Targeting the conformational transitions of MDM2 and MDMX: Insights into key residues affecting p53 recognition

The oncogenic proteins MDM2 and MDMX have distinct and critical roles in the control of the activity of the p53 tumor suppressor protein. Recently, we have used spatial coarse graining simulations to analyze the conformational transitions manifest in the p53 recognition of MDM2 and MDMX. These conformational movements are different between MDM2 and MDMX and unveil the presence of conserved and nonconserved interactions in the p53 binding cleft that may be exploited in the design of selective and dual modulators of the oncogenic proteins. In this study, we investigate the conformational profiles of apo‐ and p53‐bound states of MDM2 and MDMX using molecular dynamic simulations along a time scale of 60 ns. The analysis of the trajectories is instrumental to discuss energetical and conformational aspects of p53 recognition and to point out specific key residues whose conformational shifts have crucial roles in affecting the apo‐ and p53‐bound states of MDM2 and MDMX. Among these, in particular, linear discriminant analyses identify diverse conformations of Y99/Y100 (MDMX/MDM2) as markers of the apo‐ and p53‐bound states of the oncogenic proteins. The results of this study shed further light on different p53 recognition in MDM2 and MDMX and may prove useful for the design and identification of new potent and selective synthetic modulators of p53‐MDM2/MDMX interactions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Simulating molecular mechanisms of the MDM2-mediated regulatory interactions: a conformational selection model of the MDM2 lid dynamics

Diversity and complexity of MDM2 mechanisms govern its principal function as the cellular antagonist of the p53 tumor suppressor. Structural and biophysical studies have demonstrated that MDM2 binding could be regulated by the dynamics of a pseudo-substrate lid motif. However, these experiments and subsequent computational studies have produced conflicting mechanistic models of MDM2 function and dynamics. We propose a unifying conformational selection model that can reconcile experimental findings and reveal a fundamental role of the lid as a dynamic regulator of MDM2-mediated binding. In this work, structure, dynamics and energetics of apo-MDM2 are studied as a function of posttranslational modifications and length of the lid. We found that the dynamic equilibrium between "closed" and "semi-closed" lid forms may be a fundamental characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size. Our results revealed that these factors may regulate p53-MDM2 binding by fine-tuning the thermodynamic equilibrium between preexisting conformational states of apo-MDM2. In agreement with NMR studies, the effect of phosphorylation on MDM2 interactions was more pronounced with the truncated lid variant that favored the thermodynamically dominant closed form. The phosphomimetic mutation S17D may alter the lid dynamics by shifting the thermodynamic equilibrium towards the ensemble of "semi-closed" conformations. The dominant "semi-closed" lid form and weakened dependence on the phosphorylation seen in simulations with the complete lid can provide a rationale for binding of small p53-based mimetics and inhibitors without a direct competition with the lid dynamics. The results suggested that a conformational selection model of preexisting MDM2 states may provide a robust theoretical framework for understanding MDM2 dynamics. Probing biological functions and mechanisms of MDM2 regulation would require further integration of computational and experimental studies and may help to guide drug design of novel anti-cancer therapeutics.     

MDM4 binds ligands via a mechanism in which disordered regions become structured

MDM2 and MDM4 are proteins involved in regulating the tumour suppressor p53. MDM2/4 and p53 interact through their N-terminal domains and disrupting this interaction is a potential anticancer strategy. The MDM2-p53 interaction is structurally and biophysically well characterised, whereas equivalent studies on MDM4 are hampered by aggregation of the protein. Here we present the NMR characterization of MDM4 (14-111) both free and in complexes with peptide and small-molecule ligands. MDM4 is more dynamic in its apo state than is MDM2, with parts of the protein being unstructured. These regions become structured upon binding of a ligand. MDM4 appears to bind its ligand through conformational selection and/or an induced fit mechanism; this might influence rational design of MDM4 inhibitors.

Structured summary

MINT-7896835: p53 (uniprotkb:P04637) and MDM4 (uniprotkb:O15151) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-7896820: p53 (uniprotkb:P04637) and MDM4 (uniprotkb:O15151) bind (MI:0407) by nuclear magnetic resonance (MI:0077)     

Dynamics of cholesterol exchange in the oxysterol binding protein family

The oxysterol-binding protein-related protein (ORP) family is essential to sterol transfer and sterol-dependent signal transduction in eukaryotes. The crystal structure of one ORP family member, yeast Osh4, is known in apo and sterol-bound states. In the bound state, a 29 residue N-terminal lid region covers the opening of the cholesterol-binding tunnel, preventing cholesterol exchange. Equilibrium and steered molecular dynamics (MD) simulations of Osh4 were carried out to characterize the mechanism of cholesterol exchange. While most of the structural core was stable during the simulations, the lid was partly opened in the apo equilibrium MD simulation. Helix α7, which undergoes the largest conformational change in the crystallized bound and apo states, is conformationally coupled to the opening of the lid. The movement of α7 helps create a docking site for donor or acceptor membranes in the open state. In the steered MD simulations of cholesterol dissociation, we observed complete opening of the lid covering the cholesterol-binding tunnel. Cholesterol was found to exit the binding pocket in a step-wise process involving (i) the breaking of water-mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.     

