Homogeneous and heterogeneous tertiary structure ensembles of amyloid-β peptides

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-β 1-42 (Aβ42), the primary peptide associated with Alzheimer's disease, and fragments such as Aβ21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aβ42 and Aβ21-30 from the perspective of their classification as IDPs. Unlike the Aβ21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aβ42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.     

All-atom structure ensembles of islet amyloid polypeptides determined by enhanced sampling and experiment data restraints

Exploring the accurate structure ensembles are critical to understand the functions of intrinsically disordered proteins (IDPs). As a well-known IDP, islet amyloid polypeptide (IAPP) plays important roles in the development of human type II diabetes (T2D). The toxicity of human IAPP (hIAPP) is induced by the amyloidosis of the peptide, however, its aggregation mechanism remains ambiguous. The characterization of structure ensemble of hIAPP, as well as the differences between hIAPP and its non-amyloidogenic homologous such as rat IAPP (rIAPP), would greatly help to illuminate the amyloidosis mechanism of IAPP. In this study, the atomic structure ensembles of hIAPP and rIAPP were characterized by all-atom molecular dynamics (MD) simulations combined with enhanced sampling technology and experiment data restraints. The obtained structure ensembles were firstly compared with those determined by the conventional MD (cMD) and enhanced sampling without experiment data restraints. The results showed that the enhanced sampling and experiment data restraints would improve the simulation accuracy. The transient N-terminal α-helix structures were adopted by the sub-states of both hIAPP and rIAPP, however, the C-terminal helical structures were only present on hIAPP. The hydrophobic residues in the amyloid-core region of hIAPP are exposed to the solvent. The structure ensemble differences between hIAPP and rIAPP revealed in this work provide potential explain to the amyloidogenic mechanism and would be helpful for the design of drugs to combat T2D.     

Energy Landscapes of Protein Aggregation and Conformation Switching in Intrinsically Disordered Proteins

The protein folding problem was apparently solved recently by the advent of a deep learning method for protein structure prediction called AlphaFold. However, this program is not able to make predictions about the protein folding pathways. Moreover, it only treats about half of the human proteome, as the remaining proteins are intrinsically disordered or contain disordered regions. By definition these proteins differ from natively folded proteins and do not adopt a properly folded structure in solution. However these intrinsically disordered proteins (IDPs) also systematically differ in amino acid composition and uniquely often become folded upon binding to an interaction partner. These factors preclude solving IDP structures by current machine-learning methods like AlphaFold, which also cannot solve the protein aggregation problem, since this meta-folding process can give rise to different aggregate sizes and structures. An alternative computational method is provided by molecular dynamics simulations that already successfully explored the energy landscapes of IDP conformational switching and protein aggregation in multiple cases. These energy landscapes are very different from those of ‘simple’ protein folding, where one energy funnel leads to a unique protein structure. Instead, the energy landscapes of IDP conformational switching and protein aggregation feature a number of minima for different competing low-energy structures. In this review, I discuss the characteristics of these multifunneled energy landscapes in detail, illustrated by molecular dynamics simulations that elucidated the underlying conformational transitions and aggregation processes.     

Transient Tertiary Structures of Disordered Dynein Intermediate Chain Regulate its Interactions with Multiple Partners

