Multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes

Proteins are constantly involved in the multitude of various interactions creating sophisticated networks which define and control all (or almost all) the biological processes taking place in any living organism. Intrinsically disordered proteins or regions play a number of crucial roles in mediating protein interactions. The lack of fixed structure protruding to the high level of intrinsic dynamics and almost unrestricted flexibility at various structure levels, being the major characteristics of intrinsically disordered proteins, provides them with unprecedented advantages over the ordered proteins. The binding modes attainable by disordered proteins are highly diverse, creating a multitude of unusual complexes. Although the majority of studied to date intrinsic disorder-based complexes are ordered or static entities originating due to the global or local disorder-to-order transitions, a new development is the discovery of dynamic complexes in which intrinsically disordered proteins continue to sample an ensemble of rapidly interconverting conformations mostly devoid of structure even in their bound state. The goal of this critical review is to illustrate binding plasticity of intrinsically disordered proteins by representing a portrait gallery of the disorder-based complexes (119 references).     

Hydrogen exchange mass spectrometry for studying protein structure and dynamics

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in solution. This approach relies on the fact that exposure of a protein to D(2)O induces rapid amide H → D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a number of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissociation methods provide a solution to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful reference for practitioners, who require an overview of some recent trends in HDX/MS.     

Structure and dynamics of an unfolded protein examined by molecular dynamics simulation

The accurate characterization of the structure and dynamics of proteins in disordered states is a difficult problem at the frontier of structural biology whose solution promises to further our understanding of protein folding and intrinsically disordered proteins. Molecular dynamics (MD) simulations have added considerably to our understanding of folded proteins, but the accuracy with which the force fields used in such simulations can describe disordered proteins is unclear. In this work, using a modern force field, we performed a 200 μs unrestrained MD simulation of the acid-unfolded state of an experimentally well-characterized protein, ACBP, to explore the extent to which state-of-the-art simulation can describe the structural and dynamical features of a disordered protein. By comparing the simulation results with the results of NMR experiments, we demonstrate that the simulation successfully captures important aspects of both the local and global structure. Our simulation was ~2 orders of magnitude longer than those in previous studies of unfolded proteins, a length sufficient to observe repeated formation and breaking of helical structure, which we found to occur on a multimicrosecond time scale. We observed one structural feature that formed but did not break during the simulation, highlighting the difficulty in sampling disordered states. Overall, however, our simulation results are in reasonable agreement with the experimental data, demonstrating that MD simulations can already be useful in describing disordered proteins. Finally, our direct calculation of certain NMR observables from the simulation provides new insight into the general relationship between structural features of disordered proteins and experimental NMR relaxation properties.     

Mapping the potential energy landscape of intrinsically disordered proteins at amino Acid resolution

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.     

Disordered proteins and network disorder in network descriptions of protein structure, dynamics and function: hypotheses and a comprehensive review

During the last decade, network approaches became a powerful tool to describe protein structure and dynamics. Here we review the links between disordered proteins and the associated networks, and describe the consequences of local, mesoscopic and global network disorder on changes in protein structure and dynamics. We introduce a new classification of protein networks into 'cumulus-type', i.e., those similar to puffy (white) clouds, and 'stratus-type', i.e., those similar to flat, dense (dark) low-lying clouds, and relate these network types to protein disorder dynamics and to differences in energy transmission processes. In the first class, there is limited overlap between the modules, which implies higher rigidity of the individual units; there the conformational changes can be described by an 'energy transfer' mechanism. In the second class, the topology presents a compact structure with significant overlap between the modules; there the conformational changes can be described by 'multi-trajectories'; that is, multiple highly populated pathways. We further propose that disordered protein regions evolved to help other protein segments reach 'rarely visited' but functionally-related states. We also show the role of disorder in 'spatial games' of amino acids; highlight the effects of intrinsically disordered proteins (IDPs) on cellular networks and list some possible studies linking protein disorder and protein structure networks.     