Influence of electrostatic forces on the association kinetics and conformational ensemble of an intrinsically disordered protein

Recent work has revealed that the association of a disordered region of a protein with a folded binding partner can occur as rapidly as association between two folded proteins. This is the case for the phosphatase calcineurin (CaN) and its association with its activator calmodulin. Calmodulin binds to the intrinsically disordered regulatory domain of CaN. Previous studies have shown that electrostatic steering can accelerate the binding of folded proteins with disordered ligands. Given that electrostatic forces are strong determinants of disordered protein ensembles, the relationship between electrostatics, conformational ensembles, and quaternary interactions is unclear. Here, we employ experimental approaches to explore the impact of electrostatic interactions on the association of calmodulin with the disordered regulatory region of CaN. We find that estimated association rate constants of calmodulin with our chosen calmodulin-substrates are within the diffusion-limited regime. The association rates are dependent on the ionic strength, indicating that favorable electrostatic forces increase the rate of association. Further, we show that charged amino acids outside the calmodulin-binding site modulate the binding rate. Conformational ensembles obtained from computer simulations suggest that electrostatic interactions within the regulatory domain might bias the conformational ensemble such that the calmodulin binding region is readily accessible. Given the prevalence of charged residues in disordered protein chains, our findings are likely relevant to many protein-protein interactions.     

The Effects of Phosphomimetic Lid Mutation on the Thermostability of the N-terminal Domain of MDM2

The multidomain E3 ubiquitin ligase MDM2 catalyzes p53 ubiquitination by a “dual-site” docking mechanism whereby MDM2 binding to at least two distinct peptide motifs on p53 promotes ubiquitination. One protein-protein interaction occurs between the N-terminal hydrophobic pocket of MDM2 and the transactivation motif of p53, and the second interaction occurs between the acidic domain of MDM2 and a motif in the DNA-binding domain of p53. A flexible N-terminal pseudo-substrate or “lid” adjacent to the N-terminal hydrophobic pocket of MDM2 has a phosphorylation site, and there are distinct models proposed on how the phosphorylated lid could affect MDM2 function. Biochemical studies have predicted that phosphomimetic mutation will stabilize the lid on the surface of MDM2 and will “open” the hydrophobic pocket and stabilize the MDM2-p53 complex, while NMR studies proposed that phosphomimetic mutation “closes” the lid over the MDM2 pocket and inhibits MDM2-p53 complex formation. To resolve these discrepancies, we utilized a quantitative fluorescence-based dye binding assay to measure the thermal unfolding of wild-type (wt), ΔLid, and S17D N-terminal domains of MDM2 as a function of increasing ligand concentration. Our data reveal that S17D lid mutation increases, rather than decreases, the thermostability of the N-terminal domain of MDM2 in the absence or in the presence of ligand. ΔLid mutation, by contrast, increases MDM2 thermoinstability. This is consistent with biochemical data, using full-length MDM2, showing that the S17D mutation stabilizes the MDM2-p53 complex and increases the specific activity of the E3 ubiquitin ligase function of MDM2. These data indicate that phosphomimetic lid mutation results in an “opening,” rather than a “closing,” of the pocket of MDM2 and highlight the ability of small intrinsically disordered or unstructured peptide motifs to regulate the specific activity of a protein.     

Substrate Binding Specifically Modulates Domain Arrangements in Adenylate Kinase

The enzyme adenylate kinase (ADK) features two substrate binding domains that undergo large-scale motions during catalysis. In the apo state, the enzyme preferentially adopts a globally open state with accessible binding sites. Binding of two substrate molecules (AMP + ATP or ADP + ADP) results in a closed domain conformation, allowing efficient phosphoryl-transfer catalysis. We employed molecular dynamics simulations to systematically investigate how the individual domain motions are modulated by the binding of substrates. Two-dimensional free-energy landscapes were calculated along the opening of the two flexible lid domains for apo and holo ADK as well as for all single natural substrates bound to one of the two binding sites of ADK. The simulations reveal a strong dependence of the conformational ensembles on type and binding position of the bound substrates and a nonsymmetric behavior of the lid domains. Altogether, the ensembles suggest that, upon initial substrate binding to the corresponding lid site, the opposing lid is maintained open and accessible for subsequent substrate binding. In contrast, ATP binding to the AMP-lid induces global domain closing, preventing further substrate binding to the ATP-lid site. This might constitute a mechanism by which the enzyme avoids the formation of a stable but enzymatically unproductive state.     

Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain

Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.     

