Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding

Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well‐ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence‐monitored stopped‐flow kinetic measurements we show that the secondary structure content in helix 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.     

Interrogation of MDM2 phosphorylation in p53 activation using native chemical ligation: the functional role of Ser17 phosphorylation in MDM2 reexamined

The E3 ubiquitin ligase MDM2 functions as a crucial negative regulator of the p53 tumor suppressor protein by antagonizing p53 transactivation activity and targeting p53 for degradation. Cellular stress activates p53 by alleviating MDM2-mediated functional inhibition, even though the molecular mechanisms of stress-induced p53 activation still remain poorly understood. Two opposing models have been proposed to describe the functional and structural role in p53 activation of Ser17 phosphorylation in the N-terminal "lid" (residues 1-24) of MDM2. Using the native chemical ligation technique, we synthesized the p53-binding domain (1-109)MDM2 and its Ser17-phosphorylated analogue (1-109)MDM2 pS17 as well as (1-109)MDM2 S17D and (25-109)MDM2, and comparatively characterized their interactions with a panel of p53-derived peptide ligands using surface plasmon resonance, fluorescence polarization, and NMR and CD spectroscopic techniques. We found that the lid is partially structured in apo-MDM2 and occludes p53 peptide binding in a ligand size-dependent manner. Binding of (1-109)MDM2 by the (15-29)p53 peptide fully displaces the lid and renders it completely disordered in the peptide-protein complex. Importantly, neither Ser17 phosphorylation nor the phospho-mimetic mutation S17D has any functional impact on p53 peptide binding to MDM2. Although Ser17 phosphorylation or its mutation to Asp contributes marginally to the stability of the lid conformation in apo-MDM2, neither modification stabilizes apo-MDM2 globally or the displaced lid locally. Our findings demonstrate that Ser17 phosphorylation is functionally neutral with respect to p53 binding, suggesting that MDM2 phosphorylation at a single site is unlikely to play a dominant role in stress-induced p53 activation.     

光谱法研究金属离子与肿瘤抑制蛋白p53的DNA结合结构域的相互作用

利用荧光滴定法研究了9种金属离子与序列特异性的DNA结合结构域(p53DBD)的结合反应,其结合能力依次为Fe3+＞Zn2+＞Cu2+＞Ca2+＞Mg2+＞Ba2+＞Mn2+＞Ni2+＞Co2+.圆二色谱研究结果表明,Ba2+, Ca2+, Co2+, Mn2+及Ni2+并未引起蛋白二级结构变化; Zn2+, Mg2+及Fe3+诱导蛋白结构细微调整;而Cu2+结合导致蛋白螺旋结构大量丢失.ANS结合研究结果表明, Mg2+与Zn2+相似,诱导p53DBD蛋白表面疏水性增强,而Fe3+引起p53DBD蛋白表面疏水性降低.因此,Mg2+和Fe3+可能是影响或调节p53活性的潜在因子之一.     

Long-range modulation of chain motions within the intrinsically disordered transactivation domain of tumor suppressor p53

The tumor suppressor p53 is a hub protein with a multitude of binding partners, many of which target its intrinsically disordered N-terminal domain, p53-TAD. Partners, such as the N-terminal domain of MDM2, induce formation of local structure and leave the remainder of the domain apparently disordered. We investigated segmental chain motions in p53-TAD using fluorescence quenching of an extrinsic label by tryptophan in combination with fluorescence correlation spectroscopy (PET-FCS). We studied the loop closure kinetics of four consecutive segments within p53-TAD and their response to protein binding and phosphorylation. The kinetics was multiexponential, showing that the conformational ensemble of the domain deviates from random coil, in agreement with previous findings from NMR spectroscopy. Phosphorylations or binding of MDM2 changed the pattern of intrachain kinetics. Unexpectedly, we found that upon binding and phosphorylation chain motions were altered not only within the targeted segments but also in remote regions. Long-range interactions can be induced in an intrinsically disordered domain by partner proteins that induce apparently only local structure or by post-translational modification.     

The native ensemble and folding of a protein molten-globule: functional consequence of downhill folding

The continually emerging functional significance of intrinsic disorder and conformational flexibility in proteins has challenged the long-standing dogma of a well-defined structure contributing to a specific function. Molten-globular states, a class of proteins with significant secondary-structure but a fluid hydrophobic core, is one such example. They have however been difficult to characterize due to the complexity of experimental data and lack of computational avenues. Here, we dissect the folding mechanism of the α-helical molten-globular protein NCBD from three fundamentally different approaches: statistical-mechanical variable barrier model, C(α)-based Gō-model and explicit water all-atom molecular dynamics (MD) simulations. We find that NCBD displays the characteristics of a one-state globally downhill folder but is significantly destabilized. Using simulation techniques, we generate a highly constrained but a heterogeneous native ensemble of the molten-globule for the first time that is consistent with experimental data including small angle X-ray scattering (SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR). The resulting native ensemble populates conformations reported in other bound-forms providing direct evidence to the mechanism of conformational selection for binding multiple partners in this domain. Importantly, our simulations reveal a connection between downhill folding and large conformational flexibility in this domain that has been evolutionarily selected and functionally exploited resulting in large binding promiscuity. Finally, the multimodel approach we employ here serves as a powerful methodology to study mechanisms and suggests that the thermodynamic features of molten-globules fall within the array of folding mechanisms available to small single-domain proteins.     

