892 | Mol. BioSyst., 2017, 13, 892--900 This journal is©The Royal Society of Chemistry 2017

Cite this:Mol. BioSyst., 2017,

13, 892

Molecular dynamics simulations elucidateconformational selection and induced fitmechanisms in the binding of PD-1 and PD-L1†

Wenping Liu,‡ab Bing Huang,‡a Yashu Kuangb and Guangjian Liu*b

Blockage of the interactions between immunologic checkpoint protein PD-1 and its ligand PD-L1 showed

efficacy for cancer treatment. X-ray structures have captured static conformational snapshots of PD-1 and

revealed that the CC0 loop adopts an open conformation in the apo-protein but turns into a closed form

and interacts with PD-L1 in the complex. This structural heterogeneity brings difficulties for structure-

based drug discovery targeting PD-1. To gain insights into the role of the CC0 loop in molecular

recognition, we have undertaken a comparative study between the open and closed conformations in

apo-PD-1 and the PD-1/PD-L1 complex using molecular dynamics simulations. Results show that the

moderate stability of intramolecular hydrogen bonds between SER71 and THR120 allows the CC0 loop to

sample both the open and closed states in apo-PD-1. Binding of PD-L1 accelerates the open-to-closed

switch and locks the loop in the closed state through four newly formed intermolecular hydrogen

bonds. Thus, we suggest a complex binding mechanism between PD-1 and PD-L1 where both the

conformational selection and induced fit theories play a role.

Introduction

Cancer is a leading cause of death worldwide.1,2 The humanimmune system has an important role in controlling cancerbut cancer cells avoid immune surveillance by overexpressingnegative immunologic regulators.3 One of the immunosuppres-sion mechanisms involves interference with the immunologiccheckpoint receptor on immune cells, for example, the pro-grammed death receptor 1 (PD-1).4,5 PD-1 is a 288 amino acidtype I transmembrane protein, which is expressed in activatedT cells, B cells, and macrophages.5 Programmed death ligand 1protein (PD-L1), a type I transmembrane protein belonging tothe B7 family, can bind to PD-16 and exert immunosuppressiveeffects by inhibiting T cell proliferation and cytokine productionof IL-2 and IFN-g.7 Under normal conditions, this complexationrestrains the immune system against targeting self-antigens.However, PD-L1 is often overexpressed in tumors includinglymphoma, melanoma, lung and breast cancer, glioblastoma,ovarian and kidney tumors, and bladder cancers, protecting

cancer cells against the hosts’ immune system.8,9 Even more,the PD-1/PD-L1 interaction inhibits T lymphocyte proliferation,survival and effector functions, resulting in exhaustion andapoptosis of tumor-specific T cells.10,11

Blockage of the PD-1/PD-L1 interactions restores the attenuatedimmune response and leads to increased anti-tumor and anti-viralactivities.4,12 So PD-1 and PD-L1 become potential targets forcancer immunotherapy.4,13,14 Several antibodies or Ig fusionproteins, like MDX-1106, MK3475, CT-011 and AMP-224 target-ing PD-1, are undergoing phase I and II clinical trials forvarious cancers and showed efficacy for non-small-cell lungcancer, melanoma, and renal-cell cancer.13,15–17

A critical step in therapeutic drug design is to define the hot-spot pockets in the interface of PD-1 and PD-L1. Krzysztof M. Zaket al. reported the crystal structure of the PD-1/PD-L1 complex in2015, providing us with detailed interface information at theatomic level.18 The CC0 loop (residues MET70-ASP77) is found toadopt an open conformation with the side chains directed awayfrom the interface in apo-PD-1, while in the complex it turns intoa closed conformation through a 901 twist and 5 Å displacementand interacts with PD-L118 (Fig. 1). This crystallographic observa-tion brings confusion for structure-based drug discovery targetingPD-118 and raises the following questions: is the CC0 loop inthe open or closed conformation stable or transient? Does theopen-to-closed switch occur spontaneously (according to theconformational selection theory) or is it induced by the ligand(according to the induced fit theory)?

a School of Bioscience and Bioengineering, South China University of Technology,

Guangzhou 510006, Chinab Division of Birth Cohort Study, Guangzhou Women and Children’s Medical

Center, Guangzhou Medical University, Guangzhou 510623, China.

