Model system to study Gly-Rich PPII bundlesThe experimentally resolved 3D structure of the short isoform of the HhAFP consists of six PPII helices, where helices I, III, and V constitute a polar face while helices II, IV, and VI form the non-polar, ice-binding surface (Fig. 1A). The large HhAFP isoform is predicted by AlphaFold2 (AF2) with a similar fold containing thirteen instead of six PPII helices (Fig. 1A), which is in agreement with the model proposed by Mok et al. 13. The GrAFP structure also matches this bundle arrangement (Fig. 1A), with five parallel helices in one face packing antiparallel with a four-helical face12. The dearth of side chains in these Gly-rich sequences underlines the importance of H-bonding for bundle stabilization in these and in other Gly-rich PPII bundles such as in the recently elucidated ALK structure14 (Fig. 1A).Fig. 1: H-bonding in polyproline II helical bundle domains.A Experimentally determined structures from the Protein Data Bank (PDB) for HhAFP short isoform (PDB ID: 3BOG), GrAFP (PDB ID: 7JJV) and ALK Gly-rich domain (PDB ID: 7LS0/7LRZ), and predicted structure from AF2 for HhAFP long isoform (AF2 ID: D7PBP2). Roman numerals denote the distinct helices in each protein. The dashed red circle indicates the PPII helix cloned for the computational model. B Gly-rich PPII helical bundle computational model: red spheres represent oxygen; blue, nitrogen; gray, carbon, and white, hydrogen. Shown with a green dashed line are the potential hydrogen bonds that may exist based on the proximity between the hydrogen donor (i.e., N or C) and hydrogen acceptor (i.e., O) atoms. C Scheme utilized for the calculation of the different parameters between PPII helices in the different interfaces. Blue PPII helices give rise to the ab interface; the red ones, to the bc interface, and the green ones, to the cd interface. The interaction energies are probed at these colored interfaces, i.e., where the n-mers are split for the HBS calculations, both DFT-D and SAPT. The rest of the computational parameters are calculated directly between the colored PPII helices through the NBO and QTAIM analyses. For added clarity, the gray eye symbol indicates the interface under study for each case.In order to investigate the nature of H-bonding interactions across the different PPII bundles described above, we have designed a model system that recreates the interactions seen in the various assemblies. This model relies on replicating the PPII helical stretch spanning residues 31–39 from the central PPII helix in the polar face (i.e., helix III, as depicted in Fig. 1A) of the short HhAFP isoform. These nine residues correspond to three turns of the PPII helix that are properly aligned to form H-bonds with the helices above (helix I) and below (helix V), which lie on the same face and are spaced by 4.7 Å (Fig. 1A). The nine-residue stretch corresponding to residues 31–39 was cloned (i.e., repeated in space) four times to build a five-helical bundle. This leads to five monomeric, nine-residue helices lying within the same plane that are spaced by 4.7 Å from each other. The N- and C-termini of each PPII helix were capped with acetyl and methylamine moieties, respectively, to recreate the sequence continuity beyond the three turns that form the stretch. To isolate the pure PPII architecture from any other driving force for assembly arising from hydrophobic, polar or charged side chains, as well as to generalize the model for all types of Gly-rich PPII bundles, as those shown in Fig. 1A, all non-glycine residues, i.e., H32, N35, and N38, were mutated to glycine. Therefore, each PPII helix will contain 41 heavy atoms, giving rise to a total of 205 for the complete bundle (Fig. 1B). In order to analyze the interactions inherent to these structures, no type of solvent is included in the computational model.Cooperative bundle assembly of PPII helices and H-Bonding patternsInteraction energy calculations reveal enhanced H-Bonding strength and cooperativity in growing PPII helical bundlesBuilding on the general model representing Gly-rich PPII bundles elaborated in the previous section, we next sought to characterize the interaction energy between every pair of PPII helices in such an assembly. In this model system, H-bonds drive PPII bundle assembly as there are no nonpolar, charged or aromatic groups which would give rise to hydrophobic, charge-charge or cation-\(\pi\) interactions (Fig. 1B). Thus, the interaction energy between two monomeric helices provides a direct estimation of the H-bonding strength (HBS). On this basis, we employed DFT-D to compute the interaction energies for dimers (a···b), trimers (a···bc and ab···c), tetramers (a···bcd, ab···cd and abc···d) and pentamers (a···bcde, ab···cde, abc···de, abcd···e) of PPII helices, according