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Abstract Cis-peptide bonds are rare in proteins, and building blocks less favorable to the trans-conformer have been considered destabilizing. Although proline tolerates the cis-conformer modestly among all amino acids, for collagen, the most prevalent proline-abundant protein, all peptide bonds must be trans to form its hallmark triple-helix structure. Here, using host-guest collagen mimetic peptides (CMPs), we discover that surprisingly, even the cis-enforcing peptoid residues (N-substituted glycines) form stable triple-helices. Our interrogations establish that these peptoid residues entropically stabilize the triple-helix by pre-organizing individual peptides into a polyproline-II helix. Moreover, noting that the cis-demanding peptoid residues drastically reduce the folding rate, we design a CMP whose triple-helix formation can be controlled by peptoid cis-trans isomerization, enabling direct targeting of fibrotic remodeling in myocardial infarction in vivo. These findings elucidate the principles of peptoid cis-trans isomerization in protein folding and showcase the exploitation of cis-amide-favoring residues in building programmable and functional peptidomimetics.
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An Unbound Proline-Rich Signaling Peptide Frequently Samples Cis Conformations in Gaussian Accelerated Molecular Dynamics Simulations
Disordered proline-rich motifs are common across the proteomes of many species and are often involved in protein-protein interactions. Proline is a unique amino acid due to the covalent bond between the backbone nitrogen and the proline side chain. The resulting five-membered ring allows proline to sample the cis state about its peptide bond, which other residues cannot do as readily. Because proline-rich disordered sequences exist as ensembles that likely include structures with the proline peptide bond in cis , a robust methodology to accurately account for these conformations in the overall ensemble is crucial. Observing the cis conformations of proline in a disordered sequence is challenging both experimentally and computationally. Nitrogen-hydrogen NMR spectroscopy cannot directly observe proline residues, which lack an amide bond, and computational methods struggle to overcome the large kinetic barrier between the cis and trans states, since isomerization usually occurs on the order of seconds. In the current work, Gaussian accelerated molecular dynamics was used to overcome this free energy barrier and simulate proline isomerization in a tetrapeptide (KPTP) and in the 12-residue proline-rich SH3 binding peptide, ArkA. We found that Gaussian accelerated molecular dynamics, when combined with a lowered peptide bond dihedral angle potential energy barrier (15 kcal/mol), allowed sufficient sampling of the proline cis and trans states on a microsecond timescale. All ArkA prolines spend a significant fraction of time in cis , leading to a more compact ensemble with less polyproline II helix structure than an ArkA ensemble with all peptide bonds in trans . The ensemble containing cis prolines also matches more closely to in vitro circular dichroism data than the all- trans ensemble. The ability of the ArkA prolines to isomerize likely affects the peptide’s ability to bind its partner SH3 domain, and should be studied further. This is the first molecular dynamics simulation study of proline isomerization in a biologically relevant proline-rich sequence that we know of, and a similar protocol could be applied to study multi-proline isomerization in other proline-containing proteins to improve conformational diversity and agreement with in vitro data.
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- PAR ID:
- 10328300
- Date Published:
- Journal Name:
- Frontiers in Molecular Biosciences
- Volume:
- 8
- ISSN:
- 2296-889X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Despite the importance of proline conformational equilibria (trans versus cis amide, exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and of cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (FF = 0–3 ppm) when a trans X–Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (FF = 5–12 ppm) difference in chemical shift in a cis X–Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that the FF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e. the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e. pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small . In contrast, when the Pro is ordered (i.e. when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.more » « less
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In proteins, proline-aromatic sequences exhibit increased frequencies of cis-proline amide bonds, via proposed C–H/π interactions between the aromatic ring and either the proline ring or the backbone C–Hα of the residue prior to proline. These interactions would be expected to result in tryptophan, as the most electron-rich aromatic residue, exhibiting the highest frequency of cis-proline. However, prior results from bioinformatics studies on proteins and experiments on proline-aromatic sequences in peptides have not revealed a clear correlation between the properties of the aromatic ring and the population of cis-proline. An investigation of the effects of aromatic residue (aromatic ring properties) on the conformation of proline-aromatic sequences was conducted using three distinct approaches: (1) NMR spectroscopy in model peptides of the sequence Ac-TGPAr-NH2 (Ar = encoded and unnatural aromatic amino acids); (2) bioinformatics analysis of structures in proline-aromatic sequences in the PDB; and (3) computational investigation using DFT and MP2 methods on models of proline-aromatic sequences and interactions. C–H/π and hydrophobic interactions were observed to stabilize local structures in both the trans-proline and cis-proline conformations, with both proline amide conformations exhibiting C–H/π interactions between the aromatic ring and Hα of the residue prior to proline (Hα-trans-Pro-aromatic and Hα-cis-Pro-aromatic interactions) and/or with the proline ring (trans-ProH-aromatic and cis-ProH-aromatic interactions). These C–H/π interactions were strongest with tryptophan (Trp) and weakest with cationic histidine (HisH+). Aromatic interactions with histidine were modulated in strength by His ionization state. Proline-aromatic sequences were associated with specific conformational poses, including type I and type VI β-turns. C–H/π interactions at the pre-proline Hα, which were stronger than interactions at Pro, stabilize normally less favorable conformations, including the ζ or αL conformations at the pre-proline residue, cis-proline, and/or the g+ χ1 rotamer or αL conformation at the aromatic residue. These results indicate that proline-aromatic sequences, especially Pro-Trp sequences, are loci to nucleate turns, helices, loops, and other local structures in proteins. These results also suggest that mutations that introduce proline-aromatic sequences, such as the R406W mutation that is associated with protein misfolding and aggregation in the microtubule-binding protein tau, might result in substantial induced structure, particularly in intrinsically disordered regions of proteins.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fmolb.2021.734169
