Signatures of tRNAGlx -specificity in proteobacterial glutamyl-tRNA synthetases.

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNAGlu . Yet not all bacterial GluRSs glutamylate tRNAGlu ; many glutamylate both tRNAGlu and tRNAGln , while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu . Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx -specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx -specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu -bound GluRS were from non-proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteobacterial and non-proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNAGlx -specificity vis-a-vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we probed the structural basis of the tRNAGlx -specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D-helix contacting loop of GluRS in the modulation of tRNAGln -specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx -specificity of GluRS, this is the first report of complementary signatures of tRNAGlx -specificity in GluRS.

Molecular Dynamics and Machine Learning reveal distinguishing mechanisms of Competitive Ligands to perturb α,ß-Tubulin.

The mechanisms of action of ligands competing for the Colchicine Binding Site (CBS) of the α,ß-Tubulin are non-standard compared to the commonly witnessed ligand-induced inhibition of proteins. This is because their potencies are not solely judged by the binding affinity itself, but also by their capacity to bias the conformational states of the dimer. Regarding the latter requirement, it is observed that ligands competing for the same pocket that binds colchicine exhibit divergence in potential clinical outcomes. Molecular dynamics-based ∼5.2 µs sampling of α,ß-Tubulin complexed with four different ligands has revealed that each ligand has its customized way of influencing the complex. Primarily, it is the proportion of twisting and/or bending characteristic of modes of the intrinsic dynamics which is revealed to be 'fundamental' to tune this variation in the mechanism. The milder influence of 'bending' makes a ligand (TUB092), better classifiable under the group of vascular disrupting agents (VDAs), which are phenotypically effective on cytoskeletons; whereas a stronger impact of 'bending' makes the classical ligand Colchicine (COL) a better Anti-Mitotic agent (AMA). Two other ligands BAL27862 (2RR) and Nocodazole (NZO) fall in the intermediate zone as they fail to explicitly induce bending modes. Random Forest Classification method and K-means Clustering is applied to reveal the efficiency of Machine Learning methods in classifying the Tubulin conformations according to their ligand-specific perturbations and to highlight the significance of specific amino acid residues, mostly positioned in the α-ß and ß-ß interfaces involved in the mechanism. These key residues responsible to yield discriminative actions of the ligands are likely to be highly useful in future endeavours to design more precise inhibitors.

A novel acidic pH-dependent metacaspase governs defense-response against pathogens in tomato.

The importance of metacaspases in programmed cell death and tissue differentiation is known, but their significance in disease stress response, particularly in a crop plant, remained enigmatic. We show the tomato metacaspase expression landscape undergoes differential reprogramming during biotrophic and necrotrophic modes of pathogenesis; also, the metacaspase activity dynamics correlate with the disease progression. These stresses have contrasting effects on the expression pattern of SlMC8, a Type II metacaspase, indicating that SlMC8 is crucial for stress response. In accordance, selected biotic stress-related transcription factors repress SlMC8 promoter activity. Interestingly, SlMC8 exhibits maximum proteolysis at an acidic pH range of 5-6. Molecular dynamics simulation identified the low pH-driven protonation event of Glu246 as critical to stabilize the interaction of SlMC8 with its substrate. Mutagenesis of Glu246 to charge-neutral glutamine suppressed SlMC8's proteolytic activity, corroborating the importance of the amino acid in SlMC8 activation. The glutamic acid residue is found in an equivalent position in metacaspases having acidic pH dependence. SlMC8 overexpression leads to heightened ROS levels, cell death, and tolerance to PstDC3000, and SlMC8 repression reversed the phenomena. However, the overexpression of SlMC8 increases tomato susceptibility to necrotrophic Alternaria solani. We propose that SlMC8 activation due to concurrent changes in cellular pH during infection contributes to the basal resistance of the plant by promoting cell death at the site of infection, and the low pH dependence acts as a guard against unwarranted cell death. Our study confirms the essentiality of a low pH-driven Type II metacaspase in tomato biotic stress-response regulation.

