Primary contact sites in intrinsically unstructured proteins: the case of calpastatin and microtubule-associated protein 2

Intrinsically unstructured proteins (IUPs) exist in a disordered conformational state, often considered to be equivalent with the random-coil structure. We challenge this simplifying view by limited proteolysis, circular dichroism (CD) spectroscopy, and solid-state (1)H NMR, to show short- and long-range structural organization in two IUPs, the first inhibitory domain of calpastatin (CSD1) and microtubule-associated protein 2c (MAP2c). Proteases of either narrow (trypsin, chymotrypsin, and plasmin) or broad (subtilisin and proteinase K) substrate specificity, applied at very low concentrations, preferentially cleaved both proteins in regions, i.e., subdomains A, B, and C in CSD1 and the proline-rich region (PRR) in MAP2c, that are destined to form contacts with their targets. For CSD1, nonadditivity of the CD spectra of its two halves and suboptimal hydration of the full-length protein measured by solid-state NMR demonstrate that long-range tertiary interactions provide the structural background of this structural feature. In MAP2c, such tertiary interactions are absent, which points to the importance of local structural constraints. In fact, urea and temperature dependence of the CD spectrum of its PRR reveals the presence of the extended and rather stiff polyproline II helix conformation that keeps the interaction site exposed. These data suggest that functionally significant residual structure exists in both of these IUPs. This structure, manifest as either transient local and/or global organization, ensures the spatial exposure of short contact segments on the surface. Pertinent data from other IUPs suggest that the presence of such recognition motifs may be a general feature of disordered proteins. To emphasize the possible importance of this structural trait, we propose that these motifs be called primary contact sites in IUPs.     

The highly structured encapsidation signal of MuLV RNA is involved in the nuclear export of its unspliced RNA

p27Kip1 (p27) influences cell division by regulating nuclear cyclin-dependent kinases. Before binding, p27 is at least partially disordered and folds upon binding its Cdk/cyclin targets. 30-40% of human proteins, including p27, are predicted to contain disordered segments, and have been termed intrinsically unstructured proteins (IUPs). Unfortunately, the inherent dynamics of IUPs hamper detailed analysis of their structure/function relationships. Here, we describe the use of molecular dynamics (MD) computations and solution NMR spectroscopy to reveal that several segments of the p27 kinase inhibitory domain (p27-KID), in addition to the previously characterized helical segment, exist as highly populated, intrinsically folded structural units (IFSUs). Several IFSUs resemble structural features of bound p27-KID, while another exhibits alternative conformations. Interestingly, the highly conserved, specificity determining segment of p27 is shown to be highly disordered. Elucidation of IFSUs within p27-KID allows consideration of their influences on the thermodynamics and kinetics of Cdk/cyclin binding. The degree to which IFSUs are populated within p27-KID is surprising and suggests that other putative IUPs contain IFSUs that may be studied using similar techniques.     

Preformed structural elements feature in partner recognition by intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) are devoid of extensive structural order but often display signs of local and limited residual structure. To explain their effective functioning, we reasoned that such residual structure can be crucial in their interactions with their structured partner(s) in a way that preformed structural elements presage their final conformational state. To check this assumption, a database of 24 IUPs with known 3D structures in the bound state has been assembled and the distribution of secondary structure elements and backbone torsion angles have been analysed. The high proportion of residues in coil conformation and with phi, psi angles in the disallowed regions of the Ramachandran map compared to the reference set of globular proteins shows that IUPs are not fully ordered even in their bound form. To probe the effect of partner proteins on IUP folding, inherent conformational preferences of IUP sequences have been assessed by secondary structure predictions using the GOR, ALB and PROF algorithms. The accuracy of predicting secondary structure elements of IUPs is similar to that of their partner proteins and is significantly higher than the corresponding values for random sequences. We propose that strong conformational preferences mark regions in IUPs (mostly helices), which correspond to their final structural state, while regions with weak conformational preferences represent flexible linkers between them. In our interpretation, preformed elements could serve as initial contact points, the binding of which facilitates the reeling of the flexible regions onto the template. This finding implies that IUPs draw a functional advantage from preformed structural elements, as they enable their facile, kinetically and energetically less demanding, interaction with their physiological partner.     

