A combined atomic force microscopy imaging and docking study to investigate the complex between p53 DNA binding domain and Azurin

The tumor suppressor p53 interacts with the redox copper protein Azurin (AZ) forming a complex which is of some relevance in biomedicine and cancer therapy. To obtain information on the spatial organization of this complex when it is immobilized on a substrate, we have used tapping mode‐atomic force microscopy (TM‐AFM) imaging combined with computational docking. The vertical dimension and the bearing volume of the DNA binding domain (DBD) of p53, anchored to functionalized gold substrate through exposed lysine residues, alone and after deposing AZ, have been measured by TM‐AFM. By a computational docking approach, a three‐dimensional model for the DBD of p53, before and after addition of AZ, have been predicted. Then we have calculated the possible arrangements of these biomolecular systems on gold substrate by finding a good agreement with the related experimental distribution of the height. The potentiality of the approach combining TM‐AFM imaging and computational docking for the study of biomolecular complexes immobilized on substrates is briefly discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Modeling the interaction between the N‐terminal domain of the tumor suppressor p53 and azurin

It is known that the half life of the tumor suppressor p53 can be increased by the interaction with the bacterial protein azurin, resulting in an enhanced anti‐tumoral activity. The understanding of the molecular mechanisms on the basis of this phenomenon can open the way to new anti‐cancer strategies. Some experimental works have given evidence of an interaction between p53 and azurin (AZ); however the binding regions of the proteins are still unknown. Recently, fluorescence studies have shown that p53 partakes in the binding with the bacterial protein by its N‐terminal (NT) domain. Here we have used a computational method to get insight into this interacting mode. The model that we propose for the best complex between AZ and p53 has been obtained from a rigid‐body docking, coupled with a molecular dynamics (MD) simulation, a free energy calculation, and validated by mutagenesis analysis. We have found a high degree of geometric fit between the two proteins that are kept together by several hydrophobic interactions and numerous hydrogen bonds. Interestingly, it has emerged that AZ binds essentially to the helices H_I and H_III of the p53 NT domain, which are also interacting regions for the foremost inhibitor of p53, MDM2. Copyright © 2009 John Wiley & Sons, Ltd.     

Modelling the interaction between the p53 DNA-binding domain and the p28 peptide fragment of Azurin

Recent experimental data reveal that the peptide fragment of Azurin called p28, constituted by the amino acid residues from 50 to 77 of the whole protein, retains both the Azurin cellular penetration ability and antiproliferative activity. p28 is hypothesized to act by stabilizing the well-known tumour suppressor p53 via a pathway independent from the oncogene Mdm2, which is the main p53 down-regulator, with its anticancer potentiality being probably connected with the binding of its amino acid residues 11 to 18 to p53. However, the p28 mode of action has not been completely elucidated yet, mostly because the details of the p28 interaction with p53 are still unknown. In the present study, computational docking modelling supported by cluster analysis, molecular dynamics simulations and binding free energy calculations have been performed to model the interaction between the DNA-binding domain (DBD) of p53 and the p28 fragment. Since the folding state of p28 when interacting with p53 inside the cell is not known, both the folded and the unfolded structures of this peptide have been taken into consideration. In both the cases, we have found that p28 is able to form with DBD a complex characterized by favourable negative binding free energy, high shape complementarity, and the presence of several hydrogen bonds at the interface. These results suggest that p28 might exert its anticancer action by hampering the binding of ubiquitin ligases to DBD, susceptible to promoting the p53 proteasomal degradation.     

Molecular dynamics simulations suggest Thiosemicarbazones can bind p53 cancer mutant R175H

Cancer pathologies are associated with the unfolding and aggregation of most recurring mutations in the DNA Binding Domain (DBD) of p53 that coordinate the destabilization of protein. Substitution at the 175th codon with arginine to histidine (R175H, a mutation of large to small side-chain amino acid) destabilizes the DBD by 3 kcal/mol and triggers breasts, lung cancer, etc. Stabilizing the p53 mutant by small molecules offers an attractive drug-targeted anti-cancer therapy. The thiosemicarbazone (TSC) molecules NPC and DPT are known to act as zinc-metallochaperones to reactivate p53R175H. Here, a combination of LESMD simulations for 10 TSC conformations with a p53R175H receptor, single ligand-protein conformation MD, and ensemble docking with multiple p53R175H conformations observed during simulations is suggested to identify the potential binding site of the target protein in light of their importance for the direct TSC – p53R175H binding. NPC binds mutant R175H in the loop region L2-L3, forming pivotal hydrogen bonds with HIS175, pi?sulfur bonds with TYR163, and pi-alkyl linkages with ARG174 and PRO190, all of which are contiguous to the zinc-binding native site on p53DBD. DPT, on the other hand, was primarily targeting alternative binding sites such as the loop-helix L1/H2 region and the S8 strand. The similar structural characteristics of TSC-bound p53R175H complexes with wild-type p53DBD are thought to be attributable to involved interactions that favour binding free energy contributions of TSC ligands. Our findings may be useful in the identification of novel pockets with druggable properties.     

