Pushing the Limits of Structure-Based Models: Prediction of Nonglobular Protein Folding and Fibrils Formation with Go-Model Simulations

The development of computational efficient models is essential to obtain a detailed characterization of the mechanisms underlying the folding of proteins and the formation of amyloid fibrils. Structure-based computational models (Go-model) with Cα or all-atom resolutions have been able to successfully delineate the mechanisms of folding of several globular proteins and offer an interesting alternative to computationally intensive simulations with explicit solvent description. Here, we explore the limits of Go-model predictions by analyzing the folding of the nonglobular repeat domain proteins Notch Ankyrin and p16^INK4 and the formation of human islet amyloid polypeptide (hIAPP) fibrils. Folding trajectories of the repeat domain proteins revealed that an all-atom resolution is required to capture the folding pathways and cooperativity reported in experimental studies. The all-atom Go-model was also successful in predicting the free-energy landscape of hIAPP fibrillation, suggesting a “dock and lock” mechanism of fibril elongation. We finally explored how mutations can affect the co-assembly of hIAPP fibrils by simulating a heterogeneous system composed of wild-type and mutated hIAPP peptides. Overall, this study shows that all-atom Go-model-based simulations have the potential of discerning the effects of mutations and post-translational modifications in protein folding and association and may help in resolving the dichotomy between experimental and theoretical studies on protein folding and amyloid fibrillation

Monomeric Aβ^1–40 and Aβ^1–42 Peptides in Solution Adopt Very Similar Ramachandran Map Distributions That Closely Resemble Random Coil

The pathogenesis of Alzheimer’s disease is characterized by the aggregation and fibrillation of amyloid peptides Aβ^1–40 and Aβ^1–42 into amyloid plaques. Despite strong potential therapeutic interest, the structural pathways associated with the conversion of monomeric Aβ peptides into oligomeric species remain largely unknown. In particular, the higher aggregation propensity and associated toxicity of Aβ^1–42 compared to that of Aβ^1–40 are poorly understood. To explore in detail the structural propensity of the monomeric Aβ^1–40 and Aβ^1–42 peptides in solution, we recorded a large set of nuclear magnetic resonance (NMR) parameters, including chemical shifts, nuclear Overhauser effects (NOEs), and <i>J</i> couplings. Systematic comparisons show that at neutral pH the Aβ^1–40 and Aβ^1–42 peptides populate almost indistinguishable coil-like conformations. Nuclear Overhauser effect spectra collected at very high resolution remove assignment ambiguities and show no long-range NOE contacts. Six sets of backbone <i>J</i> couplings (³<i>J</i>_HNHα, ³<i>J</i>_C′C′, ³<i>J</i>_C′Hα, ¹<i>J</i>_HαCα, ²<i>J</i>_NCα, and ¹<i>J</i>_NCα) recorded for Aβ^1–40 were used as input for the recently developed MERA Ramachandran map analysis, yielding residue-specific backbone ϕ/ψ torsion angle distributions that closely resemble random coil distributions, the absence of a significantly elevated propensity for β-conformations in the C-terminal region of the peptide, and a small but distinct propensity for α_L at K28. Our results suggest that the self-association of Aβ peptides into toxic oligomers is not driven by elevated propensities of the monomeric species to adopt β-strand-like conformations. Instead, the accelerated disappearance of Aβ NMR signals in D₂O over H₂O, particularly pronounced for Aβ^1–42, suggests that intermolecular interactions between the hydrophobic regions of the peptide dominate the aggregation process

Improved Cross Validation of a Static Ubiquitin Structure Derived from High Precision Residual Dipolar Couplings Measured in a Drug-Based Liquid Crystalline Phase

The antibiotic squalamine forms a lyotropic liquid crystal at very low concentrations in water (0.3-3.5% w/v), which remains stable over a wide range of temperature (1-40 °C) and pH (4-8). Squalamine is positively charged, and comparison of the alignment of ubiquitin relative to 36 previously reported alignment conditions shows that it differs substantially from most of these, but is closest to liquid crystalline cetyl pyridinium bromide. High precision residual dipolar couplings (RDCs) measured for the backbone ¹H-¹⁵N, ¹⁵N-¹³C′, ¹H^α-¹³C^α, and ¹³C′-¹³C^α one-bond interactions in the squalamine medium fit well to the static structural model previously derived from NMR data. Inclusion into the structure refinement procedure of these RDCs, together with ¹H-¹⁵N and ¹H^α-¹³C^α RDCs newly measured in Pf1, results in improved agreement between alignment-induced changes in ¹³C′ chemical shift, ³<i>J</i>_HNHα values, and ¹³C^α-¹³C^β RDCs and corresponding values predicted by the structure, thereby validating the high quality of the single-conformer structural model. This result indicates that fitting of a single model to experimental data provides a better description of the average conformation than does averaging over previously reported NMR-derived ensemble representations. The latter can capture dynamic aspects of a protein, thus making the two representations valuable complements to one another

Conformational Changes induced by Flap Mutation Cluster of M46L, G48V and I54V.