Predicting the coordination geometry for Mg2+ in the p53 DNA-binding domain: insights from computational studies

Zn(2+) in the tumor-suppressor protein p53 DNA-binding domain (DBD) is essential for its structural stability and DNA-binding specificity. Mg(2+) has also been recently reported to bind to the p53DBD and influence its DNA-binding activity. In this contribution, the binding geometry of Mg(2+) in the p53DBD and the mechanism of how Mg(2+) affects its DNA-binding activity were investigated using density functional theory (DFT) calculations and molecular dynamics (MD) simulations. Various possible coordination geometries of Mg(2+) binding to histidines (His), cysteines (Cys), and water molecules were studied at the B3LYP/6-311+g** level of theory. The protonation state of Cys and the environment were taken into account to explore the factors governing the coordination geometry. The free energy of the reaction to form the Mg(2+) complexes was estimated, suggesting that the favorable binding mode changes from a four- to six-coordinated geometry as the number of the protonated Cys increases. Furthermore, MD simulations were employed to explore the binding modes of Mg(2+) in the active site of the p53DBD. The simulation results of the Mg(2+) system and the native Zn(2+) system show that the binding affinity of Mg(2+)to the p53DBD is weaker than that of Zn(2+), in agreement with the DFT calculation results and experiments. In addition, the two metal ions are found to make a significant contribution to maintain a favorable orientation for Arg248 to interact with putative DNA, which is critically important to the sequence-specific DNA-binding activity of the p53DBD. However, the effect of Mg(2+) is less marked. Additionally, analysis of the natural bond orbital (NBO) charge transfer reveals that Mg(2+) has a higher net positive charge than Zn(2+), leading to a stronger electrostatic attractive interaction between Mg(2+) and putative DNA. This may partly explain the higher sequence-independent DNA-binding affinity of p53DBD-Mg(2+) compared to p53DBD-Zn(2+) observed in experiment.     

A graph theoretical approach to the effect of mutation on the flexibility of the DNA binding domain of p53 protein

Tumor suppressor protein p53 becomes inactive due to mutation on its DNA binding core domain leading to misbehavior of this protein and preventing its interaction with DNA. In the present study, changes of the protein conformation by five hot spot mutations of T-p53C were assessed preventing the mutants wild-type (WT) behavior. While studies of this nature were undertaken both experimentally and theoretically, the focus is fundamentally on the effects of the mutation on the dynamics of the protein. Hence, the basic concept underlying this study is the change in flexibility or rigidity of the protein. It was found that stable variant T-p53C (PDB-ID: 1uol) that is structurally and functionally very close to wild-type p53 is the most rigid structure and each single carcinogenic mutation on it makes the structure more flexible. We hypothesize that these changes of the molecule’s flexibility disrupt the network of hydrogen bonds associated with the interaction of WT not only at interaction but in the internal structures of the mutants as well, which prevents them from interacting in the WT fashion loosing the anti-cancer properties of WT.     

The Mechanism of p53 Rescue by SUSP4

p53 is an important tumor‐suppressor protein deactivation of which by mdm2 results in cancers. A SUMO‐specific protease 4 (SUSP4) was shown to rescue p53 from mdm2‐mediated deactivation, but the mechanism is unknown. The discovery by NMR spectroscopy of a “p53 rescue motif” in SUSP4 that disrupts p53‐mdm2 binding is presented. This 29‐residue motif is pre‐populated with two transient helices connected by a hydrophobic linker. The helix at the C‐terminus binds to the well‐known p53‐binding pocket in mdm2 whereas the N‐terminal helix serves as an affinity enhancer. The hydrophobic linker binds to a previously unidentified hydrophobic crevice in mdm2. Overall, SUSP4 appears to use two synergizing modules, the p53 rescue motif described here and a globular‐structured SUMO‐binding catalytic domain, to stabilize p53. A p53 rescue motif peptide exhibits an anti‐tumor activity in cancer cell lines expressing wild‐type p53. A pre‐structures motif in the intrinsically disordered proteins is thus important for target recognition.     

