Molecular dynamics simulation on the allosteric analysis of the c‐di‐GMP class I riboswitch induced by ligand binding

Riboswitches are RNA molecules that regulate gene expression using conformation change, affected by binding of small molecule ligands. Although a number of ligand‐bound aptamer complex structures have been solved, it is important to know ligand‐free conformations of the aptamers in order to understand the mechanism of specific binding by ligands. In this paper, we use dynamics simulations on a series of models to characterize the ligand‐free and ligand‐bound aptamer domain of the c‐di‐GMP class I (GEMM‐I) riboswitch. The results revealed that the ligand‐free aptamer has a stable state with a folded P2 and P3 helix, an unfolded P1 helix and open binding pocket. The first Mg ions binding to the aptamer is structurally favorable for the successive c‐di‐GMP binding. The P1 helix forms when c‐di‐GMP is successive bound. Three key junctions J1/2, J2/3 and J1/3 in the GEMM‐I riboswitch contributing to the formation of P1 helix have been found. The binding of the c‐di‐GMP ligand to the GEMM‐I riboswitch induces the riboswitch's regulation through the direct allosteric communication network in GEMM‐I riboswitch from the c‐di‐GMP binding sites in the J1/2 and J1/3 junctions to the P1 helix, the indirect ones from those in the J2/3 and P2 communicating to P1 helix via the J1/2 and J1/3 media.     

pH‐dependent conformational dynamics of beta‐secretase 1: A molecular dynamics study

Beta‐secretase 1 (BACE‐1) is an aspartyl protease implicated in the overproduction of β‐amyloid fibrils responsible for Alzheimer disease. The process of β‐amyloid genesis is known to be pH dependent, with an activity peak between solution pH of 3.5 and 5.5. We have studied the pH‐dependent dynamics of BACE‐1 to better understand the pH dependent mechanism. We have implemented support for graphics processor unit (GPU) accelerated constant pH molecular dynamics within the AMBER molecular dynamics software package and employed this to determine the relative population of different aspartyl dyad protonation states in the pH range of greatest β‐amyloid production, followed by conventional molecular dynamics to explore the differences among the various aspartyl dyad protonation states. We observed a difference in dynamics between double‐protonated, mono‐protonated, and double‐deprotonated states over the known pH range of higher activity. These differences include Tyr 71‐aspartyl dyad proximity and active water lifetime. This work indicates that Tyr 71 stabilizes catalytic water in the aspartyl dyad active site, enabling BACE‐1 activity.     

Field‐based comparison of ligand and coactivator binding sites of nuclear receptors

A structure‐based comparison of the ligand‐binding domains of 35 nuclear receptors from five different subfamilies is presented. Their ligand and coactivator binding sites are characterized using knowledge‐based contact preference fields for hydrophobic and hydrophilic interactions implemented in the MOE modeling environment. Additionally, for polar knowledge‐based field points the preference for negative or positive electrostatic interactions is estimated using the Poisson‐Boltzmann equation. These molecular‐interaction fields are used to cluster the nuclear receptor family based on similarities of their binding sites. By analyzing the similarities and differences of hydrophobic and polar fields in binding pockets of related receptors it is possible to identify conserved interactions in ligand and coactivator binding pockets, which support e.g. design of specific ligands during lead optimization or virtual screening as docking filter. Examples of remarkable similarities between ligand binding sites of members from phylogenetically different nuclear receptor families (RXR, RAR, HNF4, NR5) and differences between closely related subtypes (LXR, RAR, TR) are discussed in more detail. Significant similarities and differences of coactivator binding sites are shown for NR3Cs, LXRs and PPARs. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 884–894, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A principal component analysis of the dynamics of subdomains and binding sites in human serum albumin

The conformational dynamics of human serum albumin (HSA) was investigated by principal component analysis (PCA) applied to three molecular dynamics trajectories of 200 ns each. The overlap of the essential subspaces spanned by the first 10 principal components (PC) of different trajectories was about 0.3 showing that the PCA based on a trajectory length of 200 ns is not completely convergent for this protein. The contributions of the relative motion of subdomains and of the subdomains (internal) distortion to the first 10 PCs were found to be comparable. Based on the distribution of the first 3 PC, 10 protein conformers are identified showing relative root mean square deviations (RMSD) between 2.3 and 4.6 Å. The main PCs are found to be delocalized over the whole protein structure indicating that the motions of different protein subdomains are coupled. This coupling is considered as being related to the allosteric effects observed upon ligand binding to HSA. On the other hand, the first PC of one of the three trajectories describes a conformational transition of the protein domain I that is close to that experimentally observed upon myristate binding. This is a theoretical support for the older hypothesis stating that changes of the protein onformation favorable to binding can precede the ligand complexation. A detailed all atoms PCA performed on the primary Sites 1 and 2 confirms the multiconformational character of the HSA binding sites as well as the significant coupling of their motions. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 561–572, 2014.     

Molecular simulations of β‐lactoglobulin complexed with fatty acids reveal the structural basis of ligand affinity to internal and possible external binding sites

The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β‐lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head‐group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α‐helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands. Proteins 2014; 82:2609–2619. © 2014 Wiley Periodicals, Inc.     

Interdomain dynamics and ligand binding: molecular dynamics simulations of glutamine binding protein

Periplasmic binding proteins from Gram-negative bacteria possess a common architecture, comprised of two domains linked by a hinge region, a fold which they share with the neurotransmitter-binding domains of ionotropic glutamate receptors (GluRs). Glutamine-binding protein (GlnBP) is one such protein, whose crystal structure has been solved in both open and closed forms. Multi-nanosecond molecular dynamics simulations have been used to explore motions about the hinge region and how they are altered by ligand binding. Glutamine binding is seen to significantly reduce inter-domain motions about the hinge region. Essential dynamics analysis of inter-domain motion revealed the presence of both hinge-bending and twisting motions, as has been reported for a related sugar-binding protein. Significantly, the influence of the ligand on GlnBP dynamics is similar to that previously observed in simulations of rat glutamate receptor (GluR2) ligand-binding domain. The essential dynamics analysis of GlnBP also revealed a third class of motion which suggests a mechanism for signal transmission in GluRs.     

Binding of antifusion peptides with HIVgp41 from molecular dynamics simulations: quantitative correlation with experiment

Peptides based on C-terminal regions of the human immunodeficiency virus (HIV) viral protein gp41 represent an important new class of antiviral therapeutics called peptide fusion inhibitors. In this study, computational methods were used to model the binding of six peptides that contain residues that pack into a conserved hydrophobic pocket on HIVgp41, an attractive target site for the development of small molecule inhibitors. Free energies of binding were computed using molecular mechanics Generalized Born surface area (MM-GBSA) methods from molecular dynamics (MD) simulations, which employed either explicit (TIP3P) or continuum Generalized Born (GB) water models and strong correlations between experimental and computational affinities were obtained in both cases. Energy decomposition of the TIP3P-MD results (r2 = 0.75) reveals that variation in experimental affinity is highly correlated with changes in intermolecular van der Waals energies (deltaE(vdw)) on both a local (residue-based, r2 = 0.94) and global (peptide-based, r2 = 0.84) scale. The results show that differential association of C-peptides with HIVgp41 is driven solely by changes within the conserved pocket supporting the hypothesis that this region is an important drug target site. Such strong agreement with experiment is notable given the large size of the ligands (34 amino-acids) relative to the small range of experimental affinities (2 kcal/mol) and demonstrates good sensitivity of this computational method for simulating peptide fusion inhibitors. Finally, inspection of simulation trajectories identified a highly populated pi-type hydrogen bond, which formed between Gln575 on the receptor and the aromatic ring of peptide ligand Phe631, which could have important implications for drug design.     

Evaluation of protein–ligand affinity prediction using steered molecular dynamics simulations

In computational drug design, ranking a series of compound analogs in a manner that is consistent with experimental affinities remains a challenge. In this study, we evaluated the prediction of protein–ligand binding affinities using steered molecular dynamics simulations. First, we investigated the appropriate conditions for accurate predictions in these simulations. A conic harmonic restraint was applied to the system for efficient sampling of work values on the ligand unbinding pathway. We found that pulling velocity significantly influenced affinity predictions, but that the number of collectable trajectories was less influential. We identified the appropriate pulling velocity and collectable trajectories for binding affinity predictions as 1.25 Å/ns and 100, respectively, and these parameters were used to evaluate three target proteins (FK506 binding protein, trypsin, and cyclin-dependent kinase 2). For these proteins using our parameters, the accuracy of affinity prediction was higher and more stable when Jarzynski’s equality was employed compared with the second-order cumulant expansion equation of Jarzynski’s equality. Our results showed that steered molecular dynamics simulations are effective for predicting the rank order of ligands; thus, they are a potential tool for compound selection in hit-to-lead and lead optimization processes.     

