical similar scenario fit is induced which an mechanism, adopt to favor potentially may fit induced dom- the of mechanism. being mixture fit inant induced a the is with believe selection, PKAc conformational and of and mechanism binding binding during the PKAc that the of followed evolution closely (conforma- we conformational shift Therefore, population mechanism. a selection) to PKAc to tional starting of led the speculatively states linking points three simply ending therefore, the interactions; of binding populations con- by points the ending the change while PKAc, sequently of states closed inter- and a open, mediate, exhibit binding spreading of distribution points conformational starting dynamic the wide of all that show also results dif- contains PKAc-ATP) to complex. the PKI ligand (Q by (Q or states stage classified PKAc-PKI bound initial are to ferent the (ATP states that binding The note ligand (AB)]. Please PKI second PKAc-ATP 166. (PB), to states PKAc-PKI PKI 53 to ATP distance and (UA), (UB), PKAc PKAc to [ATP to processes binding the during 6. Fig. h tal. et Chu A 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0 0 0 0 1 h togitrhi neatosbtenPIadPKAc and PKI between interactions interchain strong The aha sFse,adAPBnigSbrcs steRate-Limiting the Is Subprocess Binding ATP and Faster, Is Pathway 0 . 04 . 081 0.8 0.6 0.4 0.2 0 hr r orpsil upoessadtopossible two and subprocesses possible four are There PKI toPKAc-ATP PKI toPKAc ATP toPKAc-PKI ATP toPKAc h ouain foe O,itreit I,adcoe C states (C) closed and (I), intermediate (O), open of populations The inter bu . tts09)o h Kcadtefirst the and PKAc the of 0.95) states 0.8 about Distance 53-166 OIC Q inter inter )o the of 0) = al .Kntcdt ffu upoesso T n K binding Subprocess PKI and ATP of subprocesses four of data separately Kinetic 2. Table Asp241 of contacts the (SI complex, complex ternary ternary In S13D). the Fig. of in Appendix, process place evolution takes the situation of beginning similar the A highest complex complex. the ternary with ternary in contacts probabil- in forms probability Asp241 the significantly B). S13 However, Fig. decreases Appendix , and ). S14 contacts (SI Arg18, Fig. these Arg15, Appendix , of and (SI ity Glu331, PKAc PKI of between of Glu334 Arg19 contacts and opposite-charged Glu333, Glu332, strong many Appendix (SI In are complex complexes. binary ternary of and stages binary capture within PKAc and PKI PKI between between interactions the to capture linkage in ATP. the binding direct and on PKI ATP no of of has stage effect con- process the early no that the implying almost in complex, is ATP ternary there and However, PKI PKAc stage. between between capture tact ATP the contacts of at electrostatic existence PKI nonnative and the the Therefore, affect complex. may ternary higher in much is that complex nonna- than binary of in number contacts the opposite-charged the However, tive through 45). forces (44, biomolecular driving mechanism as in fly-casting addressed observed and widely process are tran- recognition bridges These binding. salt for step formed role siently initial important an the play contacts At (Q charged S12). binding Fig. and PKI the native , Appendix of of into (SI all classified contacts complex, were binary PKI nonnative in and than PKAc complex between ternary contacts in PKI of ing much PKAc by time. captured each a is As slowly PKI process. more ATP, of capture existence the binding the is with complexes PKI result, ternary between and difference binary in main rate the Therefore, binary. cap- in of number The the ternary. tion, in (n that mean times of the ture one-half 2), complex about ternary Table is in in binary that AB with complex and binary (UP in binding PKI in the capture, described pro- of are definitions escape evolution (the and and process escape, processes evolution one capture only of but cesses is times process many binding each the of run, on composed kinetic simulations each In kinetic separately. performed subprocess we subprocess, Binding binding the Determine PKI. of Interactions Rate Charged Oppositely Nonnative the than tial hs fA n P epciey(al ) ae nbt free both on the Based results, 2). rate (Table kinetic respectively and UP, energy and AB of those the both for in step I (UA/PB rate-limiting binding the subprocess is of binding 3B) data Fig. ATP simulated Moreover, the with above. consistent barriers is result This way. the of rate times method 18 analysis about constant that rate is obvious the is using it 3, 40), and (39, 2 Tables in listed usaelse.Teuisof units The listed. are runs PB UA AB UP P h otc asaeilsrtdt nlz h interactions the analyze to illustrated are maps contact The bind- slower much for reason the out figure further to Aiming ohtema au n h Eo ahkntcqatt n20individual 200 in quantity kinetic each of SE the and value mean the Both aha,bcuekntcrtso AadP r oe than lower are PB and UA of rates kinetic because pathway, FPT I iigt lcdt h seta neatoso each of interactions essential the elucidate to Aiming A I cap P evo aha smc ihrta hto the of that than higher much is pathway 1.03 4.28 1.43 0.71 pathway. ssmlrbtenbnr n enr.I addi- In ternary. and binary between similar is ) fPIbnigi enr sabtlwrta that than lower bit a is ternary in binding PKI of k inter ± ± ± ± UPB FPT 0.02 0.01 0.01 0.01 PNAS FPT .Teerslssgetta h kinetic the that suggest results These ). on ewe o01,nnaieopposite- nonnative 0.1), to 0 between k × × × × on UAB | 10 10 10 10 /MPT ulse nieAgs 0 2017 30, August online Published 3 5 3 3 smc ihrthan higher much is cap /FPT I A 85.72 59.24 47.57 14.82 FPT aha smr preferen- more is pathway evo MPT .Comparing Appendix). SI r picoseconds. are ± ± ± ± on cap .09.18 0.20 .134.39 0.01 .930.95 34.84 1.99 0.70 I A fPIbnigin binding PKI of ,there S13A), Fig. , aha n the and pathway k UPB FPT I P | (k ± ± ± ± evo path- E7965 UAB 0.04 0.30 1.08 1.14