E-mail: liugjcn@163.com

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7mb00036g‡ Wenping Liu and Bing Huang contributed equally to this work.

Received 13th January 2017,Accepted 13th March 2017

DOI: 10.1039/c7mb00036g

rsc.li/molecular-biosystems

MolecularBioSystems

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Figuring out the answers to these questions requires not onlyelaboration at the structural level but also an understanding ofthe dynamic aspects of the PD-1/PD-L1 interaction. Moleculardynamics (MD) simulation is well suited for studying thedynamics of proteins by exploring the possible conformationalstates and has been widely used in protein folding, assembly,allostery and protein–protein interactions.19–22 Here, to fullycharacterize the role of the CC0 loop in molecular recognitionbetween PD-1 and PD-L1, we used MD simulations to examinethe conformational dynamics of this region. In doing so, we fullysampled the conformational fluctuations of the loop beforeand after binding to PD-L1. Significant structural flexibility ofthe CC0 loop in the apo-protein was observed in both the openand closed conformations, but the binding of PD-L1 acceleratedthe conformational switch from the open to closed form andeventually locked it in the closed state. Furthermore, we identi-fied the most critical hydrogen bonds (H bonds) during thestructural rearrangement. Our results suggest a complex associa-tion mechanism where both the conformational selection andinduced fit mechanisms play a role.

MethodsSystem setup

For our MD simulations, we have used the available crystalstructures of apo-PD-1 (Protein Data Bank code 3RRQ) and thePD-1/PD-L1 complex (Protein Data Bank code 4ZQK18). In thecrystal structures, residues 85–92 of PD-1 were missing. Weused the SWISS-MODEL server to add the missing residues aswell as the hydrogen atoms.23,24

Four states of PD-1 in the open and closed conformations withor without PD-L1 have been constructed from crystal structures,designated as open-unliganded, closed-liganded, closed-unliganded,and open-liganded, as described in this section. The open-unliganded state used 3RRQ as its initial structure, in which theCC0 loop displays an open conformation. The closed-ligandedstate started from 4ZQK whose CC0 loop closes around PD-L1. Theclosed-unliganded and open-liganded structures were not avail-able and therefore have to be modeled. For the closed-unligandedstructure, the initial structure was obtained by directly removingPD-L1 from 4ZQK (Fig. 1a). For the open-liganded structure, we

superimposed 3RRQ with 4ZQK by least-squares fitting of thehydrophobic b-sheets of PD-1, and then transferred PD-L1 to theopen structure (Fig. 1b). The reconstructed models of the open-liganded PD-1/PD-L1 complex were optimized by three minimiza-tion stages: first 2000 minimization steps with all of the atoms ofPD-1 being constrained, then followed 2000 minimization stepswith only the backbone of PD-1 being constrained, and the last5000 minimization steps with all atoms unconstrained. Afterminimization, the key intermolecular contacts except the CC0

loop region were found to be preserved (ESI,† Table S1).The N- and C-terminal residues were acetylated and amidated,

respectively, to mimic the continuation of the protein chain. Thecrystallographic water molecules were retained. Each systemwas solvated with TIP3P water molecules in a rectangular box(roughly 7.6 � 5.7 � 6.7 nm) and neutralized at a 150 mM saltconcentration, containing approximately 27 000 atoms in total.The walls of the four water boxes were at least 15 Å away fromany protein atoms.

MD simulations

Two software packages, VMD 1.9.2 for visualization andmodeling25 and NAMD 2.11 for energy minimizations and MDsimulations,26 were used here. The CHARMM36 all-atom forcefield,27,28 along with the cMAP correction for the backbone, theparticle mesh Ewald algorithm for electrostatic interaction anda 12 Å cutoff for electrostatic and van der Waals interactions,were used to perform MD simulations with a periodical boundarycondition. All hydrogen bonds were constrained with the SHAKEalgorithm,29 allowing a time step of 2 fs. Each system was energy-minimized for 5000 steps with all protein atoms constrained,for 10 000 steps with only the backbone constrained andfor another 15 000 steps with all atoms free, respectively.Then each system was equilibrated thrice for 20 ns followedby three 50 ns production phases, for a simulation time of840 ns in total for the four systems. The temperature was heldat 310 K using Langevin dynamics, and the pressure was held at1 atmosphere by the Langevin piston method. The atomiccoordinates were written every 2 ps.