Dynamic behavior of the post‐SET loop region of NSD1: Implications for histone binding and drug development

NSD1 is a SET‐domain histone methyltransferase that methylates lysine 36 of histone 3. In the crystal structure of NSD1, the post‐SET loop is in an autoinhibitory position that blocks binding of the histone peptide as well as the entrance to the lysine‐binding channel. The conformational dynamics preceding histone binding and the mechanism by which the post‐SET loop moves to accommodate the target lysine is currently unknown, although potential models have been proposed. Using molecular dynamics simulations, we have identified potential conformations of the post‐SET loop differing from those of previous studies, as well as proposed a model of peptide‐bound NSD1. Our simulations illustrate the dynamic behavior of the post‐SET loop and the presence of a few distinct conformations. In every case, the post‐SET loop remains in an autoinhibitory position blocking the peptide‐binding cleft, suggesting that another interaction is required to optimally position NSD1 in an active conformation. This finding provides initial evidence for a mechanism by which NSD1 preferentially binds nucleosomal substrates.     

Substrate versus inhibitor dynamics of P‐glycoprotein

By far the most studied multidrug resistance protein is P‐glycoprotein. Despite recent structural data, key questions about its function remain. P‐glycoprotein (P‐gp) is flexible and undergoes large conformational changes as part of its function and in this respect, details not only of the export cycle, but also the recognition stage are currently lacking. Given the flexibility, molecular dynamics (MD) simulations provide an ideal tool to examine this aspect in detail. We have performed MD simulations to examine the behaviour of P‐gp. In agreement with previous reports, we found that P‐gp undergoes large conformational changes which tended to result in the nucleotide‐binding domains coming closer together. In all simulations, the approach of the NBDs was asymmetrical in agreement with previous observations for other ABC transporter proteins. To validate the simulations, we make extensive comparison to previous cross‐linking data. Our results show very good agreement with the available data. We then went on to compare the influence of inhibitor compounds bound with simulations of a substrate (daunorubicin) bound. Our results suggest that inhibitors may work by keeping the NBDs apart, thus preventing ATP‐hydrolysis. On the other hand, repeat simulations of daunorubicin (substrate) in one particular binding pose suggest that the approach of the NBDs is not impaired and that the structure would be still be competent to perform ATP hydrolysis, thus providing a model for inhibition or substrate transport. Finally we compare the latter to earlier QSAR data to provide a model for the first part of substrate transport within P‐gp. Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Signaling pathways of PDZ2 domain: a molecular dynamics interaction correlation analysis

PDZ domains are found in many signaling proteins. One of their functions is to provide scaffolds for forming membrane-associated protein complexes by binding to the carboxyl termini of their partners. PDZ domains are thought also to play a signal transduction role by propagating the information that binding has occurred to remote sites. In this study, a molecular dynamics (MD) simulation-based approach, referred to as an interaction correlation analysis, is applied to the PDZ2 domain to identify the possible signal transduction pathways. A residue correlation matrix is constructed from the interaction energy correlations between all residue pairs obtained from the MD simulations. Two continuous interaction pathways, starting at the ligand binding pocket, are identified by a hierarchical clustering analysis of the residue correlation matrix. One pathway is mainly localized at the N-terminal side of helix alpha1 and the adjacent C-terminus of loop beta1-beta2. The other pathway is perpendicular to the central beta-sheet and extends toward the side of PDZ2 domain opposite to the ligand binding pocket. The results complement previous studies based on multiple sequence analysis, NMR, and MD simulations. Importantly, they reveal the energetic origin of the long-range coupling. The PDZ2 results, as well as the earlier rhodopsin analysis, show that the interaction correlation analysis is a robust approach for determining pathways of intramolecular signal transduction.     

Nuclear magnetic resonance‐based determination of dioxygen binding sites in protein cavities

The location and ligand accessibility of internal cavities in cysteine‐free wild‐type T4 lysozyme was investigated using O₂ gas‐pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O₂ in solvent to 8.9 mM, O₂‐induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C‐terminal domain. To determine the number of O₂ binding sites and their atomic coordinates from the 1/r⁶ distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid‐search. Two O₂‐accessible sites were identified in internal cavities: One site was consistent with the xenon‐binding site in the protein in crystal, and the other site was established to be a novel ligand‐binding site. MD simulations performed at 10 and 100 mM O₂ revealed dioxygen ingress and egress as well as rotational and translational motions of O₂ in the cavities. It is therefore suggested that conformational fluctuations within the ground‐state ensemble transiently develop channels for O₂ association with the internal protein cavities.     

Bladder tissue characterization using probe‐based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction

Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor.     