PHYSICS BIOPHYSICS AND PNAS PLUS COMPUTATIONAL BIOLOGY Table 3. Kinetic data of two binding pathways mean FPTon of ATP binding to PKAc-PKI is about 20 times apo Pathway Rate that of ATP binding to PKAc. The time of evolution part for ternary is about three times that for binary; however, it will UAB (IA) 0.175 have little effect on the whole binding time. Although the mean −2 UPB (IP) 0.964 × 10 MPTcap of PB subprocess is slightly lower than that of UA sub- process, the mean times of escape (mean nesc ) of ternary are The binding pathway with PKAc-ATP as intermediate (from U via A to B stage) is denoted as UAB (I pathway). The binding pathway with PKAc-PKI much higher (more than seven times) than that of binary (shown A SI Appendix as intermediate (from U via P to B stage) is denoted as UPB (IP pathway). in , Fig. S16). As a result, it seems that ATP is cap- The units of rate are nanoseconds−1. tured by apo PKAc and PKAc-PKI with similar rates, but it is hard for ATP to reach the final successful binding pose with the existence of PKI. contribute significantly to the evolution binding process. Glu331- Both native and nonnative contacts between ATP and PKAc, 334 and Asp241 are located at “top” and “bottom” of PKAc (SI and between ATP and PKI are analyzed for the binding sub- Appendix, Fig. S14), respectively. We examined the probability processes of ATP binding to PKAc and ATP binding to PKAc- of forming contacts between PKI and the two parts of PKAc, PKI. Compared between ATP binding (SI Appendix, Fig. S17) respectively. As shown in SI Appendix, Fig. S15, the probability and PKI binding (SI Appendix, Fig. S12), there are barely any of forming contacts between the tail of PKI and C terminus of interactions between ATP and PKI during the beginning part PKAc decreases significantly in the capture stage of PKI bind- (encounter state) of PKI binding to PKAc-ATP. However, there ing in ternary complex. Consequently, it seems that the contacts are a few interactions between ATP and PKI during the begin- between the flexible C terminus of PKAc and the flexible tail of ning of ATP binding to PKAc-PKI. As shown in SI Appendix, Fig. PKI are helpful to the binding of PKI. In ternary complex, the S18A, in encounter complex, there is a native contact between existence of ATP stabilizes the C terminus of PKAc, which may ATP and Arg18 of PKI with high probability. The interactions hamper PKAc from capturing PKI. between ATP and PKI at early stage of ATP binding to PKAc- In addition, although the α helical segment of PKI (5–13) is PKI may lead to a faster capture process (attract ATP to PKAc); the high-affinity binding region, the coil tail part of PKI (14–24), however, these interactions will also lead to much more times of which is highly positively charged, accelerates the binding in the wrong binding attempt (make ATP stay at the wrong place from initial capture stage, contributing to the binding process as an successful binding) (SI Appendix, Fig. S18B). effective anchor. Essential Regions Are Different at Different Stages of Binding. PKI Blocks ATP from Finding the Right Binding Pose. The barrier To extract the crucial residues at different stages of binding of ATP binding to PKAc-PKI (PB) is much higher than that of (encounter, transition state, and bound stages), we calculated ATP binding to PKAc (UA), implying that the kinetic rate of the number of contacts formed between PKAc and ATP, and Pn ATP binding with presence of PKI is slow. As shown in Table 2, between PKAc and PKI ( j Pij ; here, Pij is the probability of

ATP to PKAcATP to PKAc-PKI PKI to PKAc PKI to PKAc-ATP A

EC state

ATP

PKI B

TS state

C

Bound state

0 max

Fig. 7. The probability score map of each residue of PKAc (15–350) in different binding subprocesses and at different stages: encounter (EC; A), transition state (TS; B), and bound (C) stages. ATP is illustrated in gray spheres, and PKI is illustrated in brown cartoons.