Prospective virtual screening for novel p53–MDM2 inhibitors using ultrafast shape recognition

The p53 protein, known as the guardian of genome, is mutated or deleted in approximately 50 % of human tumors. In the rest of the cancers, p53 is expressed in its wild-type form, but its function is inhibited by direct binding with the murine double minute 2 (MDM2) protein. Therefore, inhibition of the p53–MDM2 interaction, leading to the activation of tumor suppressor p53 protein presents a fundamentally novel therapeutic strategy against several types of cancers. The present study utilized ultrafast shape recognition (USR), a virtual screening technique based on ligand–receptor 3D shape complementarity, to screen DrugBank database for novel p53–MDM2 inhibitors. Specifically, using 3D shape of one of the most potent crystal ligands of MDM2, MI-63, as the query molecule, six compounds were identified as potential p53–MDM2 inhibitors. These six USR hits were then subjected to molecular modeling investigations through flexible receptor docking followed by comparative binding energy analysis. These studies suggested a potential role of the USR-selected molecules as p53–MDM2 inhibitors. This was further supported by experimental tests showing that the treatment of human colon tumor cells with the top USR hit, telmisartan, led to a dose-dependent cell growth inhibition in a p53-dependent manner. It is noteworthy that telmisartan has a long history of safe human use as an approved anti-hypertension drug and thus may present an immediate clinical potential as a cancer therapeutic. Furthermore, it could also serve as a structurally-novel lead molecule for the development of more potent, small-molecule p53–MDM2 inhibitors against variety of cancers. Importantly, the present study demonstrates that the adopted USR-based virtual screening protocol is a useful tool for hit identification in the domain of small molecule p53–MDM2 inhibitors.     

Y220C突变体影响p53C蛋白质构象转换的分子动力学模拟

p53是迄今发现突变频率最高的一种肿瘤抑制蛋白质,突变会导致p53抑癌功能丧失并诱导癌症的发生。绝大多数的突变发生在p53的核心DNA结合区域（p53C）,其中Y220C是研究较多的一种突变体。虽然已有研究表明该突变能够降低p53C的结构稳定性,但其影响p53C构象转换的分子机制尚不清晰。本文利用分子动力学（MD）模拟方法研究了p53C突变体Y220C（p53C-Y220C）的结构变化,发现Y220C突变主要影响Y220C cluster区域（包括残基138-164和215-238）,且Y220C突变减少了Y220C cluster的β-折叠含量。进一步分析发现,Y220C突变不仅直接破坏突变氨基酸与周围氨基酸Leu145和Thr155之间的氢键,而且降低了Y220C cluster区域的折叠片S3和S8之间的氢键数量,使Y220C突变所形成的亲水性空腔变大,加速了水分子进入该蛋白质内部,并最终导致了p53C-Y220C变性。MD模拟结果揭示了Y220C突变影响p53C结构转换的分子机制,该研究对p53C-Y220C突变体高效稳定剂的筛选和设计具有重要意义。     

A Cyclized Helix‐Loop‐Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein–Protein Interactions by Epitope and Arginine Grafting

The design of inhibitors of intracellular protein–protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix‐loop‐helix (cHLH) peptide as a scaffold for generating cell‐permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell‐permeability. To inhibit p53–HDM2 interactions, the p53 epitope was grafted onto the C‐terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53‐R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53‐R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well‐structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs.     

Electrochemical sensing of tumor suppressor protein p53–deoxyribonucleic acid complex stability at an electrified interface

Electrochemical biosensors have the unique ability to convert biological events directly into electrical signals suitable for parallel analysis. Here we utilize specific properties of constant current chronopotentiometric stripping (CPS) in the analysis of protein and DNA–protein complex nanolayers. Rapid potential changes at high negative current intensities (I_str) in CPS are utilized in the analysis of DNA–protein interactions at thiol-modified mercury electrodes. P53 core domain (p53CD) sequence-specific binding to DNA results in a striking decrease in the electrocatalytic signal of free p53. This decrease is related to changes in the accessibility of the electroactive amino acid residues in the p53CD–DNA complex. By adjusting I_str and temperature, weaker non-specific binding can be eliminated or distinguished from the sequence-specific binding. The method also reflects differences in the stabilities of different sequence-specific complexes, including those containing spacers between half-sites of the DNA consensus sequence. The high resolving power of this method is based on the disintegration of the p53CD–DNA complex by the electric field effects at a negatively charged surface and fine adjustment of the millisecond time intervals for which the complex is exposed to these effects. Picomole amounts of p53 proteins and DNA were used for the analysis at full electrode coverage but we show that even 10–20-fold smaller amounts can be analyzed. Our method cannot however take advantage of very low detection limits of the protein CPS detection because low I^_str intensities are deleterious to the p53CD–DNA complex stability at the electrode surface. These data highlight the utility of developing biosensors offering novel approaches for studying real-time macromolecular protein dynamics.