Water Distribution, Dynamics, and Interactions with Alzheimer’s β‑Amyloid Fibrils Investigated by Solid-State NMR

Water is essential for protein folding and assembly of amyloid fibrils. Internal water cavities have been proposed for several amyloid fibrils, but no direct structural and dynamical data have been reported on the water dynamics and site-specific interactions of water with the fibrils. Here we use solid-state NMR spectroscopy to investigate the water interactions of several Aβ40 fibrils. ¹H spectral lineshapes, T₂ relaxation times, and two-dimensional (2D) ¹H–¹³C correlation spectra show that there are five distinct water pools: three are peptide-bound water, while two are highly dynamic water that can be assigned to interfibrillar water and bulk-like matrix water. All these water pools are associated with the fibrils on the nanometer scale. Water-transferred 2D correlation spectra allow us to map out residue-specific hydration and give evidence for the presence of a water pore in the center of the three-fold symmetric wild-type Aβ40 fibril. In comparison, the loop residues and the intramolecular strand–strand interface have low hydration, excluding the presence of significant water cavities in these regions. The Osaka Aβ40 mutant shows lower hydration and more immobilized water than wild-type Aβ40, indicating the influence of peptide structure on the dynamics and distribution of hydration water. Finally, the highly mobile interfibrillar and matrix water exchange with each other on the time scale of seconds, suggesting that fibril bundling separates these two water pools, and water molecules must diffuse along the fibril axis before exchanging between these two environments. These results provide insights and experimental constraints on the spatial distribution and dynamics of water pools in these amyloid fibrils

Using Thioamides To Site-Specifically Interrogate the Dynamics of Hydrogen Bond Formation in β-Sheet Folding

Thioamides are sterically almost identical to their oxoamide counterparts, but they are weaker hydrogen bond acceptors. Therefore, thioamide amino acids are excellent candidates for perturbing the energetics of backbone–backbone H-bonds in proteins and hence should be useful in elucidating protein folding mechanisms in a site-specific manner. Herein, we validate this approach by applying it to probe the dynamic role of interstrand H-bond formation in the folding kinetics of a well-studied β-hairpin, tryptophan zipper. Our results show that reducing the strength of the peptide’s backbone–backbone H-bonds, except the one directly next to the β-turn, does not change the folding rate, suggesting that most native interstrand H-bonds in β-hairpins are formed only after the folding transition state

Assessment of Local Friction in Protein Folding Dynamics Using a Helix Cross-Linker

Internal friction arising from local steric hindrance and/or the excluded volume effect plays an important role in controlling not only the dynamics of protein folding but also conformational transitions occurring within the native state potential well. However, experimental assessment of such local friction is difficult because it does not manifest itself as an independent experimental observable. Herein, we demonstrate, using the miniprotein trp-cage as a testbed, that it is possible to selectively increase the local mass density in a protein and hence the magnitude of local friction, thus making its effect directly measurable via folding kinetic studies. Specifically, we show that when a helix cross-linker, <i>m</i>-xylene, is placed near the most congested region of the trp-cage it leads to a significant decrease in both the folding rate (by a factor of 3.8) and unfolding rate (by a factor of 2.5 at 35 °C) but has little effect on protein stability. Thus, these results, in conjunction with those obtained with another cross-linked trp-cage and two uncross-linked variants, demonstrate the feasibility of using a nonperturbing cross-linker to help quantify the effect of internal friction. In addition, we estimate that a <i>m</i>-xylene cross-linker could lead to an increase in the roughness of the folding energy landscape by as much as 0.4–1.0<i>k</i>_B<i>T</i>

Structural Polymorphism of Alzheimer’s β‑Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study

The amyloid-β (Aβ) peptide of Alzheimer’s disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aβ sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aβ. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aβ40 fibrils. ¹³C, ¹⁵N-labeled synthetic E22G Aβ40 peptides are studied and compared with wild-type and Osaka E22Δ Aβ40 fibrils. Under the same fibrillization conditions, Arctic Aβ40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aβ40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aβ40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aβ42. ¹³C–¹H dipolar order parameters, ¹H rotating-frame spin–lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aβ40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aβ peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis

Development of α‑Helical Calpain Probes by Mimicking a Natural Protein–Protein Interaction

We have designed a highly specific inhibitor of calpain by mimicking a natural protein–protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein–protein interactions

Exploring <i>N</i>‑Arylsulfonyl‑l‑proline Scaffold as a Platform for Potent and Selective αvβ1 Integrin Inhibitors

One small molecule inhibitor of αvβ1 integrin, <b>c8</b>, shows antifibrotic effects in multiple in vivo mouse models. Here we synthesized <b>c8</b> analogues and systematically investigate their structure–activity relationships (SAR) in αvβ1 integrin inhibition. <i>N</i>-Phenylsulfonyl-l-homoproline analogues of <b>c8</b> maintained excellent potency against αvβ1 integrin while retaining good selectivity over other RGD integrins. In addition, 2-aminopyridine or cyclic guanidine analogues were shown to be equally potent to <b>c8</b>. A rigid phenyl linker increased the potency compared to <b>c8</b>, but the selectivity over other RGD integrins diminished. These results can provide further insights on design of αvβ1 integrin inhibitors as antifibrotics

Discovery of Novel Dual Inhibitors of the Wild-Type and the Most Prevalent Drug-Resistant Mutant, S31N, of the M2 Proton Channel from Influenza A Virus

Anti-influenza drugs, amantadine and rimantadine, targeting the M2 channel from influenza A virus are no longer effective because of widespread drug resistance. S31N is the predominant and amantadine-resistant M2 mutant, present in almost all of the circulating influenza A strains as well as in the pandemic 2009 H1N1 and the highly pathogenic H5N1 flu strains. Thus, there is an urgent need to develop second-generation M2 inhibitors targeting the S31N mutant. However, the S31N mutant presents a huge challenge to drug discovery, and it has been considered undruggable for several decades. Using structural information, classical medicinal chemistry approaches, and M2-specific biological testing, we discovered benzyl-substituted amantadine derivatives with activity against both S31N and WT, among which 4-(adamantan-1-ylaminomethyl)-benzene-1,3-diol (<b>44</b>) is the most potent dual inhibitor. These inhibitors demonstrate that S31N is a druggable target and provide a new starting point to design novel M2 inhibitors that address the problem of drug-resistant influenza A infections

Stapled Voltage-Gated Calcium Channel (Ca_V) α‑Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of Ca_V Function

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein–protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein–protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop <i>meta</i>-xylyl (<i>m</i>-xylyl) stapled peptides that target a prototypic VGIC high affinity protein–protein interaction, the interaction between the voltage-gated calcium channel (Ca_V) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca_Vβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the <i>m</i>-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca_Vβ interactions and reduce the entropic penalty associated with AID binding to Ca_Vβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca_Vα₁:Ca_Vβ interaction that modulate Ca_V function in an Ca_Vβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein–protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca_V modulator design