Aggregation of zinc‐free p53 is inhibited by Hsp90 but not other chaperones

The structured DNA‐binding domain (DBD) of p53 is a well‐known client protein of the chaperone Hsp90. The p53 DBD contains a single zinc ion, coordinated by the side chains of Cys176, His179, Cys238, and Cys242; zinc coordination plays a structural role to stabilize the DBD and is required for its DNA binding. The ambiguous nature of the p53‐Hsp90 interaction, together with the stabilizing role of the zinc in the structure of the DBD, prompted us to examine the interaction of Hsp90 with zinc‐free p53 DBD. NMR spectroscopy and native gel electrophoresis did not show any apparent preference for the interaction of the destabilized zinc‐free form of p53 DBD with Hsp90. Intriguingly, however, at lower protein concentrations, closer to physiological concentrations, the addition of Hsp90, but not other chaperones such as Hsp70, Hsp40, p23, and HOP, appears to slow or prevent the aggregation of zinc‐free p53 DBD. This result suggests that part of the function of the Hsp90‐p53 interaction in the cell may be to stabilize the apoprotein in the absence of zinc.     

A model for the LexA repressor DNA complex

A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein–DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B-DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn-41, Glu-44, and Glu-45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser-39, Ala-42, and Asn-41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two β-sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half-site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed. © 1995 Wiley-Liss, Inc.     

Modulation of interaction of mutant TP53 and wild type BRCA1 by alkaloids: a computational approach towards targeting protein-protein interaction as a futuristic therapeutic intervention strategy for breast cancer impediment

Protein–protein interactions (PPI) are a new emerging class of novel therapeutic targets. In order to probe these interactions, computational tools provide a convenient and quick method towards the development of therapeutics. Keeping this in view the present study was initiated to analyse interaction of tumour suppressor protein p53 (TP53) and breast cancer associated protein (BRCA1) as promising target against breast cancer. Using computational approaches such as protein–protein docking, hot spot analyses, molecular docking and molecular dynamics simulation (MDS), stepwise analyses of the interactions of the wild type and mutant TP53 with that of wild type BRCA1 and their modulation by alkaloids were done. Protein–protein docking method was used to generate both wild type and mutant complexes of TP53-BRCA1. Subsequently, the complexes were docked using sixteen different alkaloids, fulfilling ADMET and Lipinski’s rule of five criteria, and were compared with that of a well-known inhibitor of PPI, namely nutlin. The alkaloid dicentrine was found to be the best docked alkaloid among all the docked alklaloids as well as that of nutlin. Furthermore, MDS analyses of both wild type and mutant complexes with the best docked alkaloid i.e. dicentrine, revealed higher stability of mutant complex than that of the wild one, in terms of average RMSD, RMSF and binding free energy, corroborating the results of docking. Results suggested more pronounced interaction of BRCA1 with mutant TP53 leading to increased expression of mutated TP53 thus showing a dominant negative gain of function and hampering wild type TP53 function leading to tumour progression.     

p53 DNA结合域与抗癌多肽的拉伸分子动力学研究

研究发现,取自蓝铜蛋白azurin的一段多肽p28能够进入癌细胞,结合到肿瘤抑制因子p53的DNA结合域上,进而增加p53的抗癌能力.本工作中,通过拉伸分子动力学方法,在原子尺度上研究了p28-p53 DBD复合物的解离过程.分析结果显示复合物的解离过程遵循着一定的分离顺序.对解离力的分析以及对沿着解离路径的不可逆做功的计算,使我们能够从复合物的能量地貌中提取有用的信息,而这些信息也决定了复合物的解离过程.     