E7966 | www.pnas.org/cgi/doi/10.1073/pnas.1702599114 Chu et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 h tal. et Chu aha ihitreit tt) oee,terslsshow results the however, state); P different intermediate with with pathways pathway lig- two (I two the are states inter- of There intermediate (open, process characterized. binding states are the conformational ands during three closed) and and B) mediate, and stages/basins binding P, for and four A, applied runs landscapes, (U, and energy thermodynamic developed free was the performing exploring binding By ligand dynamics. its molecular as well as dynam- PKAc ics investigating for model coarse-grained weighted A Conclusion stabil- (PIF) the fragment of “PDK1-interacting 51). increase the (50, the forming pocket” bind- to and of PKAc leading stages of ), S7 late ity Fig. the Appendix, at sharply (SI decreases ing terminus C of ation fluctu- conformational the However, pep- interactions. protein–ligand loop, Gly-rich and binding, loop, positioning PKI tide of For protein–ligand significantly. proportions for decrease and nus essential catalytic scores are loop, 7C The loop Gly-rich (Fig. interactions. positioning binding, Mg complexes ATP and ternary For loop, and S22). Fig. binary Appendix, between of difference results any barely the is There structures). (crystal state 7B). Fig. in (illustrated significantly decreases terminus Gly- C are of tion regions important and loop, the positioning con- peptide loop, binding, our rich PKI with consistent For is above. in which clusion PKAc-PKI, binding to ATP binding ATP with for of Compared scores essential. the complex, are binary terminus C and loop, Gly-rich binding, residue between contact of formation i.S21 Fig. , Appendix (SI stage encounter at that than be also analysis. can map results contact our These in increases. score found the loop the addition, positioning In of terminus. peptide C score the the of of the that of than PKAc-ATP, higher that is to region than binding higher PKI is For terminus 7A C (Fig. complexes of ternary and binary form and to binding ATP of for stability the of increase the the at to sharply leading decrease PKAc. binding, terminus of C stages the late of of the fluctuation vicinity above, and mentioned the as in rmsd However, not pocket. of is binding stage terminus ATP/PKI late termi- C the the C the at because significant the not possibly between are binding, ATP/PKI interactions and other PKAc direct of with the nus Compared PKI. PKAc, and of in loop function essential regions the positioning are for peptide loop essential ATP; are positioning loop of Mg function and the loop the for C and binding. binding, PKI ATP for for illus- crucial are is and systems loop 7 different Fig. in in binding trated for regions important the j residue of contacts formed .ZegJ ta.(93 rsa tutrso h yitltdctltcsbnto camp- of subunit catalytic myristylated the of structures Crystal (1993) al. et J, Zheng 3. .Bseee ,Eg ,Kne ,PntnlH ue 19)Popornfrs and Phosphotransferase (1993) R Huber H, Ponstingl V, Kinzel R, Engh D, Bossemeyer 4. kinase. protein camp-dependent of substrates Physiological (2001) kinase JB protein Shabb The (2002) S 2. Sudarsanam T, Hunter R, Martinez DB, Whyte G, Manning 1. stersdeo T rPI h nomto fsoe and scores of information The PKI. or ATP of residue the is tbudsae idn oei iia ota nnative in that to similar is pose binding stage, bound At ttasto tt,tesoe fms ein r uhhigher much are regions most of scores the state, transition At essential are terminus C and loop Gly-rich stage, encounter At eedn rti iaerva pnadcoe conformations. closed and open reveal kinase protein dependent rmpriehata eue rmte20asrcueo h ope ihmn2+ with complex the of structure (5-24). a pki 2.0 peptide inhibitor the and from imidodiphosphate deduced adenylyl as subunit heart catalytic porcine kinase protein from camp-dependent the of mechanism binding substrate 1573. Rev genome. human the of complement .FrPIbnigt Kc h score the PKAc, to binding PKI For S20). Fig. Appendix, SI 101:2381–2412. vrl,teGly-rich the Overall, S19. Fig. Appendix , SI A ,ctltclo,M oiinn loop, positioning Mg loop, catalytic αD, aha ihitreit state; A intermediate with pathway opaeipratrgosfor regions important are loop αF–G i .Here, ). n emnsaeabthigher bit a are terminus C and αD Science 298:1912–1934. i stersdeo Kc and PKAc, of residue the is i op h propor- The loop. αF–G and opi crucial is loop αF–G opregion. loop αF–G j ; n n termi- C and αD MOJ EMBO stenme of number the is rti Sci Protein loop αF–G 12:849–859. .FrATP For ). αF–G and 2:1559– Chem I SI P n hn ototrlSineFudto rn 2016M590268. Grant Sci- Foundation of 2013YQ170585, Science and Ministry Postdoctoral 2016YFA0203200 China 21603217, Grants and