to the scheme shown in Fig. 1C. Note that for an n-mer system, there are (n-1) interacting interfaces and thus (n-1) HBS possible values. The only interaction that will not be analyzed is abcd···e since, as de is the last interface and e is the last PPII helix, the limit of the model has been reached and it is not possible to study how the addition of monomers affects the different computational parameters. The analysis of the computed HBS values shows that as PPII helices are added to the assembly, the interaction energies between monomers are strengthened. That is, the HBS between helices a and b in a five-helix bundle, abcde, is stronger than in a four-helix bundle, abcd, which is stronger than in a three-helix bundle, abc, which in turn is stronger than in a two-helix bundle ab, (i.e., | HBS(a···b)abcde | > |HBS(a···b)abcd | > |HBS(a···b)abc | > |HBS(a···b)ab | , Fig. 2 and Table 1). In this regard, we recall that we measure the interaction energy between the first two PPII helices (called a and b) as more and more helices are added on top of them, and therefore it is not the total interaction energy of the whole assembly, but rather that between the first two PPII helices what is being quantified. The same holds true for the HBS in the rest of the interfaces as helices are added to the assembly (i.e., | HBS(b···c)abcde | > |HBS(b···c)abcd | > |HBS(b···c)abc| and |HBS(c···d)abcde | > |HBS(c···d)abcd | , Fig. 2 and Table 1). This is proof of cooperative H-bonding interactions or HBC, as seen in amyloids17,18,19,20,21,22. To uncover the source of this cooperativity, we employed various computational methods to analyze the underlying H-bonding Interactions as described in the following sections.Fig. 2: HBS between PPII helices in the different interfaces of the computational model at the M06-2X/6-31 + G(d) level of theory.The ab interface is indicated in blue; the bc interface, in red, and the cd interface, in yellow, as the color scheme depicted in Fig. 1C. Note that the HBS values decrease significantly as more PPII helices are added; this is a hallmark of HBC.Table 1 HBS between PPII helices in the different interfaces of the computational model at the M06-2X/6-31 + G(d) level of theoryIdentification of canonical and non-canonical H-Bonding patterns stabilizing Gly-Rich PPII helical bundlesHaving established that H-bonding in Gly-rich PPII assembly is cooperative, akin to amyloid assembly, we next ought to characterize which H-bonding interactions are present. We started by identifying the distinct potential stabilizing interactions by visual inspection of our model (Fig. 1B). For a PPII helix composed of three residues i, i + 1 and i + 2, which corresponds to exactly one turn of PPII helix, the carbonyl oxygen of the first residue, i, from one PPII helix interacts with the amide proton HN of the second residue, j + 1, in the next PPII helix to form a canonical CO···HN H-bond (Fig. 3A). In addition to this canonical interaction, we also identified the possible participation of the carbonyl oxygen from residue i in a non-canonical CO···HαCα interaction with the Hα from residue j in the second PPII helix. That is, the carbonyl oxygen from the first residue i in one helix participates in both the formation of a non-canonical H-bond with the Hα from the first residue j in the mating helix, and a canonical H-bond with the HN from the second residue j + 1. This non-canonical CO···HαCα interaction is denoted as a “front” H-bond (since it would be found in the same plane as each face of HhAFP) (Fig. 3A). Furthermore, we advance the existence of two additional non-canonical CO···HαCα H-bonds in Gly-rich PPII bundles that we here have denoted as an “inner” (since it would be found inside the core of HhAFP) and “outer” (since it would be found outside the core of HhAFP) (Fig. 3A). The supposed inner and outer H-bonds involve the two Hα protons of the last (i.e., third) residue in one PPII helix (i.e., residue i + 2). Particularly, in the “inner” H-bond one Hα proton would interact with the carbonyl oxygen from residue j + 2, whereas in the “outer” H-bond the second Hα proton from residue i + 2 would interact with the carbonyl oxygen from the second residue in the mating helix (i.e., j + 1, Fig. 3A). Since the results based on the HBS between PPII helices do not allow one to discern whether cooperativity holds every type of H-bond considered, we resorted to NBO and QTAIM analyses.Fig. 3: H-bonds in polyproline II helical bundle assemblies.A Possible H-bonds/interactions between PPII helices in a 2D PPII helical bundle: CO···HN (purple), CO···HαCα front (yellow), CO···HαCα inner (orange) and CO···HαCα outer (magenta). B Simplified structures for CO···HN (formamide), CO···HαCα front (acetaldehyde), CO···HN + CO···HαCα front (acetamide), CO···HαCα