Protein surface roughness accounts for binding free energy of Plasmepsin II‐ligand complexes

The calculation of absolute binding affinities for protein‐inhibitor complexes remains as one of the main challenges in computational structure‐based ligand design. The present work explored the calculations of surface fractal dimension (as a measure of surface roughness) and the relationship with experimental binding free energies of Plasmepsin II complexes. Plasmepsin II is an attractive target for novel therapeutic compounds to treat malaria. However, the structural flexibility of this enzyme is a drawback when searching for specific inhibitors. Concerning that, we performed separate explicitly solvated molecular dynamics simulations using the available high‐resolution crystal structures of different Plasmepsin II complexes. Molecular dynamics simulations allowed a better approximation to systems dynamics and, therefore, a more reliable estimation of surface roughness. This constitutes a novel approximation in order to obtain more realistic values of fractal dimension, because previous works considered only x‐ray structures. Binding site fractal dimension was calculated considering the ensemble of structures generated at different simulation times. A linear relationship between binding site fractal dimension and experimental binding free energies of the complexes was observed within 20 ns. Previous studies of the subject did not uncover this relationship. Regression model, coined FD model, was built to estimate binding free energies from binding site fractal dimension values. Leave‐one‐out cross‐validation showed that our model reproduced accurately the absolute binding free energies for our training set (R² = 0.76; <|error|> =0.55 kcal/mol; SD_error = 0.19 kcal/mol). The fact that such a simple model may be applied raises some questions that are addressed in the article.     

Theoretical investigation on the diatomic ligand migration process and ligand binding properties in non‐O2‐binding H‐NOX domain

The Nostoc sp (Ns) H‐NOX (heme‐nitric oxide or OXygen‐binding) domain shares 35% sequence identity with soluble guanylate cyclase (sGC) and exhibits similar ligand binding property with the sGC. Previously, our molecular dynamic (MD) simulation work identified that there exists a Y‐shaped tunnel system hosted in the Ns H‐NOX interior, which servers for ligand migration. The tunnels were then confirmed by Winter et al. [PNAS 2011;108(43):E 881–889] recently using x‐ray crystallography with xenon pressured conditions. In this work, to further investigate how the protein matrix of Ns H‐NOX modulates the ligand migration process and how the distal residue composition affects the ligand binding prosperities, the free energy profiles for nitric oxide (NO), carbon monooxide (CO), and O₂ migration are explored using the steered MDs simulation and the ligand binding energies are calculated using QM/MM schemes. The potential of mean force profiles suggest that the longer branch of the tunnel would be the most favorable route for NO migration and a second NO trapping site other than the distal heme pocket along this route in the Ns H‐NOX was identified. On the contrary, CO and O₂ would prefer to diffuse via the shorter branch of the tunnel. The QM/MM (quantum mechanics/molecular mechanics) calculations suggest that the hydrophobic distal pocket of Ns H‐NOX would provide an approximately vacuum environment and the ligand discrimination would be determined by the intrinsic binding properties of the diatomic gas ligand to the heme group. Proteins 2013; 81:1363–1376. © 2013 Wiley Periodicals, Inc.     

Restrained molecular dynamics simulations of HIV-1 protease: the first step in validating a new target for drug design

To test the anticorrelated relationship that was recently displayed in conventional molecular dynamics (MD) simulations, several different restrained MD simulations on a wild type and on the V82F/I84V drug-resistant mutant of HIV-1 protease were performed. This anticorrelated relationship refers to the observation that compression of the peripheral ear-to-cheek region of HIV protease (i.e., the elbow of the flap to the fulcrum and the cantilever) occurred as the active site flaps were opening, and, conversely, expansion of that ear-to-cheek region occurred as both flaps were closing. An additional examination of this anticorrelated relationship was necessary to determine whether it can be harnessed in a useful manner. Consequently, six different MD experiments were performed that incorporated pairwise distance restraints in that ear-to-cheek region (i.e., the distance between the alpha-carbons of Gly40 and Gln61 was restrained to either 7.7 or 10.5 A, in both monomers). Pushing the backbones of the ear and the cheek regions away from each other slightly did force the flaps that guard the active site to remain closed in both the wild type and the mutant systems-even though there were no ligands in the active sites. Thus, these restrained MD simulations provided evidence that the anticorrelated relationship can be exploited to affect the dynamic behavior of the flaps that guard the active site of HIV-1 protease. These simulations supported our hypothesis of the mechanism governing flap motion, and they are the first step towards validating that peripheral surface as a new target for drug design.     

