Reprogramming of glucose metabolism via PFKFB4 is critical in FGF16-driven invasion of breast cancer cells.

Fibroblast growth factors (FGFs) are expressed in both developing and adult tissues and play important roles in embryogenesis, tissue homeostasis, angiogenesis, and neoplastic transformation. Here, we report the elevated expression of FGF16 in human breast tumor and investigate its potential involvement in breast cancer progression. The onset of epithelial-mesenchymal transition (EMT), a prerequisite for cancer metastasis, was observed in human mammary epithelial cell-line MCF10A by FGF16. Further study unveiled that FGF16 alters mRNA expression of a set of extracellular matrix genes to promote cellular invasion. Cancer cells undergoing EMT often show metabolic alteration to sustain their continuous proliferation and energy-intensive migration. Similarly, FGF16 induced a significant metabolic shift toward aerobic glycolysis. At the molecular level, FGF16 enhanced GLUT3 expression to facilitate glucose transport into cells, which through aerobic glycolysis generates lactate. The bi-functional protein, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 4 (PFKFB4) was found to be a mediator in FGF16-driven glycolysis and subsequent invasion. Furthermore, PFKFB4 was found to play a critical role in promoting lactate-induced cell invasion since silencing PFKFB4 decreased lactate level and rendered the cells less invasive. These findings support potential clinical intervention of any of the members of FGF16-GLUT3-PFKFB4 axis to control the invasion of breast cancer cells.

A systematic drug repurposing approach to identify promising inhibitors from FDA-approved drugs against Nsp4 protein of SARS-CoV-2.

COVID-19 is caused by SARS-CoV-2 and responsible for the ongoing global pandemic in the world. After more than a year, we are still in lurch to combat and control the situation. Therefore, new therapeutic options to control the ongoing COVID-19 are urgently in need. In our study, we found that nonstructural protein 4 (Nsp4) of SARS-CoV-2 could be a potential target for drug repurposing. Due to availability of only the crystal structure of C-terminal domain of Nsp4 (Ct-Nsp4) and its crucial participation in viral RNA synthesis, we have chosen Ct-Nsp4 as a target for screening the 1600 FDA-approved drugs using molecular docking. Top 102 drugs were found to have the binding energy equal or less than -7.0 kcal/mol. Eribulin and Suvorexant were identified as the two most promising drug molecules based on the docking score. The dynamics of Ct-Nsp4-drug binding was monitored using 100 ns molecular dynamics simulations. From binding free energy calculation over the simulation, both the drugs were found to have considerable binding energy. The present study has identified Eribulin and Suvorexant as promising drug candidates. This finding will be helpful to accelerate the drug discovery process against COVID-19 disease.Communicated by Ramaswamy H. Sarma.

Distinct mode of membrane interaction and disintegration by diverse class of antimicrobial peptides.

The exploitation of conventional antibiotics in conjunction with the adeptness of microbes has led to the emergence of multi-drug-resistant pathogens. This has posed a severe threat to combating life-threatening infectious diseases. Antimicrobial peptides (AMP), which are considered to be the first line of defense in all living organisms, are being developed for therapeutic use. Herein, we determined the NMR solution structure of Rhesus macaque Myeloid Alpha Defensin-4 (RMAD4), a defensin AMP. Additionally, the distinct modes of membrane perturbation for two structurally dissimilar classes of AMPs was studied using biophysical methods namely, Solid-state 31P NMR, DSC and cryo-TEM. The cathelicidin - Bovine myeloid antimicrobial peptide (BMAP-28 (1-18)), which adopts a helical conformation, and the defensin RMAD4 peptide that natively folds to form ß-sheets appeared to engage differently with the bacterial membrane. The helical BMAP-28 (1-18) peptide initiates lipid segregation and membrane thinning followed by pore formation, while the ß-stranded RMAD4 peptide demonstrates fragmentation of the bilayer by the carpet or detergent-like mechanism of action. Molecular dynamics studies sufficiently corroborated these findings. The structure and mechanism of action of the AMPs studied using experimental and computational approaches are believed to help in providing a platform for the rational design of new competent and cost-effective antimicrobial peptides for therapeutic applications.

Structure and Orientation of Water and Choline Chloride Molecules around a Methane Hydrophobe: A Computer Simulation Study.