inner (acetaldehyde), CO···HαCα outer (N-methylformamide) and CO···(Hα)2Cα inner+outer (N-(2-oxoethyl)formamide) H-bonds/interactions.NBO and QTAIM analyses independently corroborate the existence and HBC of N-H···O = C and Cα-Hα···O = C inner H-bondsRegarding the NBO analysis (Tables 2 and 3 and Fig. 4), the occupancy of the σ* orbitals of the N-H bonds, q(σ*NH), and the stabilization energy corresponding to the electronic delocalization from not only the lone pairs (n) of the oxygen in those orbitals, but also from the σ and π orbitals of the C = O bonds, E(2)n(O)/σ(CO)/π(CO)→σ*(NH), indicate the progressive strengthening of the CO···HN H-bonds as the PPII helical assembly grows. This electron donation confers partial anionic character on the electron-acceptor species, then enhancing the capacity of the CONH units to act as an electron-donor in the next H-bond, which will be stronger. As for the CO···HαCα inner H-bonds, the q(σ*CαHα) and the E(2)n(O)/σ(CO)/π(CO)→σ*(CαHα) increase as PPII helices are added to the bundle, in a homologous way to what happened with CO···HN H-bonds, also involving the σ(CO) and π(CO) orbitals (Tables 2 and 3 and Fig. 4). These results are supported by the QTAIM analysis (supplementary material).Table 2 Mean occupancy (q) of the corresponding σ* orbitals for each CO···HN, CO···HαCα front, CO···HαCα inner and CO···HαCα outer H-bond/interaction between PPII helices in the different interfaces at the M06-2X/6-31 + G(d) level of theoryTable 3 Mean stabilization energy (E(2)) of the corresponding n(O)/σ(CO)/π(CO)→σ*(NH/CαHα) electron delocalization for each CO···HN, CO···HαCα front, CO···HαCα inner and CO···HαCα outer H-bond/interaction between PPII helices in the different interfaces at the M06-2X/6-31 + G(d) level of theoryFig. 4: Orbital occupancy and stabilization energies envince HBC in PPII helical bundles.A Mean occupancy (q) of the corresponding σ*(NH/CαHα) orbitals and B mean stabilization energy (E(2)) of the corresponding n(O)/σ(CO)/π(CO)→σ*(NH/CαHα) electron delocalization for each CO···HN (squares), CO···HαCα front (circles), CO···HαCα inner (triangles) and CO···HαCα outer (inverted triangles) H-bond/interaction between PPII helices. The ab interface for the different systems is indicated in blue; the bc interface, in red, and the cd interface, in green. M06-2X/6-31 + G(d) level of theory.Front H-bonds exist but show no HBC and outer H-bonds do not exist, according to NBO and QTAIM analysisFor the CO···HαCα front H-bonds, while E(2)n(O)→σ*(CαHα) slightly increases as PPII helices are added to the bundle, the q(σ*CαHα) decreases (Tables 2 and 3 and Fig. 4). This decrease of q(σ*CαHα) can be explained on the basis that the q(σ*NH) follows the opposite trend. Both types of σ* orbitals receive electrons from the same oxygen, so if the amount of electrons reaching one of the two different σ* orbitals increases as the assembly grows, the amount reaching the other σ* orbital will decrease. This phenomenon occurs even if the CONH units, which also give rise to the canonical H-bonds, have more and more electrons available to form the new H-bonds of the next interfaces, which is the reason why E(2)n(O)→σ*(CαHα) slightly increases as PPII helices are added to the assembly, since it depends on q(nO) and not on q(σ*CαHα) (Eq. 3). Finally, for the CO···HαCα outer interactions, the q(σ*CαHα) values remain practically constant as the PPII helical bundle grows and the negligible E(2)n(O)→σ*(CαHα) values do not have a clear tendency to change (Tables 2 and 3 and Fig. 4), suggesting their non-existence and hence non-cooperativity. These results are supported by QTAIM analyses (supplementary material).Contribution of the different H-bonds to PPII helical bundle stabilityThe presence of a large number of non-canonical CO···HαCα H-bonds is important for the stability of PPII helical bundles7,8. In order to quantify the extent to which the distinct H-bonds contribute to the interaction energy between PPII helices, we have followed a divide-and-conquer approach by which mimicking molecular fragments resembling all types of H-bonds are subjected to independent HBS calculations38,39,40 (Fig. 3B). Within this framework, each CO···HN canonical H-bond is represented by a pair of formamide molecules. Front and inner non-canonical CO···HαCα H-bonds are mimicked by a pair of acetaldehyde molecules with different spatial orientations. Finally, outer non-canonical CO···HαCα interactions are mimicked using pairs of N-methylformamide molecules. It is necessary to emphasize that the mimetic molecules that give rise to the different interactions maintain the same position, and therefore the same distance, as in the complete PPII helices. In the following subsections, where we employ mimetics rather than full helices, we change the