Ensemble docking of multiple protein structures: considering protein structural variations in molecular docking

One approach to incorporate protein flexibility in molecular docking is the use of an ensemble consisting of multiple protein structures. Sequentially docking each ligand into a large number of protein structures is computationally too expensive to allow large-scale database screening. It is challenging to achieve a good balance between docking accuracy and computational efficiency. In this work, we have developed a fast, novel docking algorithm utilizing multiple protein structures, referred to as ensemble docking, to account for protein structural variations. The algorithm can simultaneously dock a ligand into an ensemble of protein structures and automatically select an optimal protein structure that best fits the ligand by optimizing both ligand coordinates and the conformational variable m, where m represents the m-th structure in the protein ensemble. The docking algorithm was validated on 10 protein ensembles containing 105 crystal structures and 87 ligands in terms of binding mode and energy score predictions. A success rate of 93% was obtained with the criterion of root-mean-square deviation <2.5 A if the top five orientations for each ligand were considered, comparable to that of sequential docking in which scores for individual docking are merged into one list by re-ranking, and significantly better than that of single rigid-receptor docking (75% on average). Similar trends were also observed in binding score predictions and enrichment tests of virtual database screening. The ensemble docking algorithm is computationally efficient, with a computational time comparable to that for docking a ligand into a single protein structure. In contrast, the computational time for the sequential docking method increases linearly with the number of protein structures in the ensemble. The algorithm was further evaluated using a more realistic ensemble in which the corresponding bound protein structures of inhibitors were excluded. The results show that ensemble docking successfully predicts the binding modes of the inhibitors, and discriminates the inhibitors from a set of noninhibitors with similar chemical properties. Although multiple experimental structures were used in the present work, our algorithm can be easily applied to multiple protein conformations generated by computational methods, and helps improve the efficiency of other existing multiple protein structure(MPS)-based methods to accommodate protein flexibility.     

The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.     

Docking study and binding free energy calculation of poly (ADP-ribose) polymerase inhibitors

Recently, the massively parallel computation of absolute binding free energy with a well-equilibrated system (MP-CAFEE) has been developed. The present study aimed to determine whether the MP-CAFEE method is useful for drug discovery research. In the drug discovery process, it is important for computational chemists to predict the binding affinity accurately without detailed structural information for protein / ligand complex. We investigated the absolute binding free energies for Poly (ADP-ribose) polymerase-1 (PARP-1) / inhibitor complexes, using the MP-CAFEE method. Although each docking model was used as an input structure, it was found that the absolute binding free energies calculated by MP-CAFEE are well consistent with the experimental ones. The accuracy of this method is much higher than that using molecular mechanics Poisson-Boltzmann / surface area (MM / PBSA). Although the simulation time is quite extensive, the reliable predictor of binding free energies would be a useful tool for drug discovery projects.     

An efficient computational method for calculating ligand binding affinities

Virtual compound screening using molecular docking is widely used in the discovery of new lead compounds for drug design. However, the docking scores are not sufficiently precise to represent the protein-ligand binding affinity. Here, we developed an efficient computational method for calculating protein-ligand binding affinity, which is based on molecular mechanics generalized Born/surface area (MM-GBSA) calculations and Jarzynski identity. Jarzynski identity is an exact relation between free energy differences and the work done through non-equilibrium process, and MM-GBSA is a semimacroscopic approach to calculate the potential energy. To calculate the work distribution when a ligand is pulled out of its binding site, multiple protein-ligand conformations are randomly generated as an alternative to performing an explicit single-molecule pulling simulation. We assessed the new method, multiple random conformation/MM-GBSA (MRC-MMGBSA), by evaluating ligand-binding affinities (scores) for four target proteins, and comparing these scores with experimental data. The calculated scores were qualitatively in good agreement with the experimental binding affinities, and the optimal docking structure could be determined by ranking the scores of the multiple docking poses obtained by the molecular docking process. Furthermore, the scores showed a strong linear response to experimental binding free energies, so that the free energy difference of the ligand binding (ΔΔG) could be calculated by linear scaling of the scores. The error of calculated ΔΔG was within ≈±1.5 kcal•mol⁻¹ of the experimental values. Particularly, in the case of flexible target proteins, the MRC-MMGBSA scores were more effective in ranking ligands than those generated by the MM-GBSA method using a single protein-ligand conformation. The results suggest that, owing to its lower computational costs and greater accuracy, the MRC-MMGBSA offers efficient means to rank the ligands, in the post-docking process, according to their binding affinities, and to compare these directly with the experimental values.     

Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman,Atomic Force and Surface Plasmon Resonance spectroscopies

BackgroundMutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in > 50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding.MethodsMolecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28.ResultsWe show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge.ConclusionsThese results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function.General significanceRaman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53.     

Structural model of the mAb 806-EGFR complex using computational docking followed by computational and experimental mutagenesis

In this work, we combined computational protein-protein docking with computational and experimental mutagenesis to predict the structure of the complex formed by monoclonal antibody 806 (mAb 806) and the epidermal growth factor receptor (EGFR). We docked mAb 806, an antitumor antibody, to its epitope of EGFR residues 287-302. Potential mAb 806-EGFR orientations were generated, and computational mutagenesis was used to filter them according to their agreement with experimental mutagenesis data. Further computational mutagenesis suggested additional mutations, which were tested to arrive at a final structure that was most consistent with experimental mutagenesis data. We propose that this is the EGFR-mAb 806 structure, in which mAb 806 binds to an untethered form of the receptor, consistent with published experimental results. The steric hindrance created by the antibody near the EGFR dimer interface interferes with receptor dimerization, and we postulate this as the structural origin for the antitumor effect of mAb 806.     

Interaction of the anticancer p28 peptide with p53-DBD as studied by fluorescence,FRET, docking and MD simulations

Background

The p28 peptide, derived from the blue copper protein Azurin, exerts an anticancer action due to interaction with the tumor suppressor p53, likely interfering with its down-regulators. Knowledge of both the kinetics and topological details of the interaction, could greatly help to understand the peptide anticancer mechanism.

Automated docking of ligands to antibodies: methods and applications

Many approaches to studying protein-ligand interactions by computational docking are currently available. Given the structures of a protein and a ligand, the ultimate goal of all docking methods is to predict the structure of the resulting complex. This requires a suitable representation of molecular structures and properties, search algorithms to efficiently scan the configuration space for favorable interaction geometries, and accurate scoring functions to evaluate and rank the generated orientations. For many of the available methods, tests on experimentally known antibody-antigen or antibody-hapten complexes have appeared in the literature. In addition, some of them have been used in predictive studies on antibody-ligand interactions to provide structural insights where adequate experimental information is missing. The AutoDock program is presented as example of a method for flexibly docking ligands to antibodies. Applying parameters of the second-generation AMBER force field, three antibody-hapten complexes (AN02, DB3, NC6.8) are used as new test cases to analyze the ability of the method to reproduce experimental findings. The X-ray structures could be reconstituted and the corresponding solutions were ranked with best energy score in all cases. Docking to the free instead of the complexed NC6.8 structure indicated the limits of the rigid protein treatment, although fairly good guesses about the location of the binding site and the contact residues could still be obtained if conformational flexibility was allowed at least in the ligand.     

Zn(2+)-dependent misfolding of the p53 DNA binding domain

The DNA binding domain (DBD) of p53 folds by a complex mechanism that involves parallel pathways and multiple intermediates, both on- and off-pathway. This heterogeneity renders DBD particularly susceptible to misfolding and aggregation. The origins of parallel folding mechanisms are not well understood. DBD folding heterogeneity may be caused by the presence of the single bound Zn2+. To test that hypothesis, we carried out kinetic folding studies of DBD in its Zn2+-free form (apoDBD) and in the presence of various concentrations of free Zn2+ and the Zn2+-nitrilotriacetate (NTA) complex. Folding kinetics of apoDBD and DBD are similar, although apoDBD folds faster than DBD at some urea concentrations. The principle consequence of Zn2+ removal is to accelerate unfolding and simplify it from two exponential phases to one. Metal binding interactions are therefore not responsible for the observed complexity of the folding reaction. A slight stoichiometric excess of free Zn2+ arrests folding and traps the protein in a misfolded state in which Zn2+ is bound to nonphysiological ligands. Folding can be rescued by providing metal ions in the form of the NTA-Zn2+ complex, which simultaneously protects against misligation and provides a source of Zn2+ for regenerating the functional protein. This chemical metallochaperone strategy may be an effective means for improving folding efficiency of other metal binding proteins. The findings suggest that, in vivo, DBD must fold in an environment where free Zn2+ concentration is low and its bioavailability is carefully regulated by cellular metallochaperones.