Data analysis

Time-curves of the root mean square deviation (RMSD) of heavyatoms were used to illustrate the stabilities of the structures

Fig. 1 Initial structures used in the four simulation systems. (a) The closed-unliganded structure (silver cartoon) was overlaid on the open-unligandedstructure (green cartoon) by aligning the central hydrophobic b-sheets. (b) The closed-liganded PD-1/PD-L1 structure was overlaid on the open-liganded complex. PD-L1 in the closed-liganded complex is shown in yellow cartoon and that in the open-liganded complex is removed for clarity. Thestructural rearrangement of the CC0 loop is circled by black dotted lines.

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and the conformational changes of the CC0 loop comprehen-sively, including both the twist and displacement. When ana-lysing the RMSD for the CC0 loop, the hydrophobic sheets ofPD-1 were aligned. The Ca root mean square fluctuation (RMSF)pattern was used to mark the local structural flexibilities.Principal component analysis (PCA) of the CC0 loop was carriedout by using the Normal Mode Wizard (Version 1.0) module ofVMD 1.9.2.30 A hydrogen bond (H Bond) was defined if thedonor–acceptor distance and the bonding angle were less than3.5 Å and 30 degrees, respectively. The number of H bonds acrossthe interface of the complex and their donor–acceptor distancewere detected with in-house scripts. All visual inspection andmolecular images were completed using VMD 1.9.2.

Results and discussion

We first carried out the analysis of RMSD to rationalize oursimulations. The general picture is that the RMSD for the wholecomplex in all simulations of the four systems developed into astable plateau in the equilibration phases (ESI,† Fig. S1) indicatingsufficient stability of the trajectories for further analysis of thestructural dynamics. Variations in RMSD for PD-1 excluding theCC0 loop were also calculated, which showed little and similarfluctuations for all four systems (ESI,† Fig. S2). This meansthat the dynamics of the rest of PD-1 were not significantlyinfluenced by the conformational change of the CC0 loop and/or the binding of PD-L1. Next, we studied in detail conforma-tional fluctuations for the CC0 loop in each state as well as theaccompanying H bond disruption and formation during the 50 nslong production phase.

The details of the molecular systems used in the MD simula-tions can be found in the Methods section. Here, we haveperformed several short runs for each system rather than a singlelong one because several studies have suggested that in certaincases the former protocol can better capture the dynamics of theprotein than the latter one.31,32 The chemical timescale used hereis long enough for the local motions of the eight-residue CC0 loopand for rearranging various conformations.33,34 We have chosenRMSD and H bond measures in the present study because theyare probably the most widely used measures for the structuralcomparison of biomolecules and are sufficient to depict theconformational changes of this short loop.35,36

Both the open and closed conformations exist in apo-PD-1

To understand the stability of the CC0 loop in the open con-formation, we have performed three independent MD simula-tions of 50 ns for the open-unliganded PD-1. From the time-curves of RMSD in simulation run 1, it can be seen that the CC0

loop remained stable at the first 30 ns and then deviated fromthe initial open conformation with the RMSD between themsharply rising to 5 Å and fluctuating around this value until theend (the black line in Fig. 2a). Meanwhile, the RMSD relative tothe closed conformation decreased hastily at the same time of30 ns and then gradually decreased to about 2 Å (the red line inFig. 2a). The changes in RMSD indicated that the CC0 loop is not

stable in the open conformation in solution and is able to reachthe closed conformation in the absence of the ligand. Thisindication was confirmed in the other two runs of our simula-tions (Fig. 2b and c), as well as in the simulations of a recentstudy.37 Moreover, the open-to-closed switch was intuitivelyillustrated by a superimposition of the crystal structures withthe snapshots of PD-1 at 50 ns of run 1, 34 ns of run 2 and 28 nsof run 3 (Fig. 2d).

