Jingwei Meng

Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners

BackgroundProteins are involved in many interactions with other proteins leading to networks that regulate and control a wide variety of physiological processes. Some of these proteins, called hub proteins or hubs, bind to many different protein partners. Protein intrinsic disorder, via diversity arising from structural plasticity or flexibility, provide a means for hubs to associate with many partners (Dunker AK, Cortese MS, Romero P, Iakoucheva LM, Uversky VN: Flexible Nets: The roles of intrinsic disorder in protein interaction networks. FEBS J 2005, 272:5129-5148).ResultsHere we present a detailed examination of two divergent examples: 1) p53, which uses different disordered regions to bind to different partners and which also has several individual disordered regions that each bind to multiple partners, and 2) 14-3-3, which is a structured protein that associates with many different intrinsically disordered partners. For both examples, three-dimensional structures of multiple complexes reveal that the flexibility and plasticity of intrinsically disordered protein regions as well as induced-fit changes in the structured regions are both important for binding diversity.ConclusionsThese data support the conjecture that hub proteins often utilize intrinsic disorder to bind to multiple partners and provide detailed information about induced fit in structured regions.

TOP-IDP-Scale: A New Amino Acid Scale Measuring Propensity for Intrinsic Disorder

Intrinsically disordered proteins carry out various biological functions while lacking ordered secondary and/or tertiary structure. In order to find general intrinsic properties of amino acid residues that are responsible for the absence of ordered structure in intrinsically disordered proteins we surveyed 517 amino acid scales. Each of these scales was taken as an independent attribute for the subsequent analysis. For a given attribute value X, which is averaged over a consecutive string of amino acids, and for a given data set having both ordered and disordered segments, the conditional probabilities P(s(o) | x) and P(s(d) | x) for order and disorder, respectively, can be determined for all possible values of X. Plots of the conditional probabilities P(s(o) | x) and P(s(o) | x) versus X give a pair of curves. The area between these two curves divided by the total area of the graph gives the area ratio value (ARV), which is proportional to the degree of separation of the two probability curves and, therefore, provides a measure of the given attributes power to discriminate between order and disorder. As ARV falls between zero and one, larger ARV corresponds to the better discrimination between order and disorder. Starting from the scale with the highest ARV, we applied a simulated annealing procedure to search for alternative scale values and have managed to increase the ARV by more than 10%. The ranking of the amino acids in this new TOP-IDP scale is as follows (from order promoting to disorder promoting): W, F, Y, I, M, L, V, N, C, T, A, G, R, D, H, Q, K, S, E, P. A web-based server has been created to apply the TOP-IDP scale to predict intrinsically disordered proteins (http://www.disprot.org/dev/disindex.php).

Exploring the binding diversity of intrinsically disordered proteins involved in one‐to‐many binding

Molecular recognition features (MoRFs) are intrinsically disordered protein regions that bind to partners via disorder‐to‐order transitions. In one‐to‐many binding, a single MoRF binds to two or more different partners individually. MoRF‐based one‐to‐many protein–protein interaction (PPI) examples were collected from the Protein Data Bank, yielding 23 MoRFs bound to 2–9 partners, with all pairs of same‐MoRF partners having less than 25% sequence identity. Of these, 8 MoRFs were bound to 2–9 partners having completely different folds, whereas 15 MoRFs were bound to 2–5 partners having the same folds but with low sequence identities. For both types of partner variation, backbone and side chain torsion angle rotations were used to bring about the conformational changes needed to enable close fits between a single MoRF and distinct partners. Alternative splicing events (ASEs) and posttranslational modifications (PTMs) were also found to contribute to distinct partner binding. Because ASEs and PTMs both commonly occur in disordered regions, and because both ASEs and PTMs are often tissue‐specific, these data suggest that MoRFs, ASEs, and PTMs may collaborate to alter PPI networks in different cell types. These data enlarge the set of carefully studied MoRFs that use inherent flexibility and that also use ASE‐based and/or PTM‐based surface modifications to enable the same disordered segment to selectively associate with two or more partners. The small number of residues involved in MoRFs and in their modifications by ASEs or PTMs may simplify the evolvability of signaling network diversity.

