The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function.     

Elucidating binding mechanisms and dynamics of intrinsically disordered protein complexes using NMR spectroscopy

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Characterization of intrinsically disordered regions in proteins informed by human genetic diversity

All proteomes contain both proteins and polypeptide segments that don’t form a defined three-dimensional structure yet are biologically active—called intrinsically disordered proteins and regions (IDPs and IDRs). Most of these IDPs/IDRs lack useful functional annotation limiting our understanding of their importance for organism fitness. Here we characterized IDRs using protein sequence annotations of functional sites and regions available in the UniProt knowledgebase (“UniProt features”: active site, ligand-binding pocket, regions mediating protein-protein interactions, etc.). By measuring the statistical enrichment of twenty-five UniProt features in 981 IDRs of 561 human proteins, we identified eight features that are commonly located in IDRs. We then collected the genetic variant data from the general population and patient-based databases and evaluated the prevalence of population and pathogenic variations in IDPs/IDRs. We observed that some IDRs tolerate 2 to 12-times more single amino acid-substituting missense mutations than synonymous changes in the general population. However, we also found that 37% of all germline pathogenic mutations are located in disordered regions of 96 proteins. Based on the observed-to-expected frequency of mutations, we categorized 34 IDRs in 20 proteins (DDX3X, KIT, RB1, etc.) as intolerant to mutation. Finally, using statistical analysis and a machine learning approach, we demonstrate that mutation-intolerant IDRs carry a distinct signature of functional features. Our study presents a novel approach to assign functional importance to IDRs by leveraging the wealth of available genetic data, which will aid in a deeper understating of the role of IDRs in biological processes and disease mechanisms.     

Recent progress in NMR spectroscopy: toward the study of intrinsically disordered proteins of increasing size and complexity

Thanks to recent fast progress, NMR is now in a strategic position to provide unique atomic resolution information on a variety of different biological macromolecules in different conditions (solution, solid state, in-cell). We would like here to present recent developments that enable to focus on intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of proteins of increasing size and complexity. They have attracted the attention of the scientific community challenging well accepted ideas and concepts and demanding an expansion of the structure function paradigm.     

Apoflavodoxin (un)folding followed at the residue level by NMR

The denaturant-induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H-15N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H-15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.     

Characterization of human saposins by NMR spectroscopy

Saposins are lipid-binding and membrane-perturbing glycoproteins of the mammalian lysosomes involved in sphingolipid and membrane digestion. Although the four human saposins (Saps), A-D, are sequence-related, they are responsible for the activation of different steps in the cascade of lysosomal glycosphingolipid degradation. Saposin activity is maximal under acidic conditions, and the pH dependence of lipid and membrane binding has been assigned to conformational variability. We have employed solution NMR spectroscopy to all four (15)N-labeled human saposins at both neutral and acidic pH. Using backbone NOEs and residual dipolar couplings, the "saposin fold" comprising five alpha-helices was confirmed for Sap-A, Sap-C, and Sap-D. Structural variations within these proteins are in the order of variations between the known structures of Sap-C and NK-lysin. In contrast, Sap-B yielded spectra of very poor quality, presumably due to conformational heterogeneity and molecular association. Sap-D exists in a slow dynamic equilibrium of two conformational states with yet unknown function. At pH 4.0, where all saposins are highly unstable, Sap-C undergoes a transition to a specific dimeric state, which is likely to resemble the structure recently found in both Sap-C in a detergent environment and crystals of Sap-B.     

High resolution NMR spectroscopy of nanocrystalline proteins at ultra-high magnetic field

Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly-¹³C,¹⁵N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7, 17.6 and 21.1 Tesla (¹H frequencies of 500, 750, and 900 MHz). For two protein systems—GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline protein—line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function of field, and for DsbA, resolution in the C–C region increases by 42%, according to the number of peaks that can be uniquely picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely observed in the highest field spectra for at least two isoleucine Cδ1 sites in DsbA. These results further illustrate the benefits of high-field MAS SSNMR spectroscopy for protein structural studies.     

