Date Published: March 29, 2017
Publisher: Public Library of Science
Author(s): Rainer Bomblies, Manuel Patrick Luitz, Sandra Scanu, Tobias Madl, Martin Zacharias, Yaakov Koby Levy.
Many natural proteins are, as a whole or in part, intrinsically disordered. Frequently, such intrinsically disordered regions (IDRs) undergo a transition to a defined and often helical conformation upon binding to partner molecules. The intrinsic propensity of an IDR sequence to fold into a helical conformation already in the absence of a binding partner can have a decisive influence on the binding process and affinity. Using a combination of NMR spectroscopy and molecular dynamics (MD) simulations we have investigated the tendency of regions of Axin-1, an intrinsically disordered scaffolding protein of the WNT signaling pathway, to form helices in segments interacting with binding partners. Secondary chemical shifts from NMR measurements show an increased helical population in these regions. Systematic application of MD advanced sampling approaches on peptide segments of Axin-1 reproduces the experimentally observed tendency and allows insights into the distribution of segment conformations and free energies of helix formation. The results, however, were found to dependent on the force field water model. Recent water models specifically designed for IDRs significantly reduce the predicted helical content and do not improve the agreement with experiment.
The structure–function paradigm of molecular biology, stating that every protein exerting a function requires one specific three-dimensional form, has been under revision since the turn of the century . Evidence has accumulated that some active proteins do not adopt a single stable energy minimum at a folded structure but are intrinsically disordered in solution (intrinsically disordered proteins: IDPs) [2, 3]. Distinct sequence patterns predicted to form intrinsically disordered states in solution have been identified in genome sequences of many forms of life but are more abundant in highly evolved eukaryotes [4, 5]. About 15–45% of eukaryotic proteins have segments of significant disorder  where 30 or more consecutive residues are in a disordered state [7, 8]. The relative frequency of IDPs in the more communicative eukaryotes compared to prokaryotes is reflected in their important role in transcription, translation, cell cycle regulation and cell signaling [9–13]. Analysis of the SwissProt data bank revealed that many diseases, including cancer, malaria, human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS), deafness, obesity, cardiovascular diseases, diabetes mellitus, albinism, and prion protein related diseases, are correlated with proteins predicted to contain disordered regions .
NMR secondary chemical shifts and MD simulations provided evidence that the central Axin-1 segment is largely disordered but shows areas of helix propensity, especially in the binding regions of GSK-3β and β-catenin. The transiently folded regions identified here agree with results from the neural network predictor PONDR , where residues approximately in the ranges 380 − 400 and 450 − 480 show a reduced disorder score. Segmenting the 100 amino acid chain into peptides of 10 residues we were also able to identify regions of increased helicity in MD simulations. The used water model, however, has a significant impact on the structural ensemble of 10 amino acid peptides.
In this work we investigated the property of intrinsically disordered regions to contain transient helical population with MD simulations and NMR secondary chemical shifts. Our model system Axin-1380−490 showed intrinsically disordered behavior but increased transient helical content in the binding regions of two binding partners. This result is of significant importance for understanding the function of IDP regions in proteins. Even a small preference for adopting conformations close to the bound structure can significantly modulate the binding capacity of a protein segment. This could be a general basis for fine tuning the binding properties of IDP containing proteins. Interestingly, the predicted degree of residual helicity depended significantly on the selected water force field model. Simulations with the traditional TIP3P water model reproduced the trend of increased helicity in the binding regions quite well. However, water models explicitly parametrized for IDPs underestimated the helical content. In particular, TIP4P-D strongly disfavors collapsed, folded peptide conformations. With corrections to backbone parameters amber99SBws simulations using the TIP4P-ws were able to reproduce and even slightly overestimate the higher helical propensities of the binding regions. Hence, this model or the traditional TIP3P appear to be most appropriate for the present purpose of identifying residual helical structures in IDP segments. Our study indicates that the choice of the force field water model remains to be of critical importance for studying the properties of intrinsically disordered proteins.