Molecular principles of the interactions of disordered proteins

Thorough knowledge of the molecular principles of protein-protein recognition is essential to our understanding of protein function at the cellular level. Whereas interactions of ordered proteins have been analyzed in great detail, complexes of intrinsically unstructured/disordered proteins (IUPs) have hardly been addressed so far. Here, we have collected a database of 39 complexes of experimentally verified IUPs, and compared their interfaces with those of 72 complexes of ordered, globular proteins. The characteristic differences found between the two types of complexes suggest that IUPs represent a distinct molecular implementation of the principles of protein-protein recognition. The interfaces do not differ in size, but those of IUPs cover a much larger part of the surface of the protein than for their ordered counterparts. Moreover, IUP interfaces are significantly more hydrophobic relative to their overall amino acid composition, but also in absolute terms. They rely more on hydrophobic-hydrophobic than on polar-polar interactions. Their amino acids in the interface realize more intermolecular contacts, which suggests a better fit with the partner due to induced folding upon binding that results in a better adaptation to the partner. The two modes of interaction also differ in that IUPs usually use only a single continuous segment for partner binding, whereas the binding sites of ordered proteins are more segmented. Probably, all these features contribute to the increased evolutionary conservation of IUP interface residues. These noted molecular differences are also manifested in the interaction energies of IUPs. Our approximation of these by low-resolution force-fields shows that IUPs gain much more stabilization energy from intermolecular contacts, than from folding, i.e. they use their binding energy for folding. Overall, our findings provide a structural rationale to the prior suggestions that many IUPs are specialized for functions realized by protein-protein interactions.     

The interplay between structure and function in intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) are common in various proteomes and occupy a unique structural and functional niche in which function is directly linked to structural disorder. The evidence that these proteins exist without a well-defined folded structure in vitro is compelling, and justifies considering them a separate class within the protein world. In this paper, novel advances in the rapidly advancing field of IUPs are reviewed, with the major attention directed to the evidence of their unfolded character in vivo, the interplay of their residual structure and their various functional modes and the functional benefits their malleable structural state provides. Via all these details, it is demonstrated that in only a couple of years after its conception, the idea of protein disorder has already come of age and transformed our basic concepts of protein structure and function.     

The origin and impact of bound water around intrinsically disordered proteins

Proteins and water couple dynamically over a wide range of time scales. Motivated by their central role in protein function, protein-water dynamics and thermodynamics have been extensively studied for structured proteins, where correspondence to structural features has been made. However, properties controlling intrinsically disordered protein (IDP)-water dynamics are not yet known. We report results of megahertz-to-terahertz dielectric spectroscopy and molecular dynamics simulations of a group of IDPs with varying charge content along with structured proteins of similar size. Hydration water around IDPs is found to exhibit more heterogeneous rotational and translational dynamics compared with water around structured proteins of similar size, yielding on average more restricted dynamics around individual residues of IDPs, charged or neutral, compared with structured proteins. The on-average slower water dynamics is found to arise from excess tightly bound water in the first hydration layer, which is related to greater exposure to charged groups. The more tightly bound water to IDPs correlates with the smaller hydration shell found experimentally, and affects entropy associated with protein-water interactions, the contribution of which we estimate based on the dielectric measurements and simulations. Water-IDP dynamic coupling at terahertz frequencies is characterized by the dielectric measurements and simulations.     

Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits

It is now widely recognized that intrinsically unstructured (or disordered) proteins (IUPs or IDPs) are found in organisms from all kingdoms of life. In eukaryotes, IUPs are highly abundant and perform a wide range of biological functions, including regulation and signaling. Despite an increased level of interest in understanding the structural biology of IUPs and IDPs, questions regarding the mechanisms through which disordered proteins perform their biological function(s) remain. In other words, what are the relationships between disorder and function for IUPs? There are several excellent reviews that discuss the structural properties of IUPs and IDPs since 2005 [Receveur-Brechot, V., et al. (2006) Proteins 62, 24-45; Mittag, T., and Forman-Kay, J. D. (2007) Curr. Opin. Struct. Biol. 17, 3-14; Dyson, H. J., and Wright, P. E. (2005) Nat. Rev. Mol. Cell Biol. 6, 197-208]. Here, we briefly review general concepts pertaining to IUPs and then discuss our structural, biophysical, and biochemical studies of two IUPs, p21 and p27, which regulate the mammalian cell division cycle by inhibiting cyclin-dependent kinases (Cdks). Some segments of these two proteins are partially folded in isolation, and they fold further upon binding their biological targets. Interestingly, some portions of p27 remain flexible after binding to and inhibiting the Cdk2-cyclin A complex. This residual flexibility allows otherwise buried tyrosine residues within p27 to be phosphorylated by non-receptor tyrosine kinases (NRTKs). Tyrosine phosphorylation relieves kinase inhibition, triggering Cdk2-mediated phosphorylation of a threonine residue within the flexible C-terminus of p27. This, in turn, marks p27 for ubiquitination and proteasomal degradation, unleashing full Cdk2 activity which drives cell cycle progression. p27, thus, constitutes a conduit for transmission of proliferative signals via post-translational modifications. The term "conduit" is used here to connote a means of transmission of molecular signals which, in the case of p27, correspond to tyrosine and threonine phosphorylation, ubiquitination, and, ultimately, proteolytic degradation. Transmission of these multiple signals is enabled by the inherent flexibility of p27 which persists even after tight binding to the Cdk2-cyclin A complex. Importantly, activation of the p27 signaling conduit by oncogenic NRTKs contributes to tumorigenesis in some human cancers, including chronic myelogenous leukemia (CML) [Grimmler, M., et al. (2007) Cell 128, 269-280] and breast cancer [Chu, I., et al. (2007) Cell 128, 281-294]. Other IUPs may participate in conceptually similar molecular signaling conduits, and dysregulation of these putative conduits may contribute to other human diseases. Detailed study of these IUPs, both alone and within functional complexes, is required to test these hypotheses and to more fully understand the relationships between protein disorder and biological function.     

Structural characterization of an intrinsically unfolded mini-HBX protein from hepatitis B virus

The hepatitis B virus x protein (HBX) is expressed in HBVinfected liver cells and can interact with a wide range of cellular proteins. In order to understand such promiscuous behavior of HBX we expressed a truncated mini-HBX protein (named Tr-HBX) (residues 18-142) with 5 Cys → Ser mutations and characterized its structural features using circular dichroism (CD) spectropolarimetry, NMR spectroscopy as well as bioinformatics tools for predicting disorder in intrinsically unstructured proteins (IUPs). The secondary structural content of Tr-HBX from CD data suggests that Tr-HBX is only partially folded. The protein disorder prediction by IUPred reveals that the unstructured region encompasses its N-terminal ～30 residues of Tr-HBX. A two-dimensional (1)H-(15)N HSQC NMR spectrum exhibits fewer number of resonances than expected, suggesting that Tr-HBX is a hybrid type IUP where its folded C-terminal half coexists with a disordered N-terminal region. Many IUPs are known to be capable of having promiscuous interactions with a multitude of target proteins. Therefore the intrinsically disordered nature of Tr-HBX revealed in this study provides a partial structural basis for the promiscuous structure-function behavior of HBX.     

Structural disorder throws new light on moonlighting

A basic mechanism by which individual proteins can increase network complexity is moonlighting, whereby a given protein fulfils more than one function. Traditionally, this phenomenon is attributed to separate binding surfaces of globular, folded proteins but we suggest that intrinsically unstructured proteins (IUPs) might provide radically different mechanisms. Eleven IUPs have been identified that suggest that the structural malleability of IUPs gives rise to unprecedented cases of moonlighting by eliciting opposing (inhibiting and activating) action on different partners or even the same partner molecule. Unlike classical cases, these proteins use the same region or overlapping interaction surfaces to exert distinct effects and employ non-conventional mechanisms to switch function, enabled by their capacity to adopt different conformations upon binding. Owing to the apparent functional benefits, we expect to see many more examples of this parsimonious use of protein material in complex metabolic networks.     