and China of 91430217 Technology Grants and ence China of Foundation ence are settings and ACKNOWLEDGMENTS. steps detailed The nm. chains 5 two the than of in farther mass introduced of is to center complex the set each between poten- was distance harmonic of strong the step a if were time events, added bonds binding was MD of all tial sampling The the and (53). enhance nm, To algorithm fs. 3.0 2.0 LINCS to the set using was constrained interactions coefficient sys- nonbonded friction the for constant by the performed of cutoff with were coordinate (52), equations 4.5.5 Langevin initial with Gromacs the simulations using MD provide of the to results of 1ATP used All detailed structure tem. was more complex 15–350) obtain PKAc (residue The to (5) binding. performed Moreover, during was 43). change runs (42, conformational kinetic method of metadynamics series sim- with a thermodynamical PKI, performed and PKI, were PKAc within between ulations temperature and ATP, simulated and of PKAc parameters simu- between the for tuning constructed binary After were PKAc-PKI lations. complex) and ternary PKAc-ATP PKAc-ATP-PKI (apo, and systems complexes, different four phos- Then, Mg the charges. Each ATP. represented for (CPA) charges in charges negative shown negative (as (CPB) two charge group negative taking phate ribose one bead taking the beads one represented two beads and while CA ring, neutral adenine electrically and two sugar and bead, five by l ftePA tutrsi h D.Ec eiu fPA n K was PKI and PKAc of residue Each the PDB. on the located on bead, in based one structures developed by represented PKAc was PKAc the of of (29–31) all SBM coarse-grained weighted A Methods stability the the increasing However, consequently at binding. binding, PKAc. sharply of of of decreases stage stages terminus late late stage C the the early of the at fluctuation at not conformational significant ter- but are C binding ATP/PKI between of and interactions PKAc position- the of peptide regions, minus ATP; other positioning of with Mg function Compared and the and loop loop for ing Catalytic essential bind- binding. and are of PKI binding, loop ATP stage for for every crucial crucial is For is loop levels. Gly-rich binding bind- ing, different and that with subprocesses regions change essential ligand ing with The interactions dominant. strong being have fit selection, induced conformational mixed some the and a also, fit with suggest induced but results of pocket Our mechanism pocket. binding binding binding the the only of to not distal vicinity affect regions the can binding in transition ligand regions open addition, the the In more contain state. not to open does open binding more PKI an However, binding. has ATP which in during PKAc. occurs loop, in states Gly-rich state different the closed between change to conformational open Main from change conformational cal data. tal the that the than indicate able results above success- the from of ATP hamper addition, will In PKI binding. and ful ATP of tail the the between than rate binding higher much the that .Nryn ,CxS un h e ykL,Tyo S(97 iaycmlxo the of complex binary A (1997) SS Taylor LF, Eyck Ten Nh, Xuong S, Cox N, Narayana 6. 2.2 (1993) al. et J, Zheng 5. .Kiho R ta.(91 rsa tutr ftectltcsbntof subunit catalytic the of revealed kinase protein camp-dependent structure of features Dynamic (2003) Crystal al. et P, Akamine (1991) 8. al. et DR, Knighton 7. lo sn MFadPAmtos eso h dynami- the show we methods, PCA and RMSF using Also, aayi uui fcm-eedn rti iaeadaeoiefrhrdefines further adenosine and kinase flexibility. protein conformational camp-dependent of subunit catalytic inhibitor. peptide Crystallogr a Biol and D mnatp tallogr with complexed kinase protein dependent yaonyecytlstructure. crystal apoenzyme by kinase. protein monophosphate-dependent 414. adenosine cyclic I A IAppendix SI aha a uhlwrtasto are n a and barrier transition lower much a has pathway I opaeesnilfrtefnto fPKI. of function the for essential are loop αF–G P aha,wihi ossetwt h experimen- the with consistent is which pathway, hssuywsspotdb ainlNtrlSci- Natural National by supported was study This endcytlsrcueo h aayi uui fcamp- of subunit catalytic the of structure crystal Refined a 49:362–365. ˚ PNAS Structure . IAppendix SI k UAB o Biol Mol J | 5:921–935. ulse nieAgs 0 2017 30, August online Published 2+ saot1 ie htof that times 18 about is o a n edwt w positive two with bead one was ion .Teewr,i oa,four total, in were, There S3). Fig. , 327:159–171. I α P abnao.APwsreplaced was ATP atom. carbon I aha.Teinteractions The pathway. A aha smr favor- more is pathway Science loop αF–G γ k = UPB caCrys- Acta .The 1.0. | 253:407– E7967 All .

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