The N-terminal domain of dynein intermediate chain (N–IC) is central to the cytoplasmic dynein ‘cargo attachment subcomplex’ and regulation of motor activity. It is a prototypical intrinsically disordered protein (IDP), serving as a primarily disordered polybivalent molecular scaffold for numerous binding partners, including three dimeric dynein light chains and coiled coil domains of dynein partners dynactin p150^Glued and NudE. At the very N-terminus, a 40 amino acid single alpha helix (SAH) forms the major binding site for both p150^Glued and NudE, while a shorter nascent helix (H2) separated from SAH by a disordered linker, is necessary for tight binding to dynactin p150^Glued but not to NudE. Here we demonstrate that transient tertiary interactions in this highly dynamic protein underlie the differences in its interactions with p150^Glued and NudE. NMR paramagnetic relaxation enhancement experiments and restrained molecular dynamics simulations identify interactions between the two non-contiguous SAH and H2 helical regions, the extent of which correlates with the length and stability of H2, showing clearly that tertiary and secondary structure formation are coupled in IDPs. These interactions are significantly attenuated when N–IC is bound to NudE, suggesting that NudE binding shifts the conformational ensemble to one that is more extended and with less structure in H2. While the intrinsic disorder and flexibility in N–IC modulate its ability to serve as a binding platform for numerous partners, deviations of this protein from random-coil behavior provide a process for regulating these binding interactions and potentially the dynein motor.     

Equilibrium conformational dynamics in an RNA tetraloop from massively parallel molecular dynamics

Conformational equilibrium within the ubiquitous GNRA tetraloop motif was simulated at the ensemble level, including 10 000 independent all-atom molecular dynamics trajectories totaling over 110 µs of simulation time. This robust sampling reveals a highly dynamic structure comprised of 15 conformational microstates. We assemble a Markov model that includes transitions ranging from the nanosecond to microsecond timescales and is dominated by six key loop conformations that contribute to fluctuations around the native state. Mining of the Protein Data Bank provides an abundance of structures in which GNRA tetraloops participate in tertiary contact formation. Most predominantly observed in the experimental data are interactions of the native loop structure within the minor groove of adjacent helical regions. Additionally, a second trend is observed in which the tetraloop assumes non-native conformations while participating in multiple tertiary contacts, in some cases involving multiple possible loop conformations. This tetraloop flexibility can act to counterbalance the energetic penalty associated with assuming non-native loop structures in forming tertiary contacts. The GNRA motif has thus evolved not only to readily participate in simple tertiary interactions involving native loop structure, but also to easily adapt tetraloop secondary conformation in order to participate in larger, more complex tertiary interactions.     

Discrete Molecular Dynamics Can Predict Helical Prestructured Motifs in Disordered Proteins

Intrinsically disordered proteins (IDPs) lack a stable tertiary structure, but their short binding regions termed Pre-Structured Motifs (PreSMo) can form transient secondary structure elements in solution. Although disordered proteins are crucial in many biological processes and designing strategies to modulate their function is highly important, both experimental and computational tools to describe their conformational ensembles and the initial steps of folding are sparse. Here we report that discrete molecular dynamics (DMD) simulations combined with replica exchange (RX) method efficiently samples the conformational space and detects regions populating α-helical conformational states in disordered protein regions. While the available computational methods predict secondary structural propensities in IDPs based on the observation of protein-protein interactions, our ab initio method rests on physical principles of protein folding and dynamics. We show that RX-DMD predicts α-PreSMos with high confidence confirmed by comparison to experimental NMR data. Moreover, the method also can dissect α-PreSMos in close vicinity to each other and indicate helix stability. Importantly, simulations with disordered regions forming helices in X-ray structures of complexes indicate that a preformed helix is frequently the binding element itself, while in other cases it may have a role in initiating the binding process. Our results indicate that RX-DMD provides a breakthrough in the structural and dynamical characterization of disordered proteins by generating the structural ensembles of IDPs even when experimental data are not available.     

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics,SH3 Binding,and Phosphorylation

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein’s interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191–447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.     

Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water

The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using “realistic” potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.     

Apo-parvalbumin as an intrinsically disordered protein

Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (alpha- and beta-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in alpha-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge-charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-alpha-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20 degrees C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10-15 degrees C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (alpha-PA) or two (beta-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data.     

Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration

The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well‐understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L^?1 and 400 g L^?1 of Ficoll, a synthetic polysaccharide, using a recently‐developed strong cation exchange‐based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275–1282]. Transiently helical regions of ACTR exchanged faster in 300 g L^?1 Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L^?1 Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L^?1 Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L^?1 Ficoll is a semi‐dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468–1479. © 2017 Wiley Periodicals, Inc.     