nomenclature from “PPII helices” to “layers of molecules” and “bundle assembly (of PPII helices) growth” for “stack (of layers of molecules) growth”. The term “interface” will now signify the region where mimetic molecules interact, rather than complete PPII helices.Strengthened canonical N-H···O = C H-bonds versus electron-limited non-canonical Cα-Hα···O = C front interactions in expanding stacksRegarding canonical H-bonds, the H-bonding strength or HBS among the different interfaces between formamide moieties (Fig. 3B) is strengthened as the stack grows (Fig. 5 and Table 4). These observations are reminiscent of the complete model of PPII bundles, and evince the cooperative reinforcement in CO···HN H-bonds between PPII helices. In the same way, Fig. 5 and Table 4 also show that, when the stack of layers of the acetaldehyde molecules that mimic CO···HαCα front H-bonds (Fig. 3B) grows, the HBS of the different interfaces is strengthened. One could interpret this as evidence of the cooperative reinforcement between non-canonical front H-bonds, which is in conflict with the previous NBO and QTAIM analyses of the full-length system that seem to indicate that they do not show HBC because the carbonyl oxygen’s electron density is shared with the much more favored canonical H-bonds. Perhaps the most intuitive way to visualize the effect that CO···HN H-bonds have on CO···HαCα front H-bonds, as well as to adequately quantify both interactions, is to calculate the HBS corresponding to acetamide molecules since they mimic both H-bonds at the same time (Fig. 3B). When the stack grows, the HBS of the different interfaces is reinforced, and so does the HBS of both the canonical and non-canonical front H-bonds separately, albeit less than the sum of both H-bonds isolated at each interface (Fig. 5 and Table 4). As explained earlier, this situation can be attributed to the limited quantity of electrons that oxygens may donate to the canonical and the non-canonical front H-bonds.Fig. 5: HBC in CO···HN and CO···Hα-Cα H-bonds.Mean HBS between the molecules that reproduce each CO···HN (formamide, squares), CO···HαCα front (acetaldehyde, circles), CO···HN + CO···HαCα front (acetamide, diamonds, and dashed line), CO···HαCα inner (acetaldehyde, triangles), CO···HαCα outer (N-methylformamide, inverted triangles) and CO···(Hα)2Cα inner+outer (N-(2-oxoethyl)formamide, hourglass and dashed line) H-bond/interaction. As in the Fig. 1C color scheme, the ab interface for the different systems is indicated in blue; the bc interface, in red, and the cd interface, in green.Table 4 Mean HBS at the M06-2X/6-31 + G(d) level of theoryStrengthened non-canonical Cα-Hα···O = C inner H-bonds versus dubious non-canonical Cα-Hα···O = C outer interactions in expanding stacksConcerning non-canonical CO···HαCα inner H-bonds, while it is true that the HBS of the different interfaces between pairs of acetaldehyde molecules that mimic these interactions (Fig. 3B) become stronger upon addition of layers to the stack, it does so over a range of energy values that might fall within the uncertainty of the computational level of theory (Fig. 5 and Table 4). The values of the HBS between the N-methylformamide molecules that represent the non-canonical CO···HαCα outer interactions (Fig. 3B) are null within the error of the calculation (Fig. 5 and Table 4). Furthermore, these negligible interaction energies corresponding to the different interfaces do not show any tendency to increase or decrease, nor any evidence of cooperativity therefore, as the stack of mimicking molecules grows. Moreover, it is necessary to take into account that since these two putative non-canonical H-bonds share a common Cα, one interaction cannot be isolated from the other to obtain the HBS that would actually correspond to them. Consequently, we used pairs of N-(2-oxoethyl)formamide molecules to adequately mimic both potential H-bonds together (Fig. 3B), which gave rise to considerable HBS values showing a notable strengthening of the interactions across the different interfaces as the stack grows (Fig. 5 and Table 4). Additional QTAIM and SAPT analyses and results are shown in Supplementary Figs. 1–10 and Supplementary Tables 1–9. These interaction energy values are due almost exclusively to non-canonical inner H-bonds, not to the combination with the apparently non-existent non-canonical outer H-bonds that were initially proposed, as the previous NBO and QTAIM analyses of the full PPII bundle model showed. The findings of this section are in agreement with the NBO and QTAIM analyses of the mimicking systems.Summing interaction energy contributions in PPII helical bundles provides insight into electron delocalization effectsSince the set of different molecules of acetamide (i.e., CO···HN + HαCα front H-bonds) and