We next sought structural insights into the flexibility of theCC0 loop through analyzing its interactions with the remainderof PD-1. In the crystal structure of apo-PD-1, three H bonds existbetween MET70 and SER71 of the C strand and THR120 of the Fstrand in the antiparallel CF b sheet (Fig. 2e). It is of interest tofind that the occurrence of the open-to-closed switch was highlycorrelated with the stabilities of these H bonds. In the simula-tions, the H bonds SER71O:THR120N and SER71N:THR120OG1

were not very stable and they were disrupted (with donor–acceptor distances above 3.5 Å) when the loop adopted a closedconformation (Fig. 2f–h and ESI,† Table S2). However, theH bond MET70N:THR120O was stabilized below 3.5 Å through-out all three runs (Fig. 2f–h and ESI,† Table S2) and served as a‘‘stabilizer’’ to restrict the extent of loop deviation in the case oftotal separation. It is expected that the loss of this H bondwould increase the possibility of the formation of favorablecontacts with PD-L1. This was approved recently by the study ofRoberta Pascolutti et al., where the mutation Met70Glu inthe high-affinity consensus PD-1 allows the formation of twoH bonds and a salt bridge with Arg125 in PD-L1.37

We noticed that the closed conformation turned back to theopen form in simulation run 2 and run 3 (Fig. 2b and c). Thus, aconformational equilibrium between the open and closed states canbe expected for the CC0 loop in the unliganded PD-1. To confirmthis, we have performed three production simulations of 50 ns onthe closed-unliganded PD1 through taking PD-L1 away from thecrystal structure of the PD-1/PD-L1 complex (see Methods). Asexpected, the RMSD of the CC0 loop relative to the closed conforma-tion increased and decreased alternatively while the RMSD relative tothe open conformation changed conversely (Fig. 3a). The distancesbetween the main-chain nitrogen and oxygen atoms of SER71 andTHR120 also fluctuated correspondingly although at above 3.5 Å(Fig. 3b and ESI,† Table S2). It may be that the simulation timewas not long enough for the H bonds to reform. However, theinterconversion of the open and closed conformations of theCC0 loop could be clearly seen in the average structure ofthe simulation, which lies in the middle of the crystal struc-tures of the two states (Fig. 3c). These results were repeatedin the other two independent runs, further enhancing theirreliability (ESI,† Fig. S3).

PD-L1 stabilizes the CC0 loop in the closed conformation

As mentioned above, the CC0 loop is able to visit both the openand closed conformations in the absence of the ligand. Thismeans that PD-L1 could encounter PD-1 of either form insolution. From the crystallography experiments, the CC0 loopadopts the closed conformation around PD-L1 and forms fourintermolecular hydrogen bonds. We want to know whether the

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closed state in the complex is stable. Therefore, three MDsimulations were carried out on the closed-liganded systeminitiating from the crystal structure of the PD-1/PD-L1 complex.

Not surprisingly, the CC0 loop was very stable in the simulationsand its RMSD values relative to the open and closed conformationboth demonstrated less fluctuation (Fig. 4a–c). Most H bondsbetween the CC0 loop and PD-L1 found in the crystal structures,

especially GLN75OE1:ARG125N, GLN75NE2:ARG125O, GLN75NE2:ASP26OD1 and THR76OG1:ARG125NH1, remained stable throughoutat least one of the three runs, as reflected in the average structures(Fig. 4d–f) and donor–acceptor distances (ESI,† Fig. S4).

Overall, when PD-1 was unbound, the CC0 loop was con-formationally flexible to adopt a wide range of conformationsin addition to the open form seen in the static crystal structure

Fig. 3 Structural fluctuations in the closed-unliganded system. (a) RMSD of the CC0 loop relative to the open (black) and closed (red) conformation.(b) Donor–receptor distances for three hydrogen bonds between MET70, SER71 and THR120. (c) The averaged structure of the CC0 loop (purple) in thesimulation after 10 ns, which was aligned to crystal structures of the open (green) and closed (silver) forms. Movement of the CC0 loop from its initialclosed conformation to the open state is clearly discernible.