Identification and characterization of a flexile G-quadruplex in the distal promoter region of stemness gene REX1.

We have identified a parallel G-quadruplex (R1WT) in the distal promoter region (-821 base-pairs upstream of the TSS) of the pluripotent gene REX1. Through biophysical and biochemical approach, we have characterized the G-quadruplex (GQ) as a potential molecular switch that may control REX1 promoter activity to determine the transcriptional fate. Small- molecule interactive study of the monomeric form of R1WT (characterized as R1mut2) with TMPyP4 and BRACO-19 revealed GQ destabilization upon interaction with TMPyP4 and stabilization upon interaction with BRACO-19. This distinctive drug interactivity suggests the in cellulo R1WT to be a promising drug target. The endogenous existence of R1WT was confirmed by BG4 antibody derived chromatin immunoprecipitation experiment. Here in, we also report the endogenous interaction of GQ specific transcription factors (TFs) with R1WT region in the human chromatin of cancer cell. The wild-type G-quadruplex was found to interact with four important transcription factors, (i) specificity protein (Sp1) (ii) non-metastatic cell 2 (NM23-H2): a diphosphatase (iii) cellular nucleic acid binding protein (CNBP) and (iv) heterogenous nuclear ribonucleoprotein K (hnRNPK) in the REX1 promoter. In contrast, nucleolin protein (NCL) binding was found to be low to the said G-quadruplex. The flexibility of R1WT between folded and unfolded states, obtained from experimental and computational analysis strongly suggests R1WT to be an important gene regulatory element in the genome. It controls promoter DNA relaxation with the coordinated interaction of transcription factors, the deregulation of which seeds stemness characteristic in cancer cells for further metastatic progression.

Sequence driven interaction of amino acids in de-novo designed peptides determines c-Myc G-quadruplex unfolding inducing apoptosis in cancer cells.

c-MYC proto-oncogene harbors a putative G-quadruplex structure (Pu27) at the NHEIII1 domain, which can shuffle between transcriptional inhibitor quadruplex and transcriptionally active duplex. In cancer cells this quadruplex destabilization is preferred and NHEIII1 domain assume a duplex topology thereby inducing c-MYC overexpression and tumorigenesis. Hence, the c-MYC quadruplex acts as an excellent target for anti-cancer therapy. Though researcher have tried to develop G-quadruplex targeted small molecules, work with G-quadruplex targeting peptides is very limited. Here we present a peptide that can bind to c-MYC quadruplex, destabilize the tetrad core, and permit the formation of a substantially different structure from the quartet core seen in the canonical G-quadruplexes. Such conformation potentially acted as a roadblock for transcription factors thereby reducing cMYC expression. This event sensitizes the cancer cell to activate apoptotic cascade via the c-MYC-VEGF-A-BCL2 axis. This study provides a detailed insight into the peptide-quadruplex interface that encourages better pharmacophore design to target dynamic quadruplex structure. We believe that our results will contribute to the development, characterization, and optimization of G-quadruplex binding peptides for potential clinical application.

Mechanisms of influence of the microtubule over-stabilizing ligands on the structure and intrinsic dynamics of α,ß-Tubulin.

The intervention into the cell cycle progression by administering microtubule over-stabilizing ligands that arrest the mitotic cell division by preventing spindle dissociation, is a promising strategy to fight against cancers. The building blocks of the microtubules and the spindles, i.e. the α,ß-tubulin dimer, upon binding of such ligands, stay more comfortably in the microtubular multimeric form; the phenomenon of which is the key to the said over-stabilization. Using two such over-stabilizing ligands, Taxol and Taxotere, the present work reports the collective changes that these ligands induce on the structure and dynamics of the α,ß-tubulin dimer which could be reconciled as the molecular basis of the over-stabilization of the microtubules; the trends have been found to be statistically significant across all independent calculations on them. The ligand binding increases the coherence between the residue communities of the two opposite faces of the ß-subunit, which in a periodic arrangement in microtubule are knwon to form intermolecular contact with each other. This is likely to create an indirect cooperativity between those structural regions and this is a consequence of the reshuffling of the internal network of interactions upon ligand binding. Such reorganizations are also complemented by the increased contributions of the softer modes of the intrinsic dynamics more, which is likely to increase the plasticity of the system favourable for making structural adjustments in a multimer. Further, the ligands are able to compensate the drawback of lacking one phosphate group in protein-GDP interactions compared to the same for protein-GTP and this is in agreement with the hints form the earlier reports. The findings form a mechanistic basis of the enhanced capacity of the α,ß-tubulin dimer to get more favourably accommodated into the microtubule superstructure upon binding either of Taxol and Taxotere.