Subclassifying disordered proteins by the CH-CDF plot method.

Intrinsically disordered proteins (IDPs) are associated with a wide range of functions. We suggest that sequence-based subtypes, which we call flavors, may provide the basis for different biological functions. The problem is to find a method that separates IDPs into different flavor / function groups. Here we discuss one approach, the (Charge-Hydropathy) versus (Cumulative Distribution Function) plot or CH-CDF plot, which is based the combined use of the CH and CDF disorder predictors. These two predictors are based on significantly different inputs and methods. This CH-CDF plot partitions all proteins into 4 groups: structured, mixed, disordered, and rare. Studies of the Protein Data Bank (PDB) entries and homologous show different structural biases for each group classified by the CH-CDF plot. The mixed class has more order-promoting residues and more ordered regions than the disordered class. To test whether this partition accomplishes any functional separation, we performed gene ontology (GO) term analysis on each class. Some functions are indeed found to be related to subtypes of disorder: the disordered class is highly active in mitosis-related processes among others. Meanwhile, the mixed class is highly associated with signaling pathways, where having both ordered and disordered regions could possibly be important.

High-throughput characterization of intrinsic disorder in proteins from the Protein Structure Initiative.

The identification of intrinsically disordered proteins (IDPs) among the targets that fail to form satisfactory crystal structures in the Protein Structure Initiative represents a key to reducing the costs and time for determining three-dimensional structures of proteins. To help in this endeavor, several Protein Structure Initiative Centers were asked to send samples of both crystallizable proteins and proteins that failed to crystallize. The abundance of intrinsic disorder in these proteins was evaluated via computational analysis using predictors of natural disordered regions (PONDR®) and the potential cleavage sites and corresponding fragments were determined. Then, the target proteins were analyzed for intrinsic disorder by their resistance to limited proteolysis. The rates of tryptic digestion of sample target proteins were compared to those of lysozyme/myoglobin, apomyoglobin, and α-casein as standards of ordered, partially disordered and completely disordered proteins, respectively. At the next stage, the protein samples were subjected to both far-UV and near-UV circular dichroism (CD) analysis. For most of the samples, a good agreement between CD data, predictions of disorder and the rates of limited tryptic digestion was established. Further experimentation is being performed on a smaller subset of these samples in order to obtain more detailed information on the ordered/disordered nature of the proteins.

Differential expression of CaMKII isoforms and overall kinase activity in rat dorsal root ganglia after injury

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) decodes neuronal activity by translating cytoplasmic Ca(2+) signals into kinase activity that regulates neuronal functions including excitability, gene expression, and synaptic transmission. Four genes lead to developmental and differential expression of CaMKII isoforms (α, β, γ, δ). We determined mRNA levels of these isoforms in the dorsal root ganglia (DRG) of adult rats with and without nerve injury in order to determine if differential expression of CaMKII isoforms may contribute to functional differences that follow injury. DRG neurons express mRNA for all four isoforms, and the relative abundance of CaMKII isoforms was γ>α>β=δ, based on the CT values. Following ligation of the 5th lumbar (L5) spinal nerve (SNL), the β isoform did not change, but mRNA levels of both the γ and α isoforms were reduced in the directly injured L5 neurons, and the α isoform was reduced in L4 neurons, compared to their contemporary controls. In contrast, expression of the δ isoform mRNA increased in L5 neurons. CaMKII protein decreased following nerve injury in both L4 and L5 populations. Total CaMKII activity measured under saturating Ca(2+)/CaM conditions was decreased in both L4 and L5 populations, while autonomous CaMKII activity determined in the absence of Ca(2+) was selectively reduced in axotomized L5 neurons 21days after injury. Thus, loss of CaMKII signaling in sensory neurons after peripheral nerve injury may contribute to neuronal dysfunction and pain.