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high‐speed super‐resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy‐ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs.     

Basic folded and low-populated locally disordered conformers of SUMO-2 characterized by NMR spectroscopy at varying pressures

SUMO proteins, a group of post-translational ubiquitin-like modifiers, have target enzymes (E1 and E2) like other ubiquitin-like modifiers, e.g., ubiquitin and NEDD8, but their physiological roles are quite different. In an effort to determine the characteristic molecular design of ubiquitin-like modifiers, we have investigated the structure of human SUMO-2 in solution not only in its basic folded state but also in its higher-energy state by utilizing standard and variable-pressure NMR spectroscopy, respectively. We have determined average coordinates of the basic folded conformer at ambient pressure, which gives a backbone structure almost identical with those of ubiquitin and NEDD8. We have further investigated conformational fluctuations in a wide conformational space using variable-pressure NMR spectroscopy in the range of 30-3 kbar, by which we find a low-populated ( approximately 2.5%) alternative conformer preferentially disordered in the enzyme-binding segment. The alternative conformer is structurally very close to but markedly different in equilibrium population from those for ubiquitin and NEDD8. These results support our notion that post-translational ubiquitin-like modifiers are evolutionarily designed for function both structurally and thermodynamically in their low-populated, high-energy conformers rather than in their basic folded conformers.     

NMR spectroscopy of 113Cd(II)-substituted gene 32 protein

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein. Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains. NMR spectroscopy of the 113Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single 113Cd resonance, delta 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein. In vitro mutagenesis of Cys166 to Ser166 creates a mutant g32P that still contains 1 Zn(II)/molecule. This mutant protein when substituted with 113Cd(II) shows a 113Cd signal with a delta and a line width the same as those observed for the wild-type protein. Thus, the S-ligands to the metal ion appear to be contributed by Cys77, Cys87, and Cys90. Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the 113Cd nucleus in g32P, since a dipolar modulation from ligand protons is observed at 44.4 MHz but not at 110.9 MHz. Complexation of core 113Cd g32P with d(pA)6 or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions, respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of cell cycle entry mediated by the intrinsically disordered protein p27(Kip1)

p27(Kip1) (p27), a prototypical intrinsically disordered protein (IDP), regulates eukaryotic cell division through interactions with cyclin-dependent kinase (Cdk)/cyclin complexes. The activity, stability, and subcellular localization of p27 are regulated by phosphorylation. We illustrate how p27 integrates regulatory signals from several non-receptor tyrosine kinases (NRTKs) to activate Cdk4 and initiate cell cycle entry. Unmodified p27 potently inhibits Cdk/cyclin complexes, including Cdk4/cyclin D (IC(50), 1 nM). Some NRTKs (e.g., Abl) phosphorylate p27 on Tyr 88, which facilitates a second modification on Tyr 74 by another NRTK (e.g., Src). Importantly, this second modification causes partial reactivation of Cdk4 within ternary complexes containing doubly Tyr phosphorylated p27. Partial activation of Cdk4 initiates entry into the cell division cycle. Therefore, p27's disordered features enable NRTKs to sequentially promote a phosphorylation cascade that controls cell fate. Beyond cell cycle control, these results illustrate general concepts regarding why IDPs are well-suited for roles in signaling and regulation in biological systems.     

Fibrillation mechanism of a model intrinsically disordered protein revealed by 2D correlation deep UV resonance Raman spectroscopy

Understanding of numerous biological functions of intrinsically disordered proteins (IDPs) is of significant interest to modern life science research. A large variety of serious debilitating diseases are associated with the malfunction of IDPs including neurodegenerative disorders and systemic amyloidosis. Here we report on the molecular mechanism of amyloid fibrillation of a model IDP (YE8) using 2D correlation deep UV resonance Raman spectroscopy. YE8 is a genetically engineered polypeptide, which is completely unordered at neutral pH yet exhibits all properties of a fibrillogenic protein at low pH. The very first step of the fibrillation process involves structural rearrangements of YE8 at the global structure level without the detectable appearance of secondary structural elements. The formation of β-sheet species follows the global structural changes and proceeds via the simultaneous formation of turns and β-strands. The kinetic mechanism revealed is an important new contribution to understanding of the general fibrillation mechanism proposed for IDP.     