Taking snapshots of photoexcited molecules in disordered media by using pulsed synchrotron X-rays

Photoexcited molecules are quintessential reactants in photochemistry. Structures of these photoexcited molecules in disordered media in which a majority of photochemical reactions take place remained elusive for decades owing to a lack of suitable X-ray sources, despite their importance in understanding fundamental aspects in photochemistry. As new pulsed X-ray sources become available, short-lived excited-state molecular structures in disordered media can now be captured by using laser-pulse pump, X-ray pulse-probe techniques of third-generation synchrotron sources with time resolutions of 30-100 ps, as demonstrated by examples in this review. These studies provide unprecedented information on structural origins of molecular properties in the excited states. By using other ultrafast X-ray facilities that will be completed in the near future, time-resolution for the excited-state structure measurements should reach the femtosecond time scales, which will make "molecular movies" of bond breaking or formation, and vibrational relaxation, a reality.     

Predicting protein disorder and induced folding: from theoretical principles to practical applications

In the last years there has been an increasing amount of experimental evidence pointing out that a large number of proteins are either fully or partially disordered (unstructured). Intrinsically disordered proteins are ubiquitary proteins that fulfil essential biological functions while lacking highly populated and uniform secondary and tertiary structure under physiological conditions. Despite the large abundance of disorder, disordered regions are still poorly detected. Recognition of disordered regions in a protein is instrumental for reducing spurious sequence similarity between disordered regions and ordered ones, and for delineating boundaries of protein domains amenable to crystallization. As presently none of the available automated methods for prediction of protein disorder can be taken as fully reliable on its own, we present a brief overview of the methods currently employed highlighting their philosophy. We show a few practical examples of how they can be combined to avoid pitfalls and to achieve more reliable predictions. We also describe the currently available methods for the identification of regions involved in induced folding and provide a few practical examples in which the accuracy of predictions was experimentally confirmed.     

天然无序蛋白质:序列-结构-功能的新关系

天然无序蛋白质是一类新发现的蛋白质,它们在天然条件下没有确定的三维结构,却具有正常的生物学功能,广泛参与信号传递、DNA转录、细胞分裂和蛋白质聚集等重要的生理与病理过程.无序蛋白质的发现是对传统的蛋白质"序列-结构-功能"范式的挑战.在这篇综述里,我们首先回顾了蛋白质的传统范式以及无序蛋白质的发现过程,然后介绍无序蛋白质在结构、序列、功能等方面的特征与相互作用,并以分子识别过程为例,进一步阐述目前国际上对无序蛋白质所具有优势的一些认识与观点.我们还分析了无序蛋白质研究在生命科学和医学等领域的应用前景,并介绍了国内在无序蛋白质领域的研究现状.     

Structure-Function Insights of Jaburetox and Soyuretox: Novel Intrinsically Disordered Polypeptides Derived from Plant Ureases

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.     

Chemical shift correlations in disordered solids

We show how two-dimensional chemical shift conditional probability distributions can be extracted from experimental NMR correlation spectra of disordered solids. We show that transverse dephasing times are of central importance in determining the resolution (and sensitivity) of these probability distributions. These conditional probability distributions provide a new source of structural information characteristic of disordered solids, which is much more sensitive to structure than the individual-atom chemical shift distributions. The structural information contained in these distributions is clearly a potentially extremely rich source for understanding disorder. This is illustrated with examples of a phosphorus-containing organic compound and with a sample of disordered cellulose where the different structural allomorphs present are identified from the distributions.     

Towards proteomic approaches for the identification of structural disorder

Intrinsically unstructured/disordered proteins (IUPs) and protein domains lack a well-defined three-dimensional structure under physiological conditions. Structural disorder imparts advantages in many non-conventional functions, which poses a significant challenge to our understanding of the structure-function relationship of proteins. The general appreciation of this fact, however, is hampered by the large gap in our knowledge on IUPs, as we have biophysical data on less than 500 of them, whereas bioinformatic predictions suggest at least several thousand such proteins in the human proteome alone. Thus, proteomic-scale identification and characterization of IUPs will need to be implemented to fill this gap and advance our knowledge in this important field. In this review we give an insight into the various rationales of proteomic efforts of identifying IUPs, and survey the handful of attempts that combined enrichment of extracts for IUPs by heat- or acid treatment with a subsequent two-dimensional electrophoresis/mass spectrometry identification. Advantages and drawbacks of the various approaches are outlined in anticipation of future inventions in the field that will hopefully elevate IUP research to the truly proteomic level.     