Recent studies have reported manifold industrial applications of aqueous choline chloride (ChCl) solution as an alternative to deep eutectic solvent. ChCl also serves as a protecting co-solvent for proteins by restricting urea to approach the protein surface and thereby maintaining the water structure around the protein. However, a detailed molecular-level picture of the ChCl and water, even in the absence of urea around a representative hydrophobe is largely lacking. This motivates us to probe the effect of varying wt % of ChCl on the occupancy and orientations of the constituents around a representative solute like methane using computer simulations. Accumulation of water molecules and preferential exclusion of ChCl from the surface of methane perturb the tetrahedral geometry of water around it. We find a tangential alignment of the polar part of the ChCl molecules that interact with water, whereas its hydrophobic part is preferentially facing the methane. With an increase in ChCl wt %, a disruption in the tetrahedrality is evident for water molecules accompanied by a reduction in hydrogen bonds between water pairs in the solution. In short, ChCl induces crowding and modifies the microscopic arrangement and hydrogen bonding structure of the water around the methane and beyond.

Computational design of stapled peptide inhibitor against SARS-CoV-2 receptor binding domain.

Since its first detection in 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been the cause of millions of deaths worldwide. Despite the development and administration of different vaccines, the situation is still worrisome as the virus is constantly mutating to produce newer variants some of which are highly infectious. This raises an urgent requirement to understand the infection mechanism and thereby design therapeutic-based treatment for COVID-19. The gateway of the virus to the host cell is mediated by the binding of the receptor binding domain (RBD) of the virus spike protein to the angiotensin-converting enzyme 2 (ACE2) of the human cell. Therefore, the RBD of SARS-CoV-2 can be used as a target to design therapeutics. The α1 helix of ACE2, which forms direct contact with the RBD surface, has been used as a template in the current study to design stapled peptide therapeutics. Using computer simulation, the mechanism and thermodynamics of the binding of six stapled peptides with RBD have been estimated. Among these, the one with two lactam stapling agents has shown binding affinity, sufficient to overcome RBD-ACE2 binding. Analyses of the mechanistic detail reveal that a reorganization of amino acids at the RBD-ACE2 interface produces favorable enthalpy of binding whereas conformational restriction of the free peptide reduces the loss in entropy to result higher binding affinity. The understanding of the relation of the nature of the stapling agent with their binding affinity opens up the avenue to explore stapled peptides as therapeutic against SARS-CoV-2.

In Silico Elucidation of Molecular Picture of Water-Choline Chloride Mixture.

Choline chloride (ChCl) is a component of several deep eutectic solvents (DESs) having numerous applications. Recent studies have reported manifold promising use of aqueous choline chloride solution as an alternative to DES, where water plays the role of the hydrogen-bond donor. The characteristic physical properties of the DESs and aqueous DES originate from the "inter-" and intraspecies hydrogen-bond network formed by the constituents. However, a detailed molecular-level picture of choline chloride and water mixture is largely lacking in the literature. This motivates us to carry out extensive all-atom molecular dynamics simulations of the ChCl-water mixture of varying compositions. Our analyses clearly show an overall increase in the interspecies association with an increase in ChCl concentration. At higher concentrations, the trimethylammonium groups of choline are stabilized by a nonpolar interaction, whereas the hydroxyl groups preferentially interact with water. Chloride ions are found to be involved in two types of interactions: one where chloride ions intercalate two or more choline cations, and the other one where they are surrounded by five to six water molecules forming solvated chloride ions. However, the relative fractions of these two types of associations depend on the concentration of ChCl in the mixture. Another important structural aspect is the disruption of the hydrogen-bonded water network due to the presence of both choline cations and chloride ions. However, chloride ions participate to partially restore the tetrahedral arrangement of partners around water molecules.

Overexpression of LYK4, a lysin motif receptor with non-functional kinase domain, enhances tolerance to Alternaria brassicicola and increases trichome density in Brassica juncea.

Lysin motif receptor-like kinases (LYKs) are involved in the recognition of chitin and activation of plant immune response. In this study, we found LYK4 to be strongly induced in resistant Sinapis alba compared with susceptible Brassica juncea on challenge with Alternaria brassicicola. In silico analysis and in vitro kinase assay revealed that despite the presence of canonical protein kinase fold, B.juncea LYK4 (BjLYK4) lacks several key residues of a prototype protein kinase which renders it catalytically inactive. Transient expression analysis confirmed that fluorescently tagged BjLYK4 localizes specifically to the plasma membrane. Overexpression (OE) of BjLYK4 in B. juncea enhanced tolerance against A. brassicicola. Interestingly, the OE lines also exhibited a novel trichome dense phenotype and increased jasmonic acid (JA) responsiveness. We further showed that many chitin responsive WRKY transcription factors and JA biosynthetic genes were strongly induced in the OE lines on challenge with the pathogen. Moreover, several JA inducible trichome developmental genes constituting the WD-repeat/bHLH/MYB activator complex were also upregulated in the OE lines compared with vector control and RNA interference line. These results suggest that BjLYK4 plays an essential role in chitin-dependent activation of defense response and chitin independent trichome development likely by influencing the JA signaling pathway.