Myosin-induced volume increase of the hyper-mobile water surrounding actin filaments

Microwave dielectric spectroscopy can measure the rotational mobility of water molecules that hydrate proteins and the hydration-shell volume. Using this technique, we have recently shown that apart from typical hydrating water molecules with lowered mobility there are other water molecules around the actin filaments (F-actin) which have a much higher mobility than that of bulk water [Biophys. J. 85 (2003) 3154]. We report here that the volume of this water component (hyper-mobile water) markedly increases without significant change of the volume of the ordinary hydration shell when the myosin motor-domain (S1, myosin subfragment-1) binds to F-actin. No hyper-mobile component was found in the hydration shell of S1 itself. The present results strongly suggest that the solvent space around S1 bound to F-actin is diffusionally asymmetric, which supports our model of force generation by actomyosin proposed previously [op. cit.].     

Choice of Force Field for Proteins Containing Structured and Intrinsically Disordered Regions

Biomolecular force fields optimized for globular proteins fail to properly reproduce properties of intrinsically disordered proteins. In particular, parameters of the water model need to be modified to improve applicability of the force fields to both ordered and disordered proteins. Here, we compared performance of force fields recommended for intrinsically disordered proteins in molecular dynamics simulations of three proteins differing in the content of ordered and disordered regions (two proteins consisting of a well-structured domain and of a disordered region with and without a transient helical motif and one disordered protein containing a region of increased helical propensity). The obtained molecular dynamics trajectories were used to predict measurable parameters, including radii of gyration of the proteins and chemical shifts, residual dipolar couplings, paramagnetic relaxation enhancement, and NMR relaxation data of their individual residues. The predicted quantities were compared with experimental data obtained within this study or published previously. The results showed that the NMR relaxation parameters, rarely used for benchmarking, are particularly sensitive to the choice of force-field parameters, especially those defining the water model. Interestingly, the TIP3P water model, leading to an artificial structural collapse, also resulted in unrealistic relaxation properties. The TIP4P-D water model, combined with three biomolecular force-field parameters for the protein part, significantly improved reliability of the simulations. Additional analysis revealed only one particular force field capable of retaining the transient helical motif observed in NMR experiments. The benchmarking protocol used in our study, being more sensitive to imperfections than the commonly used tests, is well suited to evaluate the performance of newly developed force fields.     

Predicting disordered regions in proteins based on decision trees of reduced amino acid composition.

Intrinsically unstructured proteins (IUPs) are proteins lacking a fixed three dimensional structure or containing long disordered regions. IUPs play an important role in biology and disease. Identifying disordered regions in protein sequences can provide useful information on protein structure and function, and can assist high-throughput protein structure determination. In this paper we present a system for predicting disordered regions in proteins based on decision trees and reduced amino acid composition. Concise rules based on biochemical properties of amino acid side chains are generated for prediction. Coarser information extracted from the composition of amino acids can not only improve the prediction accuracy but also increase the learning efficiency. In cross-validation tests, with four groups of reduced amino acid composition, our system can achieve a recall of 80% at a 13% false positive rate for predicting disordered regions, and the overall accuracy can reach 83.4%. This prediction accuracy is comparable to most, and better than some, existing predictors. Advantages of our approach are high prediction accuracy for long disordered regions and efficiency for large-scale sequence analysis. Our software is freely available for academic use upon request.     

Sequential hydration of dry proteins: a direct difference IR investigation of sequence homologs lysozyme and alpha- lactalbumin

Direct difference ir spectra are presented as a function of hydration for lysozyme and α-lactalbumin, and detailed sequential hydration molecular events identified. Despite the strong sequence homology between the two proteins, and their expected conformational similarity, the hydration behaviour of the polar groups is different for the two proteins. Using a Hill-type analysis, we conclude that the acid groups ionize and hydrate rapidly and noncooperatively in both proteins, consistent with the known (lysozyme) and postulated (α-lactalbumin) surface chemistry. The polar group hydration shows a clear cooperativity, which is quantitatively different in the two proteins. Complementary work suggests this cooperativity relates to a hydration-induced “loosening up” of the lysozyme conformation at about 55 mol water/mol protein. α-Lactalbumin appears to “open up” more easily for hydration than does lysozyme, consistent with its lower stability against thermal and acid denaturation.     