Fig. 2 Structural fluctuations in the open-unliganded system. (a–c) The root mean square deviation (RMSD) of all Ca atoms of the CC0 loop relativeto the open (black) and closed (red) conformation in three runs. (d) Snapshots of the CC0 loop at 50 ns of run 1 (red), 34 ns of run 2 (blue) and 28 ns ofrun 3 (purple). These structures were aligned to crystal structures of the open (green) and closed (silver) forms. (e) Hydrogen bonds (dotted lines) betweenMET70, SER71 on the C strand and THR 120 on the F strand in the open-unliganded and closed-unliganded PD-1. (f–h) Donor–receptor distances for thethree hydrogen bonds shown in (e) in three runs.

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and when bound to PD-L1, it was conformationally restricted inthe closed form. It suggests that conformational selection may playa role in the molecular recognition between PD-1 and PD-L1.38

PD-L1 induces the structural rearrangement of the CC0 loop

It is of interest to note that in our simulations of the open-unliganded system, the open conformation of the CC0 loop was apredominant state in solution (Fig. 2). The energy barrier betweenthe open and closed states may be a little high due to the existenceof the H bonds SER71O:THR120N and SER71N:THR120OG1. Sucha high energy barrier could lead to a slow conformationalconversion between the open and closed states in solution. Thisslow conversion raises the interesting question of how the CC0

loop changes its conformation upon complexation.To investigate the effect of PD-L1 binding on the dynamics

of the CC0 loop, MD simulations have been performed on theopen-liganded PD-1/PD-L1 complex (see Methods). In all threeindependent runs, the RMSD of the CC0 loop relative to the openconformation increased gradually in less than 10 ns accompa-nied by a decrease in the RMSD relative to the closed conforma-tion (Fig. 5a–c), suggesting a tendency for an open-to-closedconformational change. Obviously, the tendency occurred earlierand more steadily upon PD-L1 binding in comparison to that inapo-PD-1 (Fig. 2). It means that the energy barrier between openand closed was significantly lowered by PD-L1 and supported theproposed induced-fit mechanism of ligand binding.39

A careful analysis of the structures at the end of the simula-tions showed clear structural rearrangements such as twist anddisplacement of the CC0 loop (Fig. 5d–f). Moreover, the intra-molecular H bonds SER71O:THR120N and SER71N:THR120OG1

became unstable at first and broke eventually (Fig. 5g–i).This released the constraint on the CC0 loop and the loopmoved closer to PD-L1. As a result, some new intermolecular Hbonds were formed between the loop and PD-L1, includingASN74OD1:VAL23N, ASN74ND2:THR22OG1, and SER73OG:ASP26OD1

in run 1, GLN75NE2:TYR123OH, GLN75OE1:ARG125N, andASP77OD2:LYS124NZ in run 2, and THR76O:LYS124NZ, andASP77OD2:LYS124NZ in run 3 (Fig. 5d–f). These pairs wereseparated by distances of more than 5 or 10 Å in the startingstructure of the simulations, and got closer to 3.5 Å and evenlower as the simulations progressed (Fig. 6a–c). These H bondscan offer additional stability to the PD-1/PD-L1 complex, just asshown in Fig. 6d–f, where the addition of H bonds involved inthe CC0 loop totally accounted for the increase of H bonds atthe whole interface. Although the final structures of the CC0

loop and the newly formed intermolecular H bonds in oursimulations were not completely the same as those in the crystalstructure (ESI,† Table S2), we believed that the simulated con-formations represent the intermediate states in the induced-fitconformation optimization process.

In summary, from these test calculations we confirmed thatbinding of the ligand accelerates the switch of the open con-formation to the closed one. This switch is slow in apo-PD-1,but becomes fast when PD-L1 is bound.