Effect of Stapling on the Thermodynamics of mdm2-p53 Binding.

Protein-protein interaction (PPI) is one of the key regulatory features driving biomolecular processes and hence is targeted for designing therapeutics against diseases. Small peptides are a new and emerging class of therapeutics owing to their high specificity and low toxicity. For achieving efficient targeting of the PPI, amino acid side chains are often stapled together, resulting in the rigidification of these peptides. Exploring the scope of these peptides demands a comprehensive understanding of their working principle. In this work, two stapled p53 peptides have been considered to delineate their binding mechanism with mdm2 using computational approaches. The addition of stapling agent protects the secondary structure of the peptides even in the case of thermal and chemical denaturation. Although the introduction of a stapling agent increases the hydrophobicity of the peptide, the enthalpic stabilization decreases. This is overcome by the lowering of the entropic penalty, and the overall binding affinity improves. The mechanistic insights into the benefit of peptide stapling can be adopted for further improvement of peptide therapeutics.

Microscopic structural features of water in aqueous-reline mixtures of varying compositions.

Reline, a mixture of urea and choline chloride in a 2 : 1 molar ratio, is one of the most frequently used deep eutectic solvents. Pure reline and its aqueous solution have large scale industrial use. Owing to the presence of active hydrogen bond formation sites, urea and choline cations can disrupt the hydrogen-bonded network in water. However, a quantitative understanding of the microscopic structural features of water in the presence of reline is still lacking. We carry out extensive all-atom molecular dynamics simulations to elucidate the effect of the gradual addition of co-solvents on the microscopic arrangements of water molecules. We consider four aqueous solutions of reline, between 26.3 and 91.4 wt%. A disruption of the local hydrogen-bonded structure in water is observed upon inclusion of urea and choline chloride. The extent of deviation of the water structure from tetrahedrality is quantified using the tetrahedral order parameter (qtet). Our analyses show a monotonic increase in the structural disorder as the co-solvents are added. Increase in the qtet values are observed when highly electro-negative hetero-atoms like nitrogen, oxygen of urea and choline cations are counted as partners of the central water molecules. Further insights are drawn from the characterization of the hydrogen-bonded network in water and we observe the gradual rupturing of water-water hydrogen bonds and their subsequent replacement by the water-urea hydrogen bonds. A negligible contribution from the hydrogen bonds between water and bulky choline cations has also been found. Considering all the constituents as the hydrogen bond partners we calculate the possibility of a successful hydrogen bond formation with a central water molecule. This gives a clear picture of the underlying mechanism of water replacement by urea.

Prematurity and associated future paediatric airway pathology: experience from a single tertiary paediatric ENT centre.

PURPOSE: Enhanced management of the pre-term patient has resulted in improved survival rates in increasingly premature patients. Although prematurity predisposes to congenital airway pathology, there is also increased risk of endotracheal intubation, and therefore acquired subglottic pathology. We sought to evaluate airway pathology in children outside the neonatal period with a history of prematurity to explore the relationship between prematurity and upper airway pathologies. METHODS: Data for patients undergoing elective microlaryngobronchoscopy (MLB) at our centre were collected prospectively over a 5-year period. Patients identified as premature were sub-classified by the grade of prematurity. RESULTS: 339 patients over 1 month of age underwent MLB, of which 56 (16.5%) were born prematurely. Of those with identified airway pathology, 49 (23.4%) were born prematurely, accounting for 32.6% of subglottic stenosis (n = 30), 24% of laryngomalacia (n = 13) and 19% of laryngeal cleft diagnoses (n = 16). 49 premature patients (87.5%) had one or more airway pathologies diagnosed. Multi-level airway pathology was seen in twelve premature infants (21.4%), demonstrating a statistically significant association (odds ratio 3.396; 95% CI 1.697-6.842; p value < 0.0016). Incidence of airway pathology, the severity of airway disease and multi-level airway pathology were not related to the grade of prematurity. CONCLUSIONS: Premature patients account for a significant proportion of the workload within our tertiary centre due to improving neonatal care and survival in pre-term infants. We suggest early paediatric ENT evaluation for ex-premature patients with symptoms of airway pathology, with a low threshold for MLB. Improving neonatal survival rates in ever-increasing prematurity will require the further provision of specialist paediatric ENT services to manage their ongoing care.