In silico analyses of Wnt1 nsSNPs reveal structurally destabilizing variants, altered interactions with Frizzled receptors and its deregulation in tumorigenesis.

Wnt1 is the first mammalian Wnt gene, which is discovered as proto-oncogene and in human the gene is located on the chromosome 12q13. Mutations in Wnt1 are reported to be associated with various cancers and other human diseases. The structural and functional consequences of most of the non-synonymous SNPs (nsSNPs), present in the human Wnt1 gene, are not known. In the present work, extensive bioinformatics analyses are used to screen 292 nsSNPs of Wnt1 for predicting pathogenic and harmless polymorphisms. We have identified 10 highly deleterious nsSNPs among which 7 are located within the highly conserved areas. These 10 nsSNPs are also predicted to affect the post-translational modifications of Wnt1. Further, structure based stability analyses of these 10 highly deleterious nsSNPs revealed 8 variants as highly destabilizing. These 8 highly destabilizing variants were shown to have high BC score and high RMSIP score from normal mode analyses. Based on the deformation energies, obtained from the normal mode analyses, variants like G169A, G169S, G331R and G331S were found to be unstable. Molecular Dynamics (MD) simulations revealed structural stability and fluctuation of WT Wnt1 and its prioritized variants. RMSD remained fluctuating mostly between 4 and 5 Å and occasionally between 3.5 and 5.5 Å ranges. RMSF in the CTD region (residues 330-360) of the binding pocket were lower compared to that of WT. Studying the impacts of nsSNPs on the binding interface of Wnt1 and seven Frizzled receptors have predicted substitutions which can stabilize or destabilize the binding interface. We have found that Wnt1 and FZD8-CRD is the best docked complex in our study. MD simulation based analyses of wild type Wnt1-FZD8-CRD complex and the 8 prioritized variants revealed that RMSF was higher in the unstructured regions and RMSD remained fluctuating in the region of 5 Å ± 1 Å. We have also observed differential Wnt1 gene expression pattern in normal, tumor and metastatic conditions across different tissues. Wnt1 gene expression was significantly higher in metastatic tissues of lungs, colon and skin; and was significantly lower in metastatic tissues of breast, esophagus and kidney. We have also found that Wnt1 deregulation is associated with survival outcome in patients with gastric and breast cancer. Furthermore, these computationally screened highly deleterious nsSNPs of Wnt1 can be analyzed in population based genetic studies and may help understand the Wnt1 associated diseases.

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.

Modulation of p53 binding to MDM2: computational studies reveal important roles of Tyr100.

BACKGROUND: The tumor suppressor protein p53 is regulated by the ubiquitin ligase MDM2 which down-regulates p53. In tumours with overexpressed MDM2, the p53-MDM2 interaction can be interrupted by a peptide or small molecule to stabilize p53 as a therapeutic strategy. Structural and biochemical/mutagenesis data show that p53 has 3 hydrophobic residues F19, W23 and L26 that embed into the ligand binding pocket of MDM2 which is highly plastic in nature and can modulate its size to accommodate a variety of ligands. This binding pocket is primarily dependent on the orientation of a particular residue, Y100. We have studied the role of the dynamics of Y100 in p53 recognition. RESULTS: Molecular dynamics simulations show that the Y100 side chain can be in "open" or "closed" states with only the former enabling complex formation. When both p53 and MDM2 are in near native conformations, complex formation is rapid and is driven by the formation of a hydrogen bond between W23 of p53 and L54 of MDM2 or by the embedding of F19 of p53 into MDM2. The transition of Y100 from "closed" to "open" can increase the size of the binding site. Interconversions between these two states can be induced by the N-terminal region of MDM2 or by the conformations of the p53 peptides. CONCLUSION: Molecular dynamics simulations have revealed how the binding of p53 to MDM2 is modulated by the conformational mobility of Y100 which is the gatekeeper residue in MDM2. The mobility of this residue can be modulated by the conformations of p53 and the Nterminal lid region of MDM2.