Dynamic injection behaviour of carbon monoxide nano-jets: analysis based on molecular dynamics simulation

Molecular dynamics simulation was used to characterise the dynamical injection behaviour of CO through a gold nano-injector with a Gr coating. We also varied the nozzle outlet size, system temperature, and extrusion velocity to elucidate their influence on the flow patterns, injection pressure, and flow rate of the CO nano-jets. Simulation results revealed the following important findings. (1) At 100?K, the liquefaction of a CO jet led to a wider spray angle (ø_s?=?84～96°) and allowed molecules to attach to the Gr layer, resulting in agglomeration at the orifice. (2) At 500?K and 55.824?m/s, the nebulisation of the CO nano-jet was induced, which produced a narrower spray angle (ø_s?=?47°). (3) The flow rate of CO molecules was essentially linear under the following conditions: low extrusion velocity (≤13.956?m/s), large orifice (d?=?1.5?nm), and high system temperature (≥300?K). (4) Due to the compressibility of CO molecules, the pressure inside the chamber under a high extrusion speed (≥27.912?m/s) presented a sharp increase in the middle and final extrusion stages. A delay in the pressure increase enabled the liquefaction of the extruded CO molecules, resulting in an unstable flow rate.     

A functional NMR for membrane proteins: dynamics,ligand binding,and allosteric modulation

By nature of conducting ions, transporting substrates and transducing signals, membrane channels, transporters and receptors are expected to exhibit intrinsic conformational dynamics. It is therefore of great interest and importance to understand the various properties of conformational dynamics acquired by these proteins, for example, the relative population of states, exchange rate, conformations of multiple states, and how small molecule ligands modulate the conformational exchange. Because small molecule binding to membrane proteins can be weak and/or dynamic, structural characterization of these effects is very challenging. This review describes several NMR studies of membrane protein dynamics, ligand‐induced conformational rearrangements, and the effect of ligand binding on the equilibrium of conformational exchange. The functional significance of the observed phenomena is discussed.     

Role of N‐glycosylation in EGFR ectodomain ligand binding

The epidermal growth factor receptor (EGFR) is a tyrosine kinase protein, overexpressed in several cancers. The extracellular domain of EGFR is known to be heavily glycosylated. Growth factor (mostly epidermal growth factor or EGF) binding activates EGFR. This occurs by inducing the transition from the autoinhibited tethered conformation to an extended conformation of the monomeric form of EGFR and by stabilizing the flexible preformed dimer. Activated EGFR adopts a back‐to‐back dimeric conformation after binding of another homologous receptor to its extracellular domain as the dimeric partner. Several antibodies inhibit EGFR by targeting the growth factor binding site or the dimeric interfaces. Glycosylation has been shown to be important for modulating the stability and function of EGFR. Here, atomistic MD simulations show that N‐glycosylation of the EGFR extracellular domain plays critical roles in the binding of growth factors, monoclonal antibodies, and the dimeric partners to the monomeric EGFR extracellular domain. N‐glycosylation results in the formation of several noncovalent interactions between the glycans and EGFR extracellular domain near the EGF binding site. This stabilizes the growth factor binding site, resulting in stronger interactions (electrostatic) between the growth factor and EGFR. N‐glycosylation also helps maintain the dimeric interface and plays distinct roles in binding of antibodies to spatially separated epitopes of the EGFR extracellular domain. Analysis of SNP data suggests the possibility of altered glycosylation with functional consequences. Proteins 2017; 85:1529–1549. © 2017 Wiley Periodicals, Inc.     

Heterogeneous behavior of metalloproteins toward metal ion binding and selectivity: insights from molecular dynamics studies

About one-third of the existing proteins require metal ions as cofactors for their catalytic activities and structural complexities. While many of them bind only to a specific metal, others bind to multiple (different) metal ions. However, the exact mechanism of their metal preference has not been deduced to clarity. In this study, we used molecular dynamics (MD) simulations to investigate whether a cognate metal (bound to the structure) can be replaced with other similar metal ions. We have chosen seven different proteins (phospholipase A₂, sucrose phosphatase, pyrazinamidase, cysteine dioxygenase (CDO), plastocyanin, monoclonal anti-CD4 antibody Q425, and synaptotagmin 1 C₂B domain) bound to seven different divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Ba²⁺, and Sr²⁺, respectively). In total, 49 MD simulations each of 50 ns were performed and each trajectory was analyzed independently. Results demonstrate that in some cases, cognate metal ions can be exchanged with similar metal ions. On the contrary, some proteins show binding affinity specifically to their cognate metal ions. Surprisingly, two proteins CDO and plastocyanin which are known to bind Fe²⁺ and Cu²⁺, respectively, do not exhibit binding affinity to any metal ion. Furthermore, the study reveals that in some cases, the active site topology remains rigid even without cognate metals, whereas, some require them for their active site stability. Thus, it will be interesting to experimentally verify the accuracy of these observations obtained computationally. Moreover, the study can help in designing novel active sites for proteins to sequester metal ions particularly of toxic nature.