Choline Chloride as a Nano-Crowder Protects HP-36 from Urea-Induced Denaturation: Insights from Solvent Dynamics and Protein-Solvent Interactions.

Urea at sufficiently high concentration unfolds the secondary structure of proteins leading to denaturation. In contrast, choline chloride (ChCl) and urea, in 1 : 2 molar ratio, form a deep eutectic mixture, a liquid at room temperature, protecting proteins from denaturation. In order to get a microscopic picture of this phenomenon, we perform extensive all-atom molecular dynamics simulations on a model protein, HP-36. Based on our calculation of Kirkwood-Buff integrals, we analyze the relative accumulation of urea and ChCl around the protein. Additional insights are drawn from the translational and rotational dynamics of solvent molecules and hydrogen bond auto-correlation functions. In the presence of urea, water shows slow subdiffusive dynamics around the protein owing to a strong interaction of water with the backbone atoms. Urea also shows subdiffusive motion. The addition of ChCl further slows down the dynamics of urea, restricting its accumulation around the protein backbone. Adding to this, choline cations in the first solvation shell of the protein show the strongest subdiffusive behavior. In other words, ChCl acts as a nano-crowder by excluding urea from the protein backbone and thereby slowing down the dynamics of water around the protein. This prevents the protein from denaturation and makes it structurally rigid, which is supported by the smaller radius of gyration and root mean square deviation values of HP-36.

Dissecting the thermodynamic contributions of the charged residues in the membrane anchoring of Bcl-xl C-terminal domain.

The C-terminal helix of the Bcl-xl is known to initiate the membrane insertion of the protein by anchoring into the mitochondrial outer membrane. The C-terminal charged residues of that helix, R232 and K233, are reported to have an important structural role in the process of that insertion. The present work provides a quantitative understanding of the thermodynamic contribution of these residues on the membrane insertion energy-profile, calculated from the Adaptive Biasing Force based MD simulations of 2.67 µs altogether. Interestingly, the effect of the single neutralizing mutations at the C-terminus, i.e. K233A or R232A, is easily tolerated by the peptide without impacting the nature of insertion energy-profile, indicating the efficiency of one positively charged residue to drive the insertion. Whereas a double mutant, i.e. R232A and K233A, makes a significant impact on the energy-profile by destabilizing the membrane-associated states, as well as the membrane-embedded states. The finding provides molecular-level mechanistic insight. The water-mediated interaction formed by the peptide polar side chains within the bilayer core is found to modulate the membrane response during peptide insertion and that subsequently regulates the insertion mechanism. Mutation of the C-terminal residues eventually alters such a cascade of interactions that results in an insertion through energetically more expensive pathway. Since any one of the positively charged residues at the terminal is critical to ensure the membrane insertion, it appears that the natural selection of 'two' instead of 'one' charged residue is redundant in the context of membrane anchoring but may be important for other biochemical events.

Salt Induced Structural Collapse, Swelling, and Signature of Aggregation of Two ssDNA Strands: Insights from Molecular Dynamics Simulation.

Molecular dynamics simulations elucidate the structural collapse shown by two ssDNAs of the same base sequence in the presence of either Na+ or Mg2+, starting from in vivo ionic concentration to higher concentrations. Initially, an increase in ion concentration facilitates the structural distortion of individual ssDNA and helps to bring them close, and for this, Mg2+ is better than Na+. However, further addition of ions leads to structural reswelling of the DNA strands and inhibits their proximity. The structural changes are found to be guided by the strong interaction of the cations with the phosphinyl oxygen (pn_O). Additionally, a significant difference has been noticed in the interaction of the cations with phosphoester oxygen (pe_O) depending on the nature of the ion. The sequential and nonsequential base-pair stacking is one of the major factors in the structural collapse of individual ssDNA. Overall, the present investigation highlights some of the important aspects of aggregation of two ssDNA with the same base sequence at varying cationic concentration.

Quantum theory of spin waves for helical ground states in a hollandite lattice.