A possible mechanism of PD-1/PD-L1 binding

Much of what is known about PD-1/PD-L1 binding has beenderived from structural studies of both apo-PD-1 and thecomplex. These studies have captured static conformational‘‘snapshots’’ of PD-1 with its CC0 loop at two different con-formations: an open-unliganded structure and a closed-ligandedform.18 In this study, to assess the role of those intrinsicconformational fluctuations in molecular recognition, we focuson elucidating the dynamics of the CC0 loop at the atomic levelby MD simulations. The simulation trajectory of each produc-tion run was collected together for each system to analyze thechanges in the conformational ensemble of the CC0 loop in apoand liganded PD-1. The resultant data enabled us to depicta general picture of the dynamic adaptation in the binding ofPD-1 and PD-L1.

Fig. 4 The CC0 loop in the closed-liganded system is kept stable in the closed form. (a–c) RMSD of the CC0 loop relative to the open (black) and closedconformation (red) in three runs. (d–f) The averaged structure of the CC0 loop in run 1 (red), run 2 (blue) and run 3 (purple). These structures werealigned to crystal structures of the open (green) and closed (silver) forms. The distances (Å) labeled in the figure are averaged over the entire length ofthe 50 ns trajectory.

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As shown in the distribution of RMSD relative to the closedconformation in Fig. 7a, the CC0 loop in both the open-unliganded and closed-unliganded systems visits two distinct

clusters of conformations peaking at about 2.5 Å and 5 Å, whichrepresent the closed and open state, respectively. Simulationstructures hint that conformational transitions involve two

Fig. 6 Newly formed intermolecular hydrogen bonds in the open-liganded system. (a–c) Donor–receptor distances for hydrogen bonds between theCC0 loop and PD-L1 in three runs. (d–f) The total number of intermolecular hydrogen bonds in the PD-1/PD-L1 complex (red) and those contributed bythe CC0 loop (black) in three runs.

Fig. 5 The open-to-closed switch of the CC0 loop in the open-liganded system. (a–c) RMSD of the CC0 loop relative to the open (black) and closed (red)conformation in three runs. (d–f) Structure of the CC 0 loop at the end of run 1 (red), run 2 (blue) and run 3 (purple). These structures were aligned tocrystal structures of the open (green) and closed (silver) forms. (g–i) Donor–receptor distances for three hydrogen bonds between MET70, SER71 on theC strand and THR120 on the F strand in three runs.

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intramolecular H bonds, SER71O:THR120N and SER71N:THR120OG1

(Fig. 2e and ESI,† Table S2), which might indicate a high energybarrier and lead to a slow equilibrium between different states.These data affirm the idea that the open and closed states

simultaneously exist within a dynamic ensemble in apo-PD-1.This has been suggested as a sign of the conformationalselection mechanism, as indicated in many other receptor–ligand binding systems.40–43

Fig. 7 The mechanism of PD-1/PD-L1 binding. (a) Distribution of RMSD relative to the closed conformation in various simulated systems and in thecrystal structures. (b) RMSF of the CC0 loop in four systems during MD simulations. The loop region is shown in a dashed rectangle. (c–f) Movementdirection of CC0 loop corresponding to the first eigenvectors obtained by performing PCA on MD simulation trajectories for the open-unliganded (c),open-liganded (d), closed-unliganded (e) and closed-liganded (f) systems. PD-1 was shown in a new cyan cartoon, with the Ca atoms of the CC 0 loopshown in VDW. The movement direction of each residue of the CC0 loop is indicated with yellow arrows. Colour variation from red to blue displays theincrease of B-factor, which represents the fluctuations of atoms. (g) A schematic of the reconstructed energy landscapes for the binding of PD-1 andPD-L1, and sketch maps of the CC0 loop, PD-1 and PD-L1 with hydrogen bonds among them shown in dotted lines.