Temporary secondary structures in tau,an intrinsically disordered protein

The tau protein belongs to the category of intrinsically disordered proteins, which in their native state do not have an average stable structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilising the microtubules in the axons of the neurons. On the other hand, the same tau is involved in the development of Alzheimer disease, when it aggregates in paired helical filaments forming fibrils, which form insoluble tangles. The beginning of the pathological aggregation of tau has been attributed to a local transition of protein portions from random coil to a β-sheet. These structures would very likely be transient; therefore, we performed a molecular dynamics simulation of tau to gather information on the existence of segments of tau endowed with a secondary structure. We combined the results of our simulation with small-angle X-ray scattering experimental data to extract from the dynamics a set of most probable conformations of tau. The analysis of these conformations highlights the presence of transient secondary structures such as turns, β-bridges, β-sheets and α-helices. It also shows that a large segment of the N-terminal region is found near the repeats domain in a globular-like shape.     

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1_613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.     

Structure of crambin in solution, crystal and in the trajectories of molecular dynamics simulations

The mechanisms of the three-dimensional crambin structure alterations in the crystalline environments and in the trajectories of the molecular dynamics simulations in the vacuum and crystal surroundings have been analyzed. In the crystalline state and in the solution the partial regrouping of remote intramolecular packing contacts, involved in the formation and stabilization of the tertiary structure of the crambin molecule, occurs in NMR structures. In the crystalline state it is initiated by the formation of the intermolecular contacts, the conformational influence of its appearance is distributed over the structure. The changes of the conformations and positions of the residues of the loop segments, where the intermolecular contacts of the crystal surroundings are preferably concentrated, are most observable. Under the influence of these contacts the principal change of the regular secondary structure of crambin is taking place: extension of the two-strand β structure to the three-strand structure with the participation of the single last residue N46 of the C-terminal loop. In comparison with the C-terminal loop the more profound changes are observed in the conformation and the atomic positions of the backbone atoms and in the solvent accessibility of the residues of the interhelical loop. In the solution of the ensemble of the 8 NMR structures relative accessibility to the solvent differs more noticeably also in the region of the loop segments and rather markedly in the interhelical loop. In the crambin cryogenic crystal structures the positions of the atoms of the backbone and/or side chain of 14–18 of 46 residues are discretely disordered. The disorganizations of at least 8 of 14 residues occur directly in the regions of the intermolecular contacts and another 5 residues are disordered indirectly through the intramolecular contacts with the residues of the intermolecular contacts. Upon the molecular dynamics simulation in the vacuum surrounding as in the solution of the crystalline structure of crambin the essential changes of the backbone conformation are caused by the intermolecular contacts absence, but partly masked by the structure changes owing to the nonpolar H atoms absence on the simulated structure. The intermolecular contact absence is partly manifested upon the molecular dynamics simulation of the crambin crystal with one protein molecule. Compared to the crystal structure the lengths of the interpeptide hydrogen bonds and other interresidue contacts in an average solution NMR structure are somewhat shorter and accordingly the energy of the interpeptide hydrogen bonds is better. This length shortening can occur at the stage of the refinement of the NMR structures of the crambin and other proteins by its energy minimizations in the vacuum surroundings and not exist in the solution protein structures.     