We perform spin-wave analysis of classical ground states of a model Hamiltonian proposed earlier (Mandal et al 2014 Phys. Rev. B 90 104420) for [Formula: see text] compounds. It is known that the phase diagram of the hollandite lattice (lattice of [Formula: see text] compounds) consists of four different helical phases (FH, A2H, C2H, CH phase) in the space of model parameters [Formula: see text]. The spin wave dispersion shows presence of gapless mode which interpolates between quadratic to linear depending on phases and values of J i . In most cases, the second lowest mode shows the existence of a roton-like minima mainly from [Formula: see text] to [Formula: see text] and [Formula: see text] to [Formula: see text] path and it appears at the value of [Formula: see text] for constant [Formula: see text]. Few higher modes also show similar minima. Each helical phase has its characteristic traits which can be used to determine the phases itself. The analytical expressions of eigenmodes at high symmetry points are obtained which can be utilized to extract the values of J i . Density of states, specific heat and susceptibilities at low temperature have been studied within spin-wave approximation. The specific heat shows departure from T 1.5(3) dependence found in three-dimensional unfrustrated ferromagnetic(anti-ferromagnetic) system which seems to be the signature of incommensurate helical phase. The parallel susceptibility is maximum for FH phase and minimum for CH phase at low temperature. The perpendicular susceptibility is found to be independent of temperature at very low temperature. Our study can be used to compare experiments on magnon spectrum, elastic neutron scattering, and finite temperature properties mentioned above for clean [Formula: see text] system as well as determining the values of J i .

Flexibility enables to discriminate between ligands: Lessons from structural ensembles of Bcl-xl and Mcl-1.

The proteins of Bcl-2 family, which are promising anti-cancer-drug targets, have substantial similarity in primary sequence and share homologous domains as well as similar structural folds. In spite of similarities in sequence and structures, the members of its pro- and anti- apoptotic subgroups form complexes with different type of partners with discriminating binding affinities. Understanding the origin of this discrimination is very important for designing ligands that can either selectively target a protein or could be made broad ranged as necessary. Using principal component analysis (PCA) of the available structures and from the analysis of the evolution of the binding pocket residues, the correlation has been investigated considering two important anti-apoptotic protein Bcl-xl and Mcl-1, which serve as two ideal representatives of this family. The flexibility of the receptor enables them to discriminate between the ligands or the binding partners. It has been observed that although Bcl-xl and Mcl-1 are classified as homologous proteins, through the course of evolution the binding pocket residues are highly conserved for Bcl-xl; whereas they have been substituted frequently in Mcl-1. The investigation has revealed that the Bcl-xl can adjust the backbone conformation of the binding pocket residues to a larger extent to complement with the shape of different binding partners whereas the Mcl-1 shows more variation in the side chain conformation of binding pocket residues for the same purpose.

BIM Binding Remotely Regulates BAX Activation: Insights from the Free Energy Landscapes.

Activation of the pro-apoptotic BAX protein, a BCL-2 family member, is known to trigger apoptosis by forming pores in the mitochondrial outer membrane (MOM). While in the cytosol, release of its transmembrane C-terminal helix (called α9 helix) from a well-characterized binding pocket (BC groove) and subsequent permeabilization of the MOM are understood to be the initiating events of the activation. Concerning what initiates BAX activation, so far one plausible suggestion has been that the transient attachment of BH3-only peptide at a distal site from the BC groove triggers the activation process. Yet how this pivotal step displaces α9 from the BC groove has remained poorly understood. Using a combination of standard molecular dynamics and enhanced sampling methods, the energy landscape of BIM (BH3-only peptide) induced BAX activation has been computed, and the molecular origin of those events is hereby reported in atomistic detail. The simulated transition pathway of α9 release reveals that BIM subdues the energetic cost of the process by reducing the activation energy barrier to some extent but mostly by minimizing the free energy difference between the active (α9-released) and inactive (α9-bound) states. Interestingly, the flexibility of the α9 helix itself plays a decisive role in this mechanism. The impact of BIM encounter at the distal site is found to propagate to the α9 (BC groove bound) mostly through conserved pathways of residue level interactions. Overall, the thermodynamic basis of the "hit-and-run" mechanism for activation of the BCL-2 family is presented reconciling the available biochemical observations.

The long unstructured region of Bcl-xl modulates its structural dynamics.