Intrinsically Disordered Proteins: An Update

Just over 10 years ago, in June, 1997, in the Proceedings of the IEEE International Conference on Neural Networks, we published our first predictor of intrinsically disordered protein. Since then, we have substantially improved our predictors, and more than 20 other laboratory groups have joined in efforts to improve the prediction of protein disorder. At the algorithmic level, prediction of protein intrinsic disorder is similar to the prediction of secondary structure, but, at the structural level, secondary structure and intrinsic disorder are entirely different. The secondary structure class called random coil or irregular differs from intrinsic disorder due to very different dynamic properties, with the secondary structure class being much less mobile than the region of disorder. At the biological level, unlike the prediction of secondary structure, the prediction of intrinsic disorder has been revolutionary. That is, for many years, experimentalists have provided evidence that some proteins lack fixed structure or are disordered (or unfolded) under physiological conditions. Experimentalists further are showing that, for some proteins, functions depended on the unstructured rather than structured state. However, these examples have been mostly ignored. To our knowledge, not one disordered protein or disorder-associated function is discussed in any biochemistry textbook, even though such examples began to be discovered more than 50 years ago. Disorder prediction has been important for showing that the few experimentally characterized examples represent a very large cohort that is found all across all three domains of life. We now know that many significant biological functions depend directly on, or are importantly associated with, the unfolded or partially folded state. In this paper, we will briefly review some of the key discoveries that have occurred in the last decade, and, furthermore, will make a few highly speculative projections.

Mechanisms Underlying Cooperativity in CaMKII Autophosphorylation and Substrate Phosphorylation

As a member of the calmodulin-activated kinases, Ca2+/calmodulin dependent protein kinase II (CaMKII) is a serine/threonine kinase coupled to calcium signaling. Unlike other multifunctional CaMK members, CaMKII has a unique dodecameric architecture potentially permitting cooperative forms of autoregulation and substrate phosphorylation. We observed that CaMKII phosphorylation of a peptide derived from the autoregulatory domain (AC-2) displays positive cooperativity (nH=1.8). Another T-site binding peptide substrate derived from S1303 phosphorylation site on NR2B also displayed positive cooperativity (nH=2.1). Surprisingly, a truncated form of CaMKII1-317 monomer also shows this cooperativity towards NR2B and AC-2 peptides (nH=1.6 vs 1.8, respectively). Syntide-2, a traditional substrate peptide lacking T-site interactions, does not exhibit cooperativity in substrate phosphorylation for either monomeric or multimeric CaMKII (nH=1.0; nH=1.1, respectively). These data suggest that the positive cooperativity seen with substrate phosphorylation is unique to T-site site binding substrates and may involve potential allosteric substrate interactions on the catalytic surface. Cooperativity within the holoenzyme occurring between subunits has been predicted based on the fact that Ca2+/CaM binding can induce Thr286 autophosphorylation, an intersubunit intraholenzyme reaction, whereby, neighboring subunits act as both kinase and substrate following coincident Ca2+CaM binding. Using Ca2+/CaM-independent activity (i.e. autonomous) as a measure of Thr286 autophosphorylation, CaM titration experiments revealed that both Ca2+/CaM-dependent and autonomous forms of CaMKII activity were cooperative (nH=2.1 for both) for Ca2+/CaM activation and autophosphorylation. Titrating CaM levels to ratios below 1 per holoenzyme (i.e. 12 CaMKII subunits per holoenzyme) generate submaximal autonomous activity, whereas, CaM levels at a ratio of ∼2 per CaMKII holoenzyme generate maximal autonomous activity. Thus, CaM activation of CaMKII appears to follow a cooperative model whereby neighboring subunits within the holoenzyme preferentially obtain the activator to promote autophosphorylation even in the face of limiting CaM.