HKT1;5 Transporter Gene Expression and Association of Amino Acid Substitutions With Salt Tolerance Across Rice Genotypes.

Plants need to maintain a low Na+/K+ ratio for their survival and growth when there is high sodium concentration in soil. Under these circumstances, the high affinity K+ transporter (HKT) and its homologs are known to perform a critical role with HKT1;5 as a major player in maintaining Na+ concentration. Preferential expression of HKT1;5 in roots compared to shoots was observed in rice and rice-like genotypes from real time PCR, microarray, and RNAseq experiments and data. Its expression trend was generally higher under increasing salt stress in sensitive IR29, tolerant Pokkali, both glycophytes; as well as the distant wild rice halophyte, Porteresia coarctata, indicative of its importance during salt stress. These results were supported by a low Na+/K+ ratio in Pokkali, but a much lower one in P. coarctata. HKT1;5 has functional variability among salt sensitive and tolerant varieties and multiple sequence alignment of sequences of HKT1;5 from Oryza species and P. coarctata showed 4 major amino acid substitutions (140 P/A/T/I, 184 H/R, D332H, V395L), with similarity amongst the tolerant genotypes and the halophyte but in variance with sensitive ones. The best predicted 3D structure of HKT1;5 was generated using Ktrab potassium transporter as template. Among the four substitutions, conserved presence of aspartate (332) and valine (395) in opposite faces of the membrane along the Na+/K+ channel was observed only for the tolerant and halophytic genotypes. A model based on above, as well as molecular dynamics simulation study showed that valine is unable to generate strong hydrophobic network with its surroundings in comparison to leucine due to reduced side chain length. The resultant alteration in pore rigidity increases the likelihood of Na+ transport from xylem sap to parenchyma and further to soil. The model also proposes that the presence of aspartate at the 332 position possibly leads to frequent polar interactions with the extracellular loop polar residues which may shift the loop away from the opening of the constriction at the pore and therefore permit easy efflux of the Na+. These two substitutions of the HKT1;5 transporter probably help tolerant varieties maintain better Na+/K+ ratio for survival under salt stress.

Multiple peptide conformations give rise to similar binding affinities: molecular simulations of p53-MDM2.

Molecular dynamics simulations, guided by experimental information (Zondlo et al. Biochemistry 2006, 45, 11945-11957) have been used successfully to reproduce experimental trends in binding affinities of variant p53 peptides with MDM2. Simulations reveal how the conformations of the peptides and the receptor modulate each other to optimize interactions. The conformations of the uncomplexed peptides are governed by a combination of helix and intrinsic disorder (in agreement with experiments), while in the complexed state two very different conformations can coexist. This yields very similar binding affinities, driven by either enthalpy or entropy.

Conformational changes in protein function.

Conformational changes are the hallmarks of protein dynamics and are often intimately related to protein functions. Molecular dynamics (MD) simulation is a powerful tool to study the time-resolved properties of protein structure in atomic details. In this chapter, we discuss the various applications of MD simulation to the study of protein conformational changes, and introduce several selected advanced techniques that may significantly increase the sampling efficiencies, including locally enhanced sampling (LES), and grow-to-fit molecular dynamics (G2FMD).

Physical interaction between nuclear accumulated CC-NB-ARC-LRR protein and WRKY64 promotes EDS1 dependent Fusarium wilt resistance in chickpea.