Bcl-xl protein has a long unstructured loop attached to its structured region which joins two helices. The necessity to have this unstructured segment in Bcl-xl is not yet well understood. To what extent the unstructured segment can influence the dynamics of the structured region of protein, with potential to influence the function, has been investigated in this work. Molecular dynamics simulation and principal component analysis show how the loop affects the internal motions of the protein, particularly its ligand binding pocket. Generally an unstructured region in the structure would promote flexibility resulting entropic stability but in contrary, here it narrows down the conformational space of the structured region of protein that could be hypothesized to impact the functional precision. Effects of the loop propagate to the binding pocket through structural rearrangements of polar side chains. The immediate suspicion of possible impact of phosphorylation to modulate the function of the protein is proven to be a fact, as the phosphorylated S49 and S62 located on the large unstructured region are seen to perturb the electrostatic network of the structure; an observation that validates and clarifies the role of loop as a modulator through biophysical and biochemical mechanisms. Proteins 2017; 85:1567-1579. © 2017 Wiley Periodicals, Inc.

C-terminal tail insertion of Bcl-xL in membrane occurs via partial unfolding and refolding cycle associating microsolvation.

Bcl-xL, a member of the Bcl-2 family of proteins, remains distributed over the cytosol and the mitochondrial membrane, maintaining a balance between apoptosis and the survival of the cell. Passage to the membrane is essential for its biological functions (e.g. to antagonize pro-apoptotic proteins of the Bcl2 family), which is known to be initiated by the insertion of the C-terminal segment into the membrane. This tail, composed of ∼24 residues, is reported to act as a pseudo-inhibitor of the protein itself, adapting a helical conformation. It gets released from the confinement when Bcl-xL approaches the membrane. This article reports the events associated with the insertion of the helical tail into an explicitly modeled all-atom membrane, which reveals a partial unfolding to refolding cycle of the peptide, correlating with the early insertion, to a fully inserted state. The polar interactions have been found to have a dominant role in steering the peptide towards the membrane at the desired orientation. The landscape of the potential of mean force (PMF) is consistent with the proposed mechanism. Molecular dynamics further brings the insight that the peptide insertion is associated with the encapsulation of a thin water layer around the peptide throughout the course of insertion, which motivates the protein to refold once the insertion is complete.

Phosphorylation of Leghemoglobin at S45 is Most Effective to Disrupt the Molecular Environment of Its Oxygen Binding Pocket.

In leguminous plants, nitrogenase that catalyzes anaerobic symbiotic nitrogen fixation is protected by the sequestration of O2 by Leghemoglobin (LegH). The modulation of the oxygen binding capacity of Hemoglobin (Hb) by different post-translational modifications is well studied; whereas similar studies on LegH's O2 binding are not yet benchmarked. Our results show that in vitro serine phosphorylation of recombinant LegH from Lotus japonicus, a model legume, by a homologous kinase caused a reduction in its oxygen consumption as determined by Clark type electrode. Although mass spectrometry revealed a few phosphorylated serine residues in the LegH, molecular modeling study showed that particularly S45 is the most critical one, along with S55, however the latter with lesser impact on its molecular environment responsible for oxygen consumption. Separate S45D and S55D mutants of recombinant LegH also corroborated the results obtained from molecular modeling study. Thus, this work lays groundwork for further investigation of structural and functional role of serine phosphorylation as one of the mechanisms by which oxygen consumption by LegH may possibly be regulated during nodulation.

Interactions between Bcl-xl and its inhibitors: Insights into ligand design from molecular dynamics simulation.

The Bcl-xl protein is a potential drug target for cancer, and it has a relatively flat and flexible binding pocket. ABT263 is one of the most promising molecules that inhibit Bcl-xl, and it was developed from its precursor ABT737 with suitable substitutions. However, the structural and mechanistic implications of those changes have not yet been reported. Molecular dynamics simulation has revealed that the conformational microstates of the complex of Bcl-xl and ABT263 shows heterogeneity at the binding interface with Bcl-xl in contrast to the precise interactions witnessed in case of ABT737. This occurs because not all the functional groups of ABT263 are able to anchor into the binding pocket simultaneously at the time of complexation; leaving at least one group weakly associated every time. The insight into the mechanism shows that, in spite of such mutual exclusivity, the resultant effect becomes beneficial, i.e., becomes more effective than ABT737. Going against the traditional belief, the calculations also confirm that there is no benefit of reshaping the highly flexible binding pocket to allow the ligand to be comfortably accommodated and avoid conflicting orientations of the functional groups, as the destabilization becomes active from other sources. These structural clues and in-silico tests suggest possible avenues for improving the binding affinity of ABT263 through further in-vitro and in-vivo tests.