Transient structure and SH3 interaction sites in an intrinsically disordered fragment of the hepatitis C virus protein NS5A

Understanding the molecular mechanisms involved in virus replication and particle assembly is of primary fundamental and biomedical importance. Intrinsic conformational disorder plays a prominent role in viral proteins and their interaction with other viral and host cell proteins via transiently populated structural elements. Here, we report on the results of an investigation of an intrinsically disordered 188-residue fragment of the hepatitis C virus non-structural protein 5A (NS5A), which contains a classical poly-proline Src homology 3 (SH3) binding motif, using sensitivity- and resolution-optimized multidimensional NMR methods, complemented by small-angle X-ray scattering data. Our study provides detailed atomic-resolution information on transient local and long-range structure, as well as fast time scale dynamics in this NS5A fragment. In addition, we could characterize two distinct interaction modes with the SH3 domain of Bin1 (bridging integrator protein 1), a pro-apoptotic tumor suppressor. Despite being largely disordered, the protein contains three regions that transiently adopt α-helical structures, partly stabilized by long-range tertiary interactions. Two of these transient α-helices form a noncanonical SH3-binding motif, which allows low-affinity SH3 binding. Our results contribute to a better understanding of the role of the NS5A protein during hepatitis C virus infection. The present work also highlights the power of NMR spectroscopy to characterize multiple binding events including short-lived transient interactions between globular and highly disordered proteins.     

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

The sequence–structure–function paradigm of proteins has been revolutionized by the discovery of intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). In contrast to traditional ordered proteins, IDPs/IDRs are unstructured under physiological conditions. The absence of well‐defined three‐dimensional structures in the free state of IDPs/IDRs is fundamental to their function. Folding upon binding is an important mode of molecular recognition for IDPs/IDRs. While great efforts have been devoted to investigating the complex structures and binding kinetics and affinities, our knowledge on the binding mechanisms of IDPs/IDRs remains very limited. Here, we review recent advances on the binding mechanisms of IDPs/IDRs. The structures and kinetic parameters of IDPs/IDRs can vary greatly, and the binding mechanisms can be highly dependent on the structural properties of IDPs/IDRs. IDPs/IDRs can employ various combinations of conformational selection and induced fit in a binding process, which can be templated by the target and/or encoded by the IDP/IDR. Further studies should provide deeper insights into the molecular recognition of IDPs/IDRs and enable the rational design of IDP/IDR binding mechanisms in the future.     

Molecular dynamics of the anticodon domain of yeast tRNA(Phe): codon-anticodon interaction

We have studied the effect of codon-anticodon interaction on the structure and dynamics of transfer RNAs using molecular dynamics simulations over a nanosecond time scale. From our molecular dynamical investigations of the solvated anticodon domain of yeast tRNA(Phe) in the presence and absence of the codon trinucleotides UUC and UUU, we find that, although at a gross level the structures are quite similar for the free and the bound domains, there are small but distinct differences in certain parts of the molecule, notably near the Y37 base. Comparison of the dynamics in terms of interatomic or inter-residual distance fluctuation for the free and the bound domains showed regions of enhanced rigidity in the loop region in the presence of codons. Because fluorescence experiments suggested the existence of multiple conformers of the anticodon domain, which interconvert on a much larger time scale than our simulations, we probed the conformational space using five independent trajectories of 500 ps duration. A generalized ergodic measure analysis of the trajectories revealed that at least for this time scale, all the trajectories populated separate parts of the conformational space, indicating a need for even longer simulations or enhanced sampling of the conformational space to give an unequivocal answer to this question.     

Adaptability of restrained molecular dynamics for tertiary structure prediction: application to Crotalus atrox venom phospholipase A2

In order to assess the adaptability and/or applicability of the restrained molecular dynamics (RMD) simulation for building a possible tertiary structure of a protein from the X-ray crystal structure of a family reference protein, the tertiary structure prediction of Crotalus atrox venom phospholipase A2 (PLA2) was attempted based on the X-ray crystal structure of bovine pancreatic PLA2. For the formation of secondary and tertiary structures from the fully extended starting structure, the RMD simulation with interatomic distance restraints and torsion angle restraints, which were derived from homologous amino acid sequence regions in the reference protein, was carried out until the molecular system was fully equilibrated. The predicted tertiary structure of C. atrox venom PLA2 was compared with its X-ray crystal structure, and furthermore the utility of this method was discussed by reference to the similar tertiary structure prediction of beta-trypsin from the X-ray crystal structure of an elastase.