Binding of p53-derived ligands to MDM2 induces a variety of long range conformational changes

We have used NMR to study the effects of peptide binding on the N-terminal p53-binding domain of human MDM2 (residues 25-109). There were changes in HSQC-chemical shifts throughout the domain on binding four different p53-derived peptide ligands that were significantly large to be indicative of global conformational changes. Large changes in chemical shift were observed in two main regions: the peptide-binding cleft that directly binds the p53 ligands; and the hinge regions connecting the beta-sheet and alpha-helical structures that form the binding cleft. These conformational changes reflect the adaptation of the cleft on binding peptide ligands that differ in length and amino acid composition. Different ligands may induce different conformational transitions in MDM2 that could be responsible for its function. The dynamic nature of MDM2 might be important in the design of anti-cancer drugs that are targeted to its p53-binding site.     

Conformational changes of the p53-binding cleft of MDM2 revealed by molecular dynamics simulations

Two 35-ns molecular dynamics simulations of both ligated [mouse double minute protein 2 (MDM2(p53))] and unligated (MDM2(apo)) structures of human MDM2 bound to the N-terminal domain of the tumor suppressor p53 have been performed. Analysis of the dynamics revealed that the most flexible region of MDM2 was the p53-binding cleft. When MDM2 was bound to p53, a wider and more stable topology of the cleft was obtained, while unligated MDM2 showed a narrower and highly flexible cleft. It was also found that the dynamics involved in the opening/closing motions were due to the movement of different domains of the protein, which is in agreement with recent experimental data. Considering our results, a mechanism in which p53 might be recognized and attached to MDM2 is proposed, and some implications on future directions for in silico anticancer drug design efforts are discussed. In summary, the observations made here would be very useful not only for better understanding of the biological implications of the MDM2 dynamics, but also for future efforts in anticancer drug design and discovery.     

Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved.     

Dual-site regulation of MDM2 E3-ubiquitin ligase activity

The control of p53 ubiquitination by MDM2 provides a model system to define how an E3-ligase functions on a conformationally flexible substrate. The mechanism of MDM2-mediated ubiquitination of p53 has been analyzed by deconstructing, in vitro, the MDM2-dependent ubiquitination reaction. Surprisingly, ligands binding to the hydrophobic cleft of MDM2 do not inhibit its E3-ligase function. However, peptides from within the DNA binding domain of p53 that bind the acid domain of MDM2 inhibit ubiquitination of p53, localizing a motif that harbors a key ubiquitination signal. The binding of ligands to the N-terminal hydrophobic cleft of MDM2 reactivates, in vitro and in vivo, MDM2-catalyzed ubiquitination of p53F19A, a mutant p53 normally refractory to MDM2-catalyzed ubiquitination. We propose a model in which the interaction between the p53-BOX-I domain and the N terminus of MDM2 promotes conformational changes in MDM2 that stabilize acid-domain interactions with a ubiquitination signal in the DNA binding domain of the p53 tetramer.     

Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.     

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Searching the conformational complexity and binding properties of HDAC6 through docking and molecular dynamic simulations

Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.     

Computational analysis of spiro-oxindole inhibitors of the MDM2-p53 interaction: insights and selection of novel inhibitors

Since MDM2 is an inhibitor of the p53 tumor suppressor, disrupting the MDM2-p53 interaction is a promising approach for cancer therapy. Here, we used molecular dynamics simulations followed by free energy decomposition analysis to study conformational changes in MDM2 induced by three known spiro-oxindole inhibitors. Analysis of individual energy terms suggests that van der Waals and electrostatic interactions explain much of the binding affinities of these inhibitors. Binding free energies calculated for the three inhibitors using the molecular mechanics-generalized Born surface area model were consistent with experimental data, suggesting the validity of this approach. Based on this structure-function analysis, several novel spiro-oxindole derivatives were selected and evaluated for their ability to block the MDM2-p53 interaction in vitro. These results suggest that combining in silico and experimental techniques can provide insights into the structure-function relationships of MDM2 inhibitors and guide the rational design of anticancer drugs targeting the MDM2-p53 interaction.     

Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release

The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five alpha-helices (alphaA-alphaE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known. By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK-peptide complexes. Here we report that (i) the alphaD helix is essential for long-lived DnaK-peptide complexes. For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A-D) with k(off) equal to 7.6 and 8.9 s(-1), respectively, whereas when the D-helix is deleted, creating DnaK578(A-C), k(off) jumps to 207 s(-1). (ii) The presence of the alphaB helix impacts the rate of the ATP-induced high-to-low affinity conformational change. For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s(-1), whereas, after adding back the B-helix (residues 518-554), ATP induces this conformational change in DnaK554(A-B) with a rate constant of 2.5 s(-1). Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A-B) is bound in the substrate-binding site. (iii) The deletion analysis also revealed that residues 596-638, which comprise the alphaE helix and the flexible subdomain, affect ATP binding. Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain.