Multilayered Computational Framework for Designing Peptide Inhibitors of HVEM-LIGHT Interaction.

The herpesvirus entry mediator (HVEM) and its ligand LIGHT play crucial roles in immune system regulation, including T-cell proliferation, B-cell differentiation, and immunoglobulin secretion. However, excessive T-cell activation can lead to chronic inflammation and autoimmune diseases. Thus, inhibiting the HVEM-LIGHT interaction emerges as a promising therapeutic strategy for these conditions and in preventing adverse reactions in organ transplantation. This study focused on designing peptide inhibitors, targeting the HVEM-LIGHT interaction, using molecular dynamics (MD) simulations of 65 peptides derived from HVEM. These peptides varied in length and disulfide-bond configurations, crucial for their interaction with the LIGHT trimer. By simulating 31 HVEM domain variants, including the full-length protein, we assessed conformational changes upon LIGHT binding to understand the influence of HVEM segments and disulfide bonds on the binding mechanism. Employing multitrajectory microsecond-scale, all-atom MD simulations and molecular mechanics with generalized Born and surface area (MM-GBSA) binding energy estimation, we identified promising CRD2 domain variants with high LIGHT affinity. Notably, point mutations in these variants led to a peptide with a single disulfide bond (C58-C73) and a K54E substitution, exhibiting the highest binding affinity. The importance of the CRD2 domain and Cys58-Cys73 disulfide bond for interrupting HVEM-LIGHT interaction was further supported by analyzing truncated CRD2 variants, demonstrating similar binding strengths and mechanisms. Further investigations into the binding mechanism utilized steered MD simulations at various pulling speeds and umbrella sampling to estimate the energy profile of HVEM-based inhibitors with LIGHT. These comprehensive analyses revealed key interactions and different binding mechanisms, highlighting the increased binding affinity of selected peptide variants. Experimental circular dichroism techniques confirmed the structural properties of these variants. This study not only advances our understanding of the molecular basis of HVEM-LIGHT interactions but also provides a foundation for developing novel therapeutic strategies for immune-related disorders. Furthermore, it sets a gold standard for peptide inhibitor design in drug development due to its systematic approach.

Integrating Explicit and Implicit Fullerene Models into UNRES Force Field for Protein Interaction Studies.

Fullerenes, particularly C60, exhibit unique properties that make them promising candidates for various applications, including drug delivery and nanomedicine. However, their interactions with biomolecules, especially proteins, remain not fully understood. This study implements both explicit and implicit C60 models into the UNRES coarse-grained force field, enabling the investigation of fullerene-protein interactions without the need for restraints to stabilize protein structures. The UNRES force field offers computational efficiency, allowing for longer timescale simulations while maintaining accuracy. Five model proteins were studied: FK506 binding protein, HIV-1 protease, intestinal fatty acid binding protein, PCB-binding protein, and hen egg-white lysozyme. Molecular dynamics simulations were performed with and without C60 to assess protein stability and investigate the impact of fullerene interactions. Analysis of contact probabilities reveals distinct interaction patterns for each protein. FK506 binding protein (1FKF) shows specific binding sites, while intestinal fatty acid binding protein (1ICN) and uteroglobin (1UTR) exhibit more generalized interactions. The explicit C60 model shows good agreement with all-atom simulations in predicting protein flexibility, the position of C60 in the binding pocket, and the estimation of effective binding energies. The integration of explicit and implicit C60 models into the UNRES force field, coupled with recent advances in coarse-grained modeling and multiscale approaches, provides a powerful framework for investigating protein-nanoparticle interactions at biologically relevant scales without the need to use restraints stabilizing the protein, thus allowing for large conformational changes to occur. These computational tools, in synergy with experimental techniques, can aid in understanding the mechanisms and consequences of nanoparticle-biomolecule interactions, guiding the design of nanomaterials for biomedical applications.

Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer.