Characterization of caged compounds binding to proteins by NMR spectroscopy

Photolysable caged ligands are used to investigate protein function and activity. Here, we investigate the binding properties of caged nucleotides and their photo released products to well established but evolutionary and structurally unrelated nucleotide-binding proteins, rabbit muscle creatine kinase (RMCK) and human annexin A6 (hAnxA6), using saturation transfer difference NMR spectroscopy. We detect the binding of the caged nucleotides and discuss the general implications on interpreting data collected with photolysable caged ligands using different techniques. Strategies to avoid non-specific binding of caged compound to certain proteins are also suggested.     

The 5-O-beta-D-galactofuranosyl-containing peptidophosphogalactomannan of Penicillium charlesii. Characterization of the mannan by 13C NMR spectroscopy

Penicillium charlesii secretes a galactofuranosyl and phosphodiester-containing peptidophosphogalactomannan (pPGM). A linear mannan was prepared from pPGM by treatment with 48% aqueous HF which selectively cleaves galactofuranosyl and phosphodiesters; treatment with alkaline borohydride releases the mannan from the polypeptide. Mannan from P. charlesii cultured in D-[1,2-13C2]glucose contained mannopyranosyl residues which were enriched in 13C at both C-1 and C-2 and, to a lesser extent, at C-5 and C-6. The mannan was examined with a combination of 13C NMR INADEQUATE pulse sequence and selective 13C saturation to assign the resonance frequency of anomeric carbons directly coupled to specific C-2 signals. Three species of mannosyl residues, each substituted with a glycosidic linkage at C-2, and a fourth species substituted at C-6 and not substituted at C-2 were observed. Mannan obtained from P. charlesii cultured in D-[6-13C]glucose contained mannopyranosyl residues which were enriched in 13C primarily in C-6. The mannan was examined by DEPT 13C NMR to determine the number of species which were substituted at C-6. Mannan, treated as described above, contained a 1----6-linked mannopyranosyl species. pPGM contains minor quantities of at least four other substances attached to hydroxymethyl groups of the hexosyl residues.     

Structure-function relationship in Escherichia coli translational initiation factors. Characterization of IF-3 by high resolution 1H NMR spectroscopy

Translational initiation factor-3 (IF-3) was characterized by 1H NMR spectroscopy as a function of pH and temperature and following chemical modifications. Spin-lattice relaxation times for individual resonances and bands were also measured. Several resonances were assigned to different amino acid residues by different criteria. Among these are the CH3-N of the N-terminal methionine which appears free, mobile, and very sensitive to the modification of several physicochemical parameters as well as the 3,5 and 2,6 protons of the three tyrosines (two of which play a role in the function of IF-3) which were found to be located in different magnetic environments. Two of these residues appear to be close to each other and in the vicinity of a slow reacting arginine within the tertiary structure of the factor. The properties and the titration behavior of the imidazole proton resonances suggest that the single His residue is partially buried in the protein structure. Characteristic of the IF-3 spectrum also is the presence of an abundant subset of Arg delta-CH2, Lys epsilon-CH2, and CH3 protons displaying clear cut upfield perturbations. These are probably due to the coming together of two or more apolar "fronts" which possibly arise from distant parts of the molecule and result in the close proximity between aromatic rings and aliphatic side chains. The IF-3 spectrum also includes several distinct methyl resonances significantly shifted upfield by aromatic ring currents. Overall, the characteristics of the spectrum, its relative insensitivity to temperature and ionic strength, and the existence of extensive cross-relaxation phenomena indicate that IF-3 has a highly folded tertiary structure with abundant hydrophobic regions. In spite of some heterogeneity in the distribution of the side chain environments, no indication was found for the existence of distinct domains or, at least, of extensive regions with higher mobility.     

Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy

Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.