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Wide-line 1H NMR intensity and differential scanning calorimetry measurements were carried out on the intrinsically disordered 73-residue full transactivation domain (TAD) of the p53 tumor suppressor protein and two peptides: one a wild type p53 TAD peptide with a helix pre-structuring property, and a mutant peptide with a disabled helix-forming propensity. Measurements were carried out in order to characterize their water and ion binding characteristics. By quantifying the number of hydrate water molecules, we provide a microscopic description for the interactions of water with a wild-type p53 TAD and two p53 TAD peptides. The results provide direct evidence that intrinsically disordered proteins (IDPs) and a less structured peptide not only have a higher hydration capacity than globular proteins, but are also able to bind a larger amount of charged solute ions. [BMB Reports 2016; 49(9): 497-501].

Many intrinsically unstructured/unfolded proteins (IUPs) contain transient local secondary structures even though they are "unstructured" in a tertiary sense. These local secondary structures are named "pre-structured motifs (PreSMos)" and in fact are the specificity determinants for IUP-target binding, i.e., the active sites in IUPs. Using high-resolution NMR we have delineated a PreSMo active site in the intrinsically unfolded mid-domain (residues 201-300) of SUMO-specific protease 4 (SUSP4). This 29-residue motif which we termed a p53 rescue motif can protect p53 from mdm2 quenching by binding to the p53-helix binding pocket in mdm2(3-109). Our work demonstrates that the PreSMo approach is quite effective in providing a structural rationale for interactions of p53-mdm2- SUSP4 and opens a novel avenue for designing mdm2- inhibiting anticancer compounds. [BMB Reports 2017; 50(10): 485-486].

Protein engineering method to study the mutation effects on muscle acylphosphatase (AcP) has been actively applied to describe kinetics and thermodynamics associated with AcP aggregation as well as folding processes. Despite the extensive mutation experiments, the molecular origin and the structural motifs for aggregation and folding kinetics as well as thermodynamics of AcP have not been rationalized at the atomic resolution. To this end, we have investigated the mutation effects on the structures and thermodynamics for the aggregation and folding of AcP by using the combination of fully atomistic, explicit-water molecular dynamics simulations, and three-dimensional reference interaction site model theory. The results indicate that the A30G mutant with the fastest experimental aggregation rate displays considerably decreased α1-helical contents as well as disrupted hydrophobic core compared to the wild-type AcP. Increased solvation free energy as well as hydrophobicity upon A30G mutation is achieved due to the dehydration of hydrophilic side chains in the disrupted α1-helix region of A30G. In contrast, the Y91Q mutant with the slowest aggregation rate shows a non-native H-bonding network spanning the mutation site to hydrophobic core and α1-helix region, which rigidifies the native state protein conformation with the enhanced α1-helicity. Furthermore, Y91Q exhibits decreased solvation free energy and hydrophobicity compared to wild type due to more exposed and solvated hydrophilic side chains in the α1-region. On the other hand, the experimentally observed slower folding rates in both mutants are accompanied by decreased helicity in α2-helix upon mutation. We here provide the atomic-level structures and thermodynamic quantities of AcP mutants and rationalize the structural origin for the changes that occur upon introduction of those mutations along the AcP aggregation and folding processes.

The eIF4E-binding protein 1 (4EBP1) has long been known to be completely unstructured without any secondary structures, which contributed significantly to the proposal of the induced fit mechanism for target binding of intrinsically disordered proteins. We show here that 4EBP1 is not completely unstructured, but contains a pre-structured helix.

Background IDPs function without relying on three-dimensional structures. No clear rationale for such a behavior is available yet. PreSMos are transient secondary structures observed in the target-free IDPs and serve as the target-binding “active” motifs in IDPs. Prolines are frequently found in the flanking regions of PreSMos. Contribution of prolines to the conformational stability of the helical PreSMos in IDPs is investigated. Methods MD simulations are performed for several IDP segments containing a helical PreSMo and the flanking prolines. To measure the influence of flanking-prolines on the structural content of a helical PreSMo calculations were done for wild type as well as for mutant segments with Pro → Asp, His, Lys, or Ala. The change in the helicity due to removal of a proline was measured both for the PreSMo region and for the flanking regions. Results The α-helical content in ~ 70% of the helical PreSMos at the early stage of simulation decreases due to replacement of an N-terminal flanking proline by other residues whereas the helix content in nearly all PreSMos increases when the same replacements occur at the C-terminal flanking region. The helix destabilizing/terminating role of the C-terminal flanking prolines is more pronounced than the helix promoting effect of the N-terminal flanking prolines. General significance This work represents a novel example demonstrating that a proline is encoded in an IDP with a defined purpose. The helical PreSMos presage their target-bound conformations. As they most likely mediate IDP-target binding via conformational selection their helical content can be an important feature for IDP function.