The PD-1/PD-L1 complex belongs to the group of inhibitory immune checkpoints and plays a critical role in immune regulation. The PD-1/PD-L1 axis is also responsible for immune evasion of cancer cells, and this complex is one of the main targets of immunotherapies used in oncology. Treatment using immune checkpoint inhibitors is mainly based on antibodies. This approach has great therapeutic potential; however, it also has major drawbacks and can induce immune-related adverse events. Thus, there is a strong need for alternative, non-antibody-based therapies using small molecules, peptides, or peptidomimetics. In the present study, we designed, synthesized, and evaluated a set of PD-1-targeting peptides based on the sequence and structure of PD-L1. The binding of these peptides to PD-1 was investigated using SPR and ELISA. We also assessed their ability to compete with PD-L1 for binding to PD-1 and their inhibitory properties against the PD-1/PD-L1 complex at the cellular level. The best results were obtained for the peptide PD-L1(111-127)(Y112C-I126C), named (L11), which displaced PD-L1 from binding to PD-1 in the competitive assay and inhibited the formation of the PD-1/PD-L1 complex. The (L11) peptide also exhibited strong affinity for PD-1. NMR studies revealed that (L11) does not form a well-defined secondary structure; however, MD simulation indicated that (L11) binds to PD-1 at the same place as PD-L1. After further optimization of the structure, the peptide inhibitor obtained in this study could also be used as a potential therapeutic compound targeting the PD-1/PD-L1 axis.

Energy landscapes for proteins described by the UNRES coarse-grained potential.

The self-assembly of proteins is encoded in the underlying potential energy surface (PES), from which we can predict structure, dynamics, and thermodynamic properties. However, the corresponding analysis becomes increasingly challenging with larger protein sizes, due to the computational time required, which grows significantly with the number of atoms. Coarse-grained models offer an attractive approach to reduce the computational cost. In this Feature Article, we describe our implementation of the UNited RESidue (UNRES) coarse-grained potential in the Cambridge energy landscapes software. We have applied this framework to explore the energy landscapes of four proteins that exhibit native states involving different secondary structures. Here we have tested the ability of the UNRES potential to represent the global energy landscape of proteins containing up to 100 amino acid residues. The resulting potential energy landscapes exhibit good agreement with experiment, with low-lying minima close to the PDB geometries and to results obtained using the all-atom AMBER force field. The new program interfaces will allow us to investigate larger biomolecules in future work, using the UNRES potential in combination with all the methodology available in the computational energy landscapes framework.

Pragmatic Coarse-Graining of Proteins: Models and Applications.

The molecular details involved in the folding, dynamics, organization, and interaction of proteins with other molecules are often difficult to assess by experimental techniques. Consequently, computational models play an ever-increasing role in the field. However, biological processes involving large-scale protein assemblies or long time scale dynamics are still computationally expensive to study in atomistic detail. For these applications, employing coarse-grained (CG) modeling approaches has become a key strategy. In this Review, we provide an overview of what we call pragmatic CG protein models, which are strategies combining, at least in part, a physics-based implementation and a top-down experimental approach to their parametrization. In particular, we focus on CG models in which most protein residues are represented by at least two beads, allowing these models to retain some degree of chemical specificity. A description of the main modern pragmatic protein CG models is provided, including a review of the most recent applications and an outlook on future perspectives in the field.

Toward Consistent Physics-Based Modeling of Local Backbone Structures and Chirality Change of Proteins in Coarse-Grained Approaches.

A reliable representation of local interactions is critical for the accuracy of modeling protein structure and dynamics at both the all-atom and coarse-grained levels. The development of local (mainly torsional) potentials was focused on careful parametrization of the predetermined (usually Fourier) formulas rather than on their physics-based derivation. In this Perspective we discuss the state-of-the-art methods for modeling local interactions, including the scale-consistent theory developed in our laboratory, which implies that the coarse-grained torsional potentials inseparably depend on the virtual-bond angles adjacent to a given dihedral and that multitorsional terms should be considered. We extend the treatment to split the residue-based torsional potentials into the site-based regular and improper torsional potentials. These considerations are illustrated with the revised torsional potentials and improper-torsional potentials involving the l-alanine residue and the improper-torsional potential corresponding to serine-residue enantiomerization. Applications of the new approach in coarse-grained modeling and revising all-atom force fields are discussed.

Extension of the SUGRES-1P Coarse-Grained Model of Polysaccharides to Heparin.

Heparin is an unbranched periodic polysaccharide composed of negatively charged monomers and involved in key biological processes, including anticoagulation, angiogenesis, and inflammation. Its structure and dynamics have been studied extensively using experimental as well as theoretical approaches. The conventional approach of computational chemistry applied to the analysis of biomolecules is all-atom molecular dynamics, which captures the interactions of individual atoms by solving Newton's equation of motion. An alternative is molecular dynamics simulations using coarse-grained models of biomacromolecules, which offer a reduction of the representation and consequently enable us to extend the time and size scale of simulations by orders of magnitude. In this work, we extend the UNIfied COarse-gRaiNed (UNICORN) model of biological macromolecules developed in our laboratory to heparin. We carried out extensive tests to estimate the optimal weights of energy terms of the effective energy function as well as the optimal Debye-Hückel screening factor for electrostatic interactions. We applied the model to study unbound heparin molecules of polymerization degree ranging from 6 to 68 residues. We compare the obtained coarse-grained heparin conformations with models obtained from X-ray diffraction studies of heparin. The SUGRES-1P force field was able to accurately predict the general shape and global characteristics of heparin molecules.

UNRES-GPU for physics-based coarse-grained simulations of protein systems at biological time- and size-scales.

SUMMARY: The UNited RESisdue (UNRES) package for coarse-grained simulations, which has recently been optimized to treat large protein systems, has been implemented on Graphical Processor Units (GPUs). An over 100-time speed-up of the GPU code (run on an NVIDIA A100) with respect to the sequential code and an 8.5 speed-up with respect to the parallel Open Multi-Processing (OpenMP) code (run on 32 cores of 2 AMD EPYC 7313 Central Processor Units (CPUs)) has been achieved for large proteins (with size over 10 000 residues). Due to the averaging over the fine-grain degrees of freedom, 1 time unit of UNRES simulations is equivalent to about 1000 time units of laboratory time; therefore, millisecond time scale of large protein systems can be reached with the UNRES-GPU code. AVAILABILITY AND IMPLEMENTATION: The source code of UNRES-GPU along with the benchmarks used for tests is available at https://projects.task.gda.pl/eurohpcpl-public/unres.

Long-time scale simulations of virus-like particles from three human-norovirus strains.

The dynamics of the virus like particles (VLPs) corresponding to the GII.4 Houston, GII.2 SMV, and GI.1 Norwalk strains of human noroviruses (HuNoV) that cause gastroenteritis was investigated by means of long-time (about 30 µs in the laboratory timescale) molecular dynamics simulations with the coarse-grained UNRES force field. The main motion of VLP units turned out to be the bending at the junction between the P1 subdomain (that sits in the VLP shell) and the P2 subdomain (that protrudes outside) of the major VP1 protein, this resulting in a correlated wagging motion of the P2 subdomains with respect to the VLP surface. The fluctuations of the P2 subdomain were found to be more pronounced and the P2 domain made a greater angle with the normal to the VLP surface for the GII.2 strain, which could explain the inability of this strain to bind the histo-blood group antigens (HBGAs).

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function.

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52PPQ54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52PPQ54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.

Optimization of parallel implementation of UNRES package for coarse-grained simulations to treat large proteins.

We report major algorithmic improvements of the UNRES package for physics-based coarse-grained simulations of proteins. These include (i) introduction of interaction lists to optimize computations, (ii) transforming the inertia matrix to a pentadiagonal form to reduce computing and memory requirements, (iii) removing explicit angles and dihedral angles from energy expressions and recoding the most time-consuming energy/force terms to minimize the number of operations and to improve numerical stability, (iv) using OpenMP to parallelize those sections of the code for which distributed-memory parallelization involves unfavorable computing/communication time ratio, and (v) careful memory management to minimize simultaneous access of distant memory sections. The new code enables us to run molecular dynamics simulations of protein systems with size exceeding 100,000 amino-acid residues, reaching over 1 ns/day (1 µs/day in all-atom timescale) with 24 cores for proteins of this size. Parallel performance of the code and comparison of its performance with that of AMBER, GROMACS and MARTINI 3 is presented.

Mechanical Stability of Ribonuclease A Heavily Depends on the Redox Environment.

Disulfide bonds are covalent bonds that connect nonlocal fragments of proteins, and they are unique post-translational modifications of proteins. They require the oxidizing environment to be stable, which occurs for example during oxidative stress; however, in a cell the reductive environment is maintained, lowering their stability. Despite many years of research on disulfide bonds, their role in the protein life cycle is not fully understood and seems to strictly depend on a system or process in which they are involved. In this article, coarse-grained UNited RESidue (UNRES), and all-atom Assisted Model Building with Energy Refinement (AMBER) force fields were applied to run a series of steered molecular dynamics (SMD) simulations of one of the most studied, but still not fully understood, proteins─ribonuclease A (RNase A). SMD simulations were performed to study the mechanical stability of RNase A in different oxidative-reductive environments. As disulfide bonds (and any other covalent bonds) cannot break/form in any classical all-atom force field, we applied additional restraints between sulfur atoms of reduced cysteines which were able to mimic the breaking of the disulfide bonds. On the other hand, the coarse-grained UNRES force field enables us to study the breaking/formation of the disulfide bonds and control the reducing/oxidizing environment owing to the presence of the designed distance/orientation-dependent potential. This study reveals that disulfide bonds have a strong influence on the mechanical stability of RNase A only in a highly oxidative environment. However, the local stability of the secondary structure seems to play a major factor in the overall stability of the protein. Both our thermal unfolding and mechanical stretching studies show that the most stable disulfide bond is Cys65-Cys72. The breaking of disulfide bonds Cys26-Cys84 and Cys58-Cys110 is associated with large force peaks. They are structural bridges, which are mostly responsible for stabilizing the RNase A conformation, while the presence of the remaining two bonds (Cys65-Cys72 and Cys40-Cys95) is most likely connected with the enzymatic activity rather than the structural stability of RNase A in the cytoplasm. Our results prove that disulfide bonds are indeed stabilizing fragments of the proteins, but their role is strongly redox environment-dependent.

Design, synthesis and biological evaluation of PD-1 derived peptides as inhibitors of PD-1/PD-L1 complex formation for cancer therapy.

Over the past few years, many molecules such as monoclonal antibodies, affibodies, nanobodies, and small compounds have been designed and tested as inhibitors of PD-1/PD-L1 complex formation. Some of them have been successfully implemented into clinical oncology practice. However, the majority of these compounds have disadvantages and limitations, such as high production price, potential for immunogenicity and/or prolonged clearance. Thus, new inhibitors of the PD-1/PD-L1 immune checkpoints are needed. Recently, peptides emerged as potential novel approach for blocking receptor/ligand interaction. In the presented studies we have designed, synthesised and tested peptides, which are potential inhibitors of the PD-1/PD-L1 axis. The amino acid sequences of the designed peptides were based on the binding sites of PD-1 to PD-L1, as determined by the crystal structure of the protein complex and also based on MM/GBSA analysis. Interactions of the peptides with PD-L1 protein were confirmed using SPR, while their inhibitory properties were studied using cell-based PD-1/PD-L1 immune checkpoint blockade assays. The characterization of the peptides has shown that the peptides PD-1(119-142)T120C-E141C, PD-1(119-142)C123-S137C and PD-1(122-138)C123-S137C strongly bind to PD-L1 protein and disrupt the interaction of the proteins. PD-1(122-138)C123-S137C peptide was shown to have the best inhibitory potential from the panel of peptides. Its 3D NMR structure was determined and the binding site to PD-L1 was established using molecular modelling methods. Our results indicate that the PD-1 derived peptides are able to mimic the PD-1 protein and inhibit PD-1/PD-L1 complex formation.

Coarse-grained modeling of the calcium, sodium, magnesium and potassium cations interacting with proteins.