N-(2-oxoethyl)formamide (i.e., CO···(Hα)2Cα inner+outer H-bonds/interactions) shown in Fig. 3B are not a bona fide representation of the complete PPII bundle, it would not be expected that the sum of the different contributions to HBS would give the total HBS between full-length PPII helices. Indeed, Table 4 shows that the sum of the the CO···HN and the different CO···HαCα interaction energies produce HBS values initially smaller than the ones obtained when calculating the total HBS between full-length PPII helices shown in Table 1. However, as layers are added to the stack, the HBS values calculated as the sum of contributions exceed the global ones (Tables 4 and 1), demonstrating once again the presence of HBC in PPII helices and suggesting that in complete PPII helices, electron density would be delocalized not only in adjacent helices, but also along the peptide chain itself, generating macrodipoles previously reported for these protein secondary structures41,42.Summary of interactionsThe computational parameters studied here are summarized in Table S10 as the mean variation for each of the proposed H-bonds of the system’s five PPII helices. One should interpret this mean variation as the average of the differences between the computed parameter X for the last and first value for the interfaces ab (Xabcde – Xab), bc (Xabcde – Xabc) and cd (Xabcde – Xabcd). The NBO and QTAIM analyses, as well as the DFT-D calculation of interaction energies (HBS), lead us to suggest the existence and cooperativity of the CO···HN and CO···HαCα inner H-bonds; the existence, but non-cooperativity, of the CO···HαCα front H-bonds, and the non-existence, and therefore non-cooperativity, of the CO···HαCα outer H-bonds. The distance between the H-bond acceptor, the carbonyl oxygen, and the H-bond donor (HN or Hα) is another way to gauge the strengthening and cooperativity, or the lack, of these four types of possible H-bonds. In particular, non-canonical CO···HαCα outer H-bonds, whose existence cannot be established here, feature the longest distances (Table S11 and Fig. S11). Moreover, the shortest distances are seen for the CO···HN and CO···HαCα inner H-bonds and these distances decrease further as the bundle grows (Table S11 and Fig. S11), which is consistent with their HBC. In the case of non-canonical CO···HαCα front H-bonds, intermediate H-bonding distances without any trend to increase or decrease as the assembly grows are obtained (Table S11 and Fig. S11); this is consistent with the lack of HBC seen in the NBO, QTAIM and DFT-D results.SAPT calculations are somewhat more precise, much more computationally costly, and provide insight into the contributions of the distinct interaction energy components to HBC. Our SAPT results, obtained at a higher level of theory than the rest of the methods, confirm that HBC exists in the cases already detected by DFT-D, NBO and QTAIM calculations, and evidence that it arises mostly from electrostatic effects and not induction or dispersion (Supplementary Tables S5, S6, S7 and Figs. S3, S5, S6, S7, S8, S9, S10). We refer the interested reader to the supplementary material for a specialized, in-depth description of the different interactions analyzed in this study.Corroboration of the computational modelTo further corroborate these findings, we have examined the experimental and computational NMR shift changes produced by H-bond formation. In solution, Gly-rich peptides have a tendency to adopt the extended PPII helix conformation over random coil or other structures43,44,45. Nevertheless, these situations cannot be easily distinguished spectroscopically since the two 1Hα chemical shifts of glycine residues are degenerate. However, we discovered that the two 1Hα of glycine residues in PPII bundles do display different chemical shifts, providing the first set of conformational chemical shifts, Δδ, for Gly-rich PPII bundles7. Experimental conformational shifts for 13Cα and 13CO of Δδ(Cα)exp = −0.59 (±0.65) ppm and Δδ(CO)exp = −0.20 (±1.12) ppm were obtained, while 1Hα nuclei in CO···HαCα H-bonded feature a characteristic conformational shift of Δδ(Hα)exp = −0.59 (±0.32) ppm. In our computational model (Fig. 1B), conformational chemical shifts for these nuclei were predicted to test our model’s ability to serve as a bona fide atomistic representation of PPII bundles. By comparing the corresponding shift for a nuclei in an extended PPII, isolated conformation with respect to that PPII in a bundle, the following DFT-GIAO (Gauge-Including-Atomic-Orbital) theoretical conformational shifts were obtained: Δδ(Cα)theo = −0.59 ppm, Δδ(CO)theo = −0.31 ppm and Δδ(Hα)theo = −0.61 ppm, for H-bonded nuclei. The similarity of the experimental and computational conformational shifts provides additional support for the existence of these H-bonds.