On the molecular mechanism of ion specific Hofmeister series

Hofmeister series ranks the ability of salt ions in influencing a variety of properties and processes in aqueous solutions.In this review,we reexamine how these ions and some other small molecules affect water structure and thermodynamic properties,such as surface tension and protein backbone solvation.We illustrate the difficulties in interpreting the thermodynamic information based on structural and dynamic arguments.As an alternative,we show that the solvation properties of ions and proteins/small molecules can be used to explain the salt effects on the thermodynamic properties of the solutions.Our analysis shows that the often neglected cation-anion cooperativity plays a very important role in these effects.We also argue that the change of hydrogen donor/acceptor equilibrium by added cosolutes/cosolvents can be used to explain their effects on protein secondary structure denaturation/protection:those increase hydrogen donor concentrations such as urea and salts with strongly solvated cations/weakly hydrated anions tend to dissolve protein backbone acting as secondary structure denaturants,whereas those lack of hydrogen donors but rich in acceptors have the opposite effect.     

Regional stability changes in oxidized and reduced cytochrome c located by hydrogen exchange and mass spectrometry

Amide hydrogen exchange rates are highly sensitive to protein structure and may, therefore, be used to detect and characterize structural changes in proteins. Specific regions within folded proteins undergoing structural change can often be identified if localized amide hydrogen exchange rates are determined by nuclear magnetic resonance (NMR). The ability to measure localized amide hydrogen exchange rates by proteolytic fragmentation followed by mass spectrometric analysis opens the possibility to also identify localized structural changes in proteins by mass spectrometry. If successful, this approach offers considerable advantage over NMR in speed, sensitivity, protein solubility, and ability to study large proteins. This possibility has been investigated by determining the amide hydrogen exchange rates in oxidized and reduced cytochrome c by protein fragmentation/mass spectrometry. The fundamental difference in these forms of cytochrome c is the oxidation state of the iron, which other studies have shown results in only minor structural changes in the protein. In the present study, the largest differences in hydrogen exchange rates were found for peptide amide hydrogens located distant from the Nand C-termini, indicating that the structure in these regions is most affected by the oxidation state of the iron. These results are consistent with previous studies of oxidized and reduced cytochrome c, suggesting that hydrogen exchange and mass spectrometry may be generally useful for locating subtle changes in protein structure.     

Proteus: a random forest classifier to predict disorder-to-order transitioning binding regions in intrinsically disordered proteins

The focus of the computational structural biology community has taken a dramatic shift over the past one-and-a-half decades from the classical protein structure prediction problem to the possible understanding of intrinsically disordered proteins (IDP) or proteins containing regions of disorder (IDPR). The current interest lies in the unraveling of a disorder-to-order transitioning code embedded in the amino acid sequences of IDPs/IDPRs. Disordered proteins are characterized by an enormous amount of structural plasticity which makes them promiscuous in binding to different partners, multi-functional in cellular activity and atypical in folding energy landscapes resembling partially folded molten globules. Also, their involvement in several deadly human diseases (e.g. cancer, cardiovascular and neurodegenerative diseases) makes them attractive drug targets, and important for a biochemical understanding of the disease(s). The study of the structural ensemble of IDPs is rather difficult, in particular for transient interactions. When bound to a structured partner, an IDPR adapts an ordered conformation in the complex. The residues that undergo this disorder-to-order transition are called protean residues, generally found in short contiguous stretches and the first step in understanding the modus operandi of an IDP/IDPR would be to predict these residues. There are a few available methods which predict these protean segments from their amino acid sequences; however, their performance reported in the literature leaves clear room for improvement. With this background, the current study presents ‘Proteus’, a random forest classifier that predicts the likelihood of a residue undergoing a disorder-to-order transition upon binding to a potential partner protein. The prediction is based on features that can be calculated using the amino acid sequence alone. Proteus compares favorably with existing methods predicting twice as many true positives as the second best method (55 vs. 27%) with a much higher precision on an independent data set. The current study also sheds some light on a possible ‘disorder-to-order’ transitioning consensus, untangled, yet embedded in the amino acid sequence of IDPs. Some guidelines have also been suggested for proceeding with a real-life structural modeling involving an IDPR using Proteus.     

Calculation of residual dipolar couplings from disordered state ensembles using local alignment

Residual dipolar couplings (RDCs) have been observed in disordered states of several proteins. While their nonuniform values were initially surprising, it has been shown that reasonable approximation of experimental RDCs can be obtained using simple statistical coil models and assuming global alignment of each structure, provided that many thousands of conformers are averaged. Here we show that, by using short local alignment tensors, we can achieve good agreement between experimental and simulated RDCs with far fewer structures than required when using global alignment. This makes the possibility of using RDCs as direct restraints in structural calculations of disordered proteins much more feasible. In addition, it provides insight into the nature of RDCs in disordered states, suggesting that they are primarily reporting on local structure.     

Mass spectrometry-based structural proteomics

Structural proteomics is the application of protein chemistry and modern mass spectrometric techniques to problems such as the characterization of protein structures and assemblies and the detailed determination of protein-protein interactions. The techniques used in structural proteomics include crosslinking, photoaffinity labeling, limited proteolysis, chemical protein modification and hydrogen/deuterium exchange, all followed by mass spectrometric analysis. None of these methods alone can provide complete structural information, but a "combination" of these complementary approaches can be used to provide enough information for answering important biological questions. Structural proteomics can help to determine, for example, the detailed structure of the interfaces between proteins that may be important drug targets and the interactions between proteins and ligands. In this review, we have tried to provide a brief overview of structural proteomics methodologies, illustrated with examples from our laboratory and from the literature.     

Unraveling the nature of the segmentation clock: Intrinsic disorder of clock proteins and their interaction map

Vertebrate segmentation has been proved to be under a strict temporal control governed by a biological clock, known as the segmentation clock. The present experimental evidence suggests that the segmentation clock initiates and maintains its periodic cycle by the periodic activation or inhibition of the Notch signaling pathway as well as the periodic autoregulation of the cyclic genes themselves. In this paper, we investigate the structural and evolutionary properties of the Notch pathway proteins involved in the mice segmentation clock and computationally identify the interaction map within the Notch signaling pathway. The results of our analysis strongly indicate that most of the pathway proteins are intrinsically disordered and that the mechanism of their interaction likely involves helical molecular recognition elements, short loosely structured segments within disordered regions which are directly involved in protein-protein interactions. Predicted interactions are in agreement with gene knock-out studies available in the literature.     

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 lipoproteins of cyanobacterial photosystem II

Photosystem II (PSII) complexes from cyanobacteria and plants perform water splitting and plastoquinone reduction and yet have a different complement of lumenal extrinsic proteins. Whereas PSII from all organisms has the PsbO extrinsic protein, crystal structures of PSII from cyanobacteria have PsbV and PsbU while green algae and higher plants instead contain the extrinsic PsbP and PsbQ subunits. Proteomic studies in Synechocystis sp. PCC 6803 identified three further extrinsic proteins in the thylakoid lumen that are associated with cyanobacterial PSII and these are predicted to attach to the thylakoid membrane via a lipidated N-terminus. These proteins are cyanobacterial homologues to the PsbP and PsbQ subunits as well as to Psb27, an additional extrinsic protein associated with "inactive" photosystems that lack the other extrinsic polypeptides. The PsbQ homologue is not present in Prochlorococcus species but otherwise these proteins have been identified in most cyanobacteria although our phylogenetic analyses identified some strains that lack an apparent motif for lipidation in one or other of these subunits. Over the past decade the physiological function of these additional lipoproteins has been investigated in several cyanobacterial strains and recently the structures for each have been solved. This review will evaluate the physiological and structural results obtained for these lipid-attached extrinsic proteins and in silico protein docking of these proteins to PSII centers will be presented.