Metal ions play important biological roles, e.g., activation or deactivation of enzymatic reactions and signal transduction. Moreover, they can stabilize protein structure, or even be actively involved in the protein folding process. Therefore, accurate treatment of the ions is crucial to model and investigate biological phenomena properly. In this work the coarse-grained UNRES (UNited RESidue) force field was extended to include the interactions between proteins and four alkali or alkaline earth metal cations of biological significance, i.e., calcium, magnesium, sodium and potassium. Additionally, chloride anions were introduced as counter-ions. Parameters were derived from all-atom simulations and incorporate water in an implicit manner. The new force field was tested on the set of the proteins and was able to reproduce the ion-binding preferences.

Mitochondrial potassium channels: A novel calcitriol target.

BACKGROUND: Calcitriol (an active metabolite of vitamin D) modulates the expression of hundreds of human genes by activation of the vitamin D nuclear receptor (VDR). However, VDR-mediated transcriptional modulation does not fully explain various phenotypic effects of calcitriol. Recently a fast non-genomic response to vitamin D has been described, and it seems that mitochondria are one of the targets of calcitriol. These non-classical calcitriol targets open up a new area of research with potential clinical applications. The goal of our study was to ascertain whether calcitriol can modulate mitochondrial function through regulation of the potassium channels present in the inner mitochondrial membrane. METHODS: The effects of calcitriol on the potassium ion current were measured using the patch-clamp method modified for the inner mitochondrial membrane. Molecular docking experiments were conducted in the Autodock4 program. Additionally, changes in gene expression were investigated by qPCR, and transcription factor binding sites were analyzed in the CiiiDER program. RESULTS: For the first time, our results indicate that calcitriol directly affects the activity of the mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa) from the human astrocytoma (U-87 MG) cell line but not the mitochondrial calcium-independent two-pore domain potassium channel (mitoTASK-3) from human keratinocytes (HaCaT). The open probability of the mitoBKCa channel in high calcium conditions decreased after calcitriol treatment and the opposite effect was observed in low calcium conditions. Moreover, using the AutoDock4 program we predicted the binding poses of calcitriol to the calcium-bound BKCa channel and identified amino acids interacting with the calcitriol molecule. Additionally, we found that calcitriol influences the expression of genes encoding potassium channels. Such a dual, genomic and non-genomic action explains the pleiotropic activity of calcitriol. CONCLUSIONS: Calcitriol can regulate the mitochondrial large-conductance calcium-regulated potassium channel. Our data open a new chapter in the study of non-genomic responses to vitamin D with potential implications for mitochondrial bioenergetics and cytoprotective mechanisms.

Modeling the Structure, Dynamics, and Transformations of Proteins with the UNRES Force Field.

The physics-based united-residue (UNRES) model of proteins ( www.unres.pl ) has been designed to carry out large-scale simulations of protein folding. The force field has been derived and parameterized based on the principles of statistical-mechanics, which makes it independent of structural databases and applicable to treat nonstandard situations such as, proteins that contain D-amino-acid residues. Powered by Langevin dynamics and its replica-exchange extensions, UNRES has found a variety of applications, including ab initio and database-assisted protein-structure prediction, simulating protein-folding pathways, exploring protein free-energy landscapes, and solving biological problems. This chapter provides a summary of UNRES and a guide for potential users regarding the application of the UNRES package in a variety of research tasks.

Theory and Practice of Coarse-Grained Molecular Dynamics of Biologically Important Systems.

Molecular dynamics with coarse-grained models is nowadays extensively used to simulate biomolecular systems at large time and size scales, compared to those accessible to all-atom molecular dynamics. In this review article, we describe the physical basis of coarse-grained molecular dynamics, the coarse-grained force fields, the equations of motion and the respective numerical integration algorithms, and selected practical applications of coarse-grained molecular dynamics. We demonstrate that the motion of coarse-grained sites is governed by the potential of mean force and the friction and stochastic forces, resulting from integrating out the secondary degrees of freedom. Consequently, Langevin dynamics is a natural means of describing the motion of a system at the coarse-grained level and the potential of mean force is the physical basis of the coarse-grained force fields. Moreover, the choice of coarse-grained variables and the fact that coarse-grained sites often do not have spherical symmetry implies a non-diagonal inertia tensor. We describe selected coarse-grained models used in molecular dynamics simulations, including the most popular MARTINI model developed by Marrink's group and the UNICORN model of biological macromolecules developed in our laboratory. We conclude by discussing examples of the application of coarse-grained molecular dynamics to study biologically important processes.