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In the conformational selection model, PD-L1 binds to andstabilizes PD-1 whose CC0 loop is already in a closed conforma-tion. However, when PD-L1 encounters apo-PD-1 of open con-formations, it would seem that these conformations mustinteract with PD-L1 to form a stable complex within whichthe open form can change to the closed one. This tendencywas clearly observed in our simulations of the open-ligandedsystem, where the loop twisted and moved towards PD-L1 andalso provided the side chains that engage PD-L1, forming acomplementary contact surface (Fig. 5). Such features suggest atwo-step, sequential reaction mechanism: open conformationsquickly bind to PD-L1 forming the initial encounter complex;the complex then undergoes ‘‘catalyzed’’ structural rearrange-ments to intermediate structures, and subsequently, to thefinal complex as seen in the crystal structure and in oursimulations of the closed-liganded system (Fig. 4). Comparisonof the open-unliganded, open-liganded and closed-ligandedsimulations regarding the probability distribution of RMSDrelative to the closed conformation validated this suggestionthrough the moving of the peak from 5 Å to 4 Å and finally to3 Å (Fig. 7a). Obviously, addition of PD-L1 alters the distribu-tion of states significantly in a manner consistent with inducedfit.44,45 This was further confirmed by RMSF and PCA analyses.As shown in Fig. 7b, the CC0 loop in the open-liganded systemis more flexible with higher RMSF values than that in theopen-unliganded system because the intramolecular H bonds,especially the H bond SER71N:THR120OG1, are impaired, whilethe newly formed intermolecular H bonds are not stable yet(ESI,† Table S2). With no doubts, the CC0 loop in the closed-liganded system has the lowest RMSF values due to the stableintermolecular H bonds with PD-L1 (Fig. 7b). Fig. 7c–f describethe movement direction of the CC0 loop by the first eigenvectorsobtained by PCA analysis and suggest that PD-L1 bindingresults in a notable switch of the CC0 loop to its closedconformation. Fig. 7g presents a schematic of the reconstructedenergy landscapes for the binding of PD-1 and PD-L1. It is clearthat PD-L1 influences the barrier heights associated with theopen-to-closed transition, as exemplified by the observationthat the transition in the open-liganded system is much faster(less than 10 ns, Fig. 5) than that in the open-unligandedsystem (more than 20 ns, Fig. 2).

This work suggests a complex binding mechanism whereboth conformational selection and induced fit play significantroles, which has been reported for many other protein–proteinbinding systems.35,46–48 It is difficult to directly assess the roleof each of these two players. However, the balance betweenthe two might change under different conditions due to thedifferent entropic/enthalpic balance. For example, if the tempera-ture is high enough or a mutation occurs to break the H bondsbetween SER71 and THR120, the CC0 loop in the unligandedsystems should sample more configurational space of the closedforms and conformational selection will play a more importantrole. The reason for this deduction can be explained by thefact that residues SER71 and THR120 locate at the end of theCF b-hairpin, which is usually frayed and is most susceptibleto solvation invasion.49,50

Conclusion

Taken together, our results give a comprehensive picture of thelocal dynamics of the CC0 loop and the mechanism of bindingbetween PD-1 and PD-L1 in atomic detail. In addition, wecoupled the dynamic ensemble of the CC0 loop with the stabilityof H bonds. Two intramolecular H bonds between SER71 andTHR120 are moderately stable and their disruption expands theconformational space accessible to the closed state; while fourintermolecular H bonds between the loop and PD-L1 are highlystable and their formation induces or stabilizes the closed stateto generate a stable complex. As far as we know, this is thefirst time that the PD-1/PD-L1 interaction process has beendynamically reproduced in silico.

Some of our results, although needing to be further elucidatedby more research studies, have important ramifications in drugdiscovery and for gaining mechanistic insights into protein func-tionality. First, the conformational ensemble of the CC0 loop canbe used to improve structure-based approaches in drug discovery.Candidate drugs should be docked against representative struc-tures of the ensemble rather than only the crystal structure of theopen or closed state, in the hopes of identifying a conformationwith a high affinity for the ligand.15,33–35 Second, our observationsfurther suggested the importance of the flexibility of proteins.51–53

Flexible structures in the interface, such as the CC0 loop, mightplay important roles in fine-tuning the interactions between PD-1and PD-L1 according to the cellular environment. Mutations inthe key sites found in our simulations would alter the flexibilityas well as the function of PD-1.

Acknowledgements

This work was supported by the National Natural Science Founda-tion of China (Grant No. 31500591) and the Natural ScienceFoundation of Guangdong Province (Grant No. 2015A030310106).All simulations were supported by National Super ComputerCenter in Guangzhou.

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