<p>A. Flap mutation cluster of M46L, G48V and I54V in PR^S17/DRV compared to PR/DRV. B. Curling of flap due to flap mutation cluster in inhibitor-free structures of PR^S17 (green ribbon), PR (grey ribbon) and PR20 (salmon ribbon). The conformation of flap tip in the circled portion is shown in sticks for inhibitor-free PR^S17 (green carbon), PR (grey carbon) and PR20 (salmon carbon). C. Distance in (Å) between the flap tip residue Ile51 and the catalytic Asp25 in inhibitor-free PR^S17, PR and PR20. D. Distance (Å) between the flap tip residue Ile51 and Thr80 in the three inhibitor-free structures.</p

NMR Analysis of Solution Conformation of Inhibitor-free PR^S17 _D25N.

<p>A. ¹H-¹⁵N TROSY-HSQC spectrum of the inhibitor-free PR^S17_D25N recorded in 20 mM sodium phosphate pH 5.7 at 20°C. B. ¹⁵N NOE measured under the same conditions for the inhibitor-free PR^S17_D25N (black) and PR20_D25N (green) as a function of the residue number. C. Comparison between the ¹D_NH RDCs measured for the inhibitor-free PR^S17_D25N in a dilute solution of squalamine with those predicted from the crystal structures of the inhibitor-free PR^S17 (red dots) and PR^S17/DRV (gray dots). All the NMR experiments were recorded at 600 MHz.</p

Conformational Changes in Hinge Loop Mutations E35D, M36I and S37N.

<p>A. Conformational changes in the hinge loop of PR^S17/DRV complex in comparison to PR/DRV. B. Comparison of hinge loop between PR^S17/DRV and PR20/DRV. C. Hinge loop conformation of PR^S17/DRV, PR/DRV and PR20/DRV in subunit B. Wild-type PR, PR^S17 and PR20 carbons are shown in grey, green and salmon, respectively.</p

Effects of Distal mutations A71V, L90M and I93L.

<p>A. L90M mutation in PR^S17 (green) induces shortened C-H…O interaction between Met90 and catalytic Asp25 in comparison to wild-type PR (gray). I93L mutation in PR^S17 results in loss of van der Waals contact observed between Ile93 and Leu 90 of wild-type PR. B. Distal mutations A71V and I93L in PR^S17 are associated with shift in 70’s β strand by ~1 Å and loss of the ion pair between His69 and the carboxylate tail of the second subunit.</p

Crystallographic Data Collection and Refinement Statistics.

<p>Crystallographic Data Collection and Refinement Statistics.</p

Hydrogen Bond Interactions of Darunavir with PR^S17 and Effect of Leu10 Mutations.

<p>A. Hydrogen bond interactions of DRV with PR^S17. PR^S17 is in stick representation with green carbons while DRV is shown in ball and stick with white carbons. For the sake of clarity only one conformation of DRV and Ile50 of PR^S17/DRV are shown. The hydrogen bond interactions of the second DRV are essentially identical. The hydrogen bonds are shown as broken lines. B. L10I mutation in PR^S17 does not break the inter-monomer ion pair between Arg8 and Asp29′ unlike L10F in PR20. Wild-type PR is shown with grey carbons, PR^S17 as green carbons and PR20 as salmon carbons. The van der Waals contacts are represented by (-·-) line. The minor conformation of Arg8 in wild-type PR and PR20 and its interactions with Asp29′ are omitted for clarity.</p

Two Alternate Conformations of Darunavir and the Active Site Mutation V82S in PR^S17/DRV Dimer.

<p>A. F_o-F_c omit map contoured at 3σ level shows Ser82 and DRV have two alternate conformations in both subunits. B. Sites of 17 mutations are mapped on PR^S17 (pale green cartoon representation) with bound DRV shown as orange sticks. V82S mutation, proximal to the active site, is shown as a green sphere in each subunit. The mutations in the hinge loop cluster are colored as red spheres while the flap mutation cluster is represented as blue spheres. Note that K20R interacts with residues in the hinge loop although it is not contiguous in sequence with this region as described later. The distal mutations perturbing active site aspartates are colored as magenta spheres and remaining mutations are shown as pink spheres. C. Sequence alignment of PR^S17 with PR and PR20. Mutations introduced in WT PR to restrict autoproteolysis (Q7K, L33I and L63I) and avoid cysteine-thiol oxidation (C67A and C95A) are indicted by asterisks. Residues identical to PR in PR20 and PR^S17 are omitted. The 17 mutations of PR^S17 are colored similar to 1B.</p