Modeling protein structures with the coarse-grained UNRES force field in the CASP14 experiment.

The UNited RESidue (UNRES) force field was tested in the 14th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP14), in which larger oligomeric and multimeric targets were present compared to previous editions. Three prediction modes were tested (i) ab initio (the UNRES group), (ii) contact-assisted (the UNRES-contact group), and (iii) template-assisted (the UNRES-template group). For most of the targets, the contact restraints were derived from the server models top-ranked by the DeepQA method, while the DNCON2 method was used for 11 targets. Our consensus-fragment procedure was used to run template-assisted predictions. Each group also processed the Nuclear Magnetic Resonance (NMR)- and Small Angle X-Ray Scattering (SAXS)-data assisted targets. The average Global Distance Test Total Score (GDT_TS) of the 'Model 1' predictions were 29.17, 39.32, and 56.37 for the UNRES, UNRES-contact, and UNRES-template predictions, respectively, increasing by 0.53, 2.24, and 3.76, respectively, compared to CASP13. It was also found that the GDT_TS of the UNRES models obtained in ab initio mode and in the contact-assisted mode decreases with the square root of chain length, while the exponent in this relationship is 0.20 for the UNRES-template group models and 0.11 for the best performing AlphaFold2 models, which suggests that incorporation of database information, which stems from protein evolution, brings in long-range correlations, thus enabling the correction of force-field inaccuracies.

Prediction of protein assemblies, the next frontier: The CASP14-CAPRI experiment.

We present the results for CAPRI Round 50, the fourth joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of twelve targets, including six dimers, three trimers, and three higher-order oligomers. Four of these were easy targets, for which good structural templates were available either for the full assembly, or for the main interfaces (of the higher-order oligomers). Eight were difficult targets for which only distantly related templates were found for the individual subunits. Twenty-five CAPRI groups including eight automatic servers submitted ~1250 models per target. Twenty groups including six servers participated in the CAPRI scoring challenge submitted ~190 models per target. The accuracy of the predicted models was evaluated using the classical CAPRI criteria. The prediction performance was measured by a weighted scoring scheme that takes into account the number of models of acceptable quality or higher submitted by each group as part of their five top-ranking models. Compared to the previous CASP-CAPRI challenge, top performing groups submitted such models for a larger fraction (70-75%) of the targets in this Round, but fewer of these models were of high accuracy. Scorer groups achieved stronger performance with more groups submitting correct models for 70-80% of the targets or achieving high accuracy predictions. Servers performed less well in general, except for the MDOCKPP and LZERD servers, who performed on par with human groups. In addition to these results, major advances in methodology are discussed, providing an informative overview of where the prediction of protein assemblies currently stands.

Investigation of Phosphorylation-Induced Folding of an Intrinsically Disordered Protein by Coarse-Grained Molecular Dynamics.

Apart from being the most common mechanism of regulating protein function and transmitting signals throughout the cell, phosphorylation has an ability to induce disorder-to-order transition in an intrinsically disordered protein. In particular, it was shown that folding of the intrinsically disordered protein, eIF4E-binding protein isoform 2 (4E-BP2), can be induced by multisite phosphorylation. Here, the principles that govern the folding of phosphorylated 4E-BP2 (pT37pT46 4E-BP218-62) are investigated by analyzing canonical and replica exchange molecular dynamics trajectories, generated with the coarse-grained united-residue force field, in terms of local and global motions and the time dependence of formation of contacts between Cαs of selected pairs of residues. The key residues involved in the folding of the pT37pT46 4E-BP218-62 are elucidated by this analysis. The correlations between local and global motions are identified. Moreover, for a better understanding of the physics of the formation of the folded state, the experimental structure of the pT37pT46 4E-BP218-62 is analyzed in terms of a kink (heteroclinic standing wave solution) of a generalized discrete nonlinear Schrödinger equation. It is shown that without molecular dynamics simulations the kinks are able to identify not only the phosphorylated sites of protein, the key players in folding, but also the reasons for the weak stability of the pT37pT46 4E-BP218-62.