Fusarium wilt is one of the most serious diseases affecting chickpea (Cicer arietinum L.). Here, we identified a putative Resistance Gene Analog (CaRGA) from chickpea, encoding a coiled-coil (CC) nucleotide-binding oligomerization domain (NB-ARC) containing leucine-rich repeat (LRR) protein (CC-NLR protein) that confers resistance against Fusarium oxysporum f. sp. ciceri race1 (Foc1). Over-expression and silencing of CaRGA in chickpea resulted in enhanced resistance and hyper-susceptibility, respectively against Foc1. Furthermore, defense response to Foc1 depends on CC-NLR interaction with WRKY64 transcription factor. CaRGA mediated wilt resistance largely compromised when WRKY64 was silenced. We also determined in planta intramolecular interactions and self-association of chickpea CC-NLR protein. The study shows CC domain suppressing auto-activation of the full-length CC-NLR protein in the absence of pathogen through self-inhibitory intramolecular interaction with NB-ARC domain, which is attenuated by self-interactions to LRR domain. Chickpea CC-NLR protein forms homocomplexes and then interacts with WRKY64. CC-NLR protein further phosphorylates WRKY64 thereby, ubiquitination and proteasome mediated degradation are protected. Phosphorylated WRKY64 with increased stability binds to EDS1 promoter and stimulates its transcription that induces in planta ectopic cell-death. The detailed analysis of CC-NLR and WRKY interactions provide a better understanding of the immune regulation by NLR proteins under biotic stresses.

Folding transition-state and denatured-state ensembles of FSD-1 from folding and unfolding simulations.

Characterization of the folding transition-state ensemble and the denatured-state ensemble is an important step toward a full elucidation of protein folding mechanisms. We report herein an investigation of the free-energy landscape of FSD-1 protein by a total of four sets of folding and unfolding molecular dynamics simulations with explicit solvent. The transition-state ensemble was initially identified from unfolding simulations at 500 K and was verified by simulations at 300 K starting from the ensemble structures. The denatured-state ensemble and the early-stage folding were studied by a combination of unfolding simulations at 500 K and folding simulations at 300 K starting from the extended conformation. A common feature of the transition-state ensemble was the substantial formation of the native secondary structures, including both the alpha-helix and beta-sheet, with partial exposure of the hydrophobic core in the solvent. Both the native and non-native secondary structures were observed in the denatured-state ensemble and early-stage folding, consistent with the smooth experimental melting curve. Interestingly, the contact orders of the transition-state ensemble structures were similar to that of the native structure and were notably lower than those of the compact structures found in early-stage folding, implying that chain and topological entropy might play significant roles in protein folding. Implications for FSD-1 folding mechanisms and the rate-limiting step are discussed. Analyses further revealed interesting non-native interactions in the denatured-state ensemble and early-stage folding and the possibility that destabilization of these interactions could help to enhance the stability and folding rate of the protein.

Unfolding dynamics of the protein ubiquitin: insight from simulation.

The temperature-induced unfolding pathway of ubiquitin has been investigated by molecular dynamics simulation at four different temperatures. It has been observed that the sequences of the unfolding events are same at all the temperatures. However, the time scale of the dynamics at different temperatures are different. The transition states at various temperatures also possess similar secondary structural elements. The intermediate conformations visited by the protein at different temperatures can help determination of the transition states. The well known " state" of ubiquitin, hitherto found to be stable only in methanol water mixture, have been observed to be a common transient intermediate conformation in the unfolding path of the protein in water. Our observation about the similarities of the unfolding process at different temperatures strongly recommend for a defined pathway for the unfolding process.

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.

Structure, dynamics, and energetics of water at the surface of a small globular protein: a molecular dynamics simulation.

The dynamics of water around a biomolecular surface has attracted a lot of attention recently. We report here protein-solvent simulation studies of the small globular protein ubiquitin (human). The simulations are run unconstrained, without freezing the bonds. The mean square displacements of the water oxygen atoms show a sublinear trend with time. The diffusion coefficient data indicate that the water in the first hydration layer behaves like water at a temperature that is roughly 12 degrees C lower than the average temperature of the system (27 degrees C). Both the dipolar second-rank relaxation and the survival time correlation function of the water layers show two decay constants, indicating contributions from fast and slow dynamics. A calculation of the interaction energy between the water layers and protein indicates that the interaction energy sharply decreases beyond 4 A from the protein surface.