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of homo- or heteropentameric ligand-gated ion channels. Understanding the physiological role of each nAChR subtype and the key residues responsible for normal and pathological states is important. alpha-Conotoxin neuropeptides are highly selective probes capable of discriminating different subtypes of nAChRs. In this study, we performed homology modeling to generate the neuronal alpha 3, beta 2 and beta 4 subunits using the x-ray structure of the alpha 1 subunit as a template. The structures of the extracellular domains containing ligand binding sites in the alpha 3 beta 2 and alpha 3 beta 4 nAChR subtypes were constructed using MD simulations and ligand docking processes in their free and ligand-bound states using alpha-conotoxin GIC, which exhibited the highest alpha 3 beta 2 vs. alpha 3 beta 4 discrimination ratio. The results provide a reasonable structural basis for such a discriminatory ability, supporting the idea that the present strategy can be used for future investigations on nAChR-ligand complexes. [BMB Reports 2012; 45(5): 275-280]

The antiparallel and parallel beta sheets are two of the most abundant secondary structures found in proteins. Although various spectroscopic methods have been used to distinguish these two different structures, the linear spectroscopic measurements could not provide incisive information for distinguishing an antiparallel beta sheet from a parallel beta sheet. After carrying out quantum-chemistry calculations and model simulations, we show that the polarization-controlled two-dimensional (2D) IR photon echo spectroscopy can be of critical use in distinguishing these two different beta sheets. Particularly, the ratio between the diagonal peak and the cross peak is found to be strongly dependent on the quasi-2D array of the amide I local-mode transition dipole vectors. The relative intensities of the cross peaks in the 2D difference spectrum of an antiparallel beta sheet are significantly larger than those of the diagonal peaks, whereas the cross-peak amplitudes in the 2D difference spectrum of a parallel beta sheet are much weaker than the main diagonal-peak amplitudes. A detailed discussion on the origin of the diagonal- and cross-peak intensity distributions of both the antiparallel and parallel beta sheets is presented by examining vibrational exciton delocalization, relative angles between two different normal-mode transition dipoles, and natures of the cross peaks in the 2D difference spectrum.

Background A candidate oncogene GIG47, previously known as a neudesin with a neurotrophic activity, was identified by applying the differential expression analysis method. Methods As a first step to understand the molecular role of GIG47, we analyzed the expression profile of GIG47 in multiple human cancers including the breast cancer and characterized its function related to human carcinogenesis. Based on this oncogenic role of GIG47, we then embarked on determining the high-resolution structure of GIG47. We have applied multidimensional heteronuclear NMR methods to GIG47. Results GIG47 was over-expressed in primary breast tumors as well as other human tumors including carcinomas of the uterine cervix, malignant lymphoma, colon, lung, skin, and leukemia. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 cells. The ectopic expression of GIG47 in MCF7 cells promoted the invasiveness in the presence of 50% serum. In addition, it also resulted in the increased tumorigenicity in in vivo tumor formation assay. The tumorigenesis mechanism involving GIG47 might be mediated by the activation of MAPK and PI3K pathways. These results indicate that GIG47 plays a role in the breast tumorigenesis, thus representing a novel target for the treatment of breast cancer. To facilitate the development of GIG47-targeted therapeutics, we determined the structural configuration of GIG47. The high-resolution structure of GIG47 was obtained by combination of NMR and homology modeling. The overall structure of GIG47 has four α-helices and 6 β-strands, arranged in a β1-α1-β2-β3-α2-β4-α3-α4-β5-β6 topology. There is a potential heme/steroid binding pocket formed between two helices α2 and α3. Conclusion The determined three-dimensional structure of GIG47 may facilitate the development of potential anti-cancer agents.