Structure and dynamics of the N-terminal half of hepatitis C virus core protein: An intrinsically unstructured protein

Hepatitis C virus core protein plays an important role in the assembly and packaging of the viral genome. We have studied the structure of the N-terminal half of the core protein (C82) which was shown to be sufficient for the formation of nucleocapsid-like particle (NLP) in vitro and in yeast. Structural bioinformatics analysis of C82 suggests that it is mostly unstructured. Circular dichroism and structural NMR data indicate that C82 lacks secondary structure. Moreover, NMR relaxation data shows that C82 is highly disordered. These results indicate that the N-terminal half of the HCV core protein belongs to the growing family of intrinsically unstructured proteins (IUP). This explains the tendency of the hepatitis C virus core protein to interact with several host proteins, a well-documented characteristic of IUPs.     

Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets

All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.     

A novel two-dimensional electrophoresis technique for the identification of intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 m urea electrophoresis of heat-treated proteins where IUPs are expected to run into the diagonal, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. This behavior was born out by a collection of 10 known IUPs and four globular proteins. By running Escherichia coli and Saccharomyces cerevisiae extracts, several novel IUPs were also identified by mass spectrometric analysis of spots at or near the diagonal. By comparing this novel method to several other techniques, such as the PONDR(R) predictor, hydrophobicity-net charge plot, CD analysis, and gel filtration chromatography, it was shown to provide dependable global assessment of disorder even in dubious cases. Overall the reproducibility and ease of performance of this technique may promote the proteomic scale recognition and characterization of protein disorder.     

The dynamics of protein hydration water: a quantitative comparison of molecular dynamics simulations and neutron-scattering experiments

We present results from an extensive molecular dynamics simulation study of water hydrating the protein Ribonuclease A, at a series of temperatures in cluster, crystal, and powder environments. The dynamics of protein hydration water appear to be very similar in crystal and powder environments at moderate to high hydration levels. Thus, we contend that experiments performed on powder samples are appropriate for discussing hydration water dynamics in native protein environments. Our analysis reveals that simulations performed on cluster models consisting of proteins surrounded by a finite water shell with free boundaries are not appropriate for the study of the solvent dynamics. Detailed comparison to available x-ray diffraction and inelastic neutron-scattering data shows that current generation force fields are capable of accurately reproducing the structural and dynamical observables. On the time scale of tens of picoseconds, at room temperature and high hydration, significant water translational diffusion and rotational motion occur. At low hydration, the water molecules are translationally confined but display appreciable rotational motion. Below the protein dynamical transition temperature, both translational and rotational motions of the water molecules are essentially arrested. Taken together, these results suggest that water translational motion is necessary for the structural relaxation that permits anharmonic and diffusive motions in proteins. Furthermore, it appears that the exchange of protein-water hydrogen bonds by water rotational/librational motion is not sufficient to permit protein structural relaxation. Rather, the complete exchange of protein-bound water molecules by translational displacement seems to be required.     

Water in barnacle muscle. IV. Factors contributing to reduced self-diffusion.

The relative self-diffusion coefficients D/Do, of water in various solutions, in fresh barnacle muscle fibers, and in membrane-damaged fibers equilibrated with several media have been estimated from NMR relaxation rates in the presence of applied field gradients. A model has been developed to account for the contributions to the observed reduction in D/Do from small organic solutes, and from the hydration and obstruction effect of both soluble macromolecules and myofilament proteins. Intracellular ions do not affect D/Do, but all tested organic solutes do. Solute effects are additive. When artificially combined in the proportions found in barnacle muscle ultracentrifugate (measured D/Do = 0.77), organic acids, small nitrogenous solutes, and proteins give D/Do = 0.77. After correcting the D/Do measured in fibers for this value, we calculate the myofilament hydration, Hm, in fresh muscle to be 0.65 g H2O/g macromolecule. Only in membrane-damaged fibers, highly swollen by salt-rich media, was this significantly increased. Because our earlier NMR relaxation measurements indicate only 0.07 g H2O bound/g myofilament protein, we conclude that the "hydration" water measured by reduction of D/Do cannot be described by stationary layers of water molecules; instead, we propose that nonpolar groups on the proteins cause extensive, hydrophobically-induced interactions among a large fraction of solvent molecules, slowing their translational motion.     

Measuring enzyme hydration in nonpolar organic solvents using NMR

A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for alpha-chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. (c) 1995 John Wiley & Sons, Inc.