Anomalous behavior of water around sodium dodecyl sulphate micelles.

The dynamics, structural properties, and energetics of hydration water around a sodium dodecyl sulphate micelle have been investigated using molecular dynamics simulation. A clear revelation of the slow dynamics of the hydration water has been made by separate measurements of the rotational and translational properties. Calculated diffusion coefficients fall within the range of experimentally observed quantities. The water-micelle head group (MHG) hydrogen bond is more stable (by an amount approximately 7.0 kcal/mol) compared to the water-water hydrogen bond. The difference in stability of the water monomers forming different numbers of hydrogen bonds (n=0,1,2) with the MHG has clearly been shown from the analyses of their rotational relaxation, residence times, as well as the energy of interaction with different components of the system. The singly hydrogen-bonded water species is the most abundant and stable. The entropy plays the key role in controlling the relative abundance of the different species.

Why is F19Ap53 unable to bind MDM2? Simulations suggest crack propagation modulates binding.

Why doesn't the F19A mutant of p53 bind to MDM2? Binding thermodynamics have suggested that the loss of packing interactions upon mutating Phe into Ala sidechain results in destabilizing the binding free energy between p53 and MDM2. Does this mutation also modulate the initial recognition between p53 and MDM2? We look at atomistic computer simulations of the process of the initial encounter between wild type p53 peptide and its F19A mutant with the N-terminal domain of MDM2. These simulations show that binding is characterized by a complex multistep process. It starts with the capture of F19 of wild type p53 by certain residues in the MDM2 binding pocket. This initial step anchors the peptide onto the surface of MDM2, and with the consequent reduction in the search space of the peptide, the peptide docks into the partially occluded surface of MDM2. This is similar to a crack forming in an otherwise occluded hydrophobic cavity in MDM2, and the peptide, docked through F19, modulates the propagation of this crack, which subsequently results in the stepwise docking of the rest of the peptide through insertions of W23 and L26. The lack of the bulky sidechain of F in the F19A mutant results in the absence of the initial "grasp" complex, and hence the mutant peptide diffuses randomly on the surface of MDM2 without binding. This is the first such demonstration of the possibility that a "kinetic" effect may partly underlie the destabilized thermodynamics of binding of F19A and is a feature that appears to be conserved in evolution. The observations by Wallace et al. (Mol Cell 2006; 23:251-63) that despite the inability of F19A to bind at the N-terminal domain of MDM2, it gets ubiquitinated, can now be partly understood based on a mechanism whereby the occupation of the binding pocket by ligands/peptides induces, via crack propagation and the dynamics of gatekeeper Y100, the ubiquitination signal for interactions between the acidic domain of MDM2 and the DNA binding domain of p53.

Chemical states of the N-terminal "lid" of MDM2 regulate p53 binding: simulations reveal complexities of modulation.

Phosphorylation of S17 in the N-terminal "lid" of MDM2 (residues 1-24) is proposed to regulate the binding of p53. The lid is composed of an intrinsically disordered peptide motif that is not resolved in the crystal structure of the MDM2 N-terminal domain. Molecular dynamics simulations of MDM2 provide novel insight into how the lid undergoes complex dynamics depending on its phosphorylation state that have not been revealed by NMR analyses. The difference in charges between the phosphate and the phosphomimetic 'Asp' and the change in shape from tetrahedral to planar are manifested in differences in strengths and durations of interactions that appear to modulate access of the binding site to ligands and peptides differentially. These findings unveil the complexities that underlie protein-protein interactions and reconcile some differences between the biochemical and NMR data suggesting that lid mutation or deletion can change the specific activity of MDM2 and provide concepts for future approaches to evaluate the effects of S17 modification on p53 binding.