To understand CV1 dimerization, we first sought to define the contribution of individual amino acid residues to dimerization. To do so, we prepared variants of CV1 in which each non-alanine, non-cysteine residue was individually substituted with alanine. In total, ten variants were prepared: T2A, F3A, N5A, L6A, W7Y, R8A, L9A, L10A, Q12A, and N13A. For Trp7, we opted to substitute with tyrosine instead of alanine to allow determination of all peptide concentrations by UV-Vis spectrophotometry. The R8A variant displayed limited aqueous solubility that prevented further study.
Analysis of bisthiol variants by CD spectroscopy revealed that all variants except L6A exhibited partial α-helical folding in sodium phosphate buffer. Aside from the L6A variant, all CD spectra were consistent with α-helical folding, displaying dual minima at approximately 208 and 222 nm (Fig. 1). Based on a calculated minimum mean residue ellipticity (MRE) of approximately − 27000 deg·cm2/dmol at 222 nm for an ideal 14-residue α-helix (Shepherd et al., 2005; Wang et al., 2006), the original CV1 bisthiol peptide is estimated to be approximately 30% α-helical in 10 mM sodium phosphate buffer at 25 ℃, which is consistent with previous results (Victorio & Sawyer, 2023). Estimated α-helix content ranged from 25% for the L10A variant to approximately 45% for the N13A variant at 25°C. In contrast, the L6A variant has a maximum around 196–197 nm and a broad minimum between 220 and 230 nm, indicating a non-α-helical structure that is distinct from a random coil (Bishop et al., 2001).
Oxidation of bisthiol monomers under mild conditions produced dimers as the major product for many, but not all, variants. Dimerization was performed as previously described using a peptide concentration of 0.88 mM in 5 mM aqueous NH4HCO3 buffer containing 20% (v/v) DMSO as oxidant. Based on HPLC quantification, reactions with CV1 and three variants (N5A, Q12A, N13A) produced the dimer as the exclusive product. The W7Y variant also displayed significant preference for the dimer product (> 90%) over the competing intramolecular disulfide species. For four remaining variants – T2A, F3A, L9A, and L10A – a mixture of dimer and intramolecular disulfide products was observed. For the L6A variant, only the intramolecular disulfide product was observed upon oxidation, which is consistent with previous results suggesting the necessity of α-helical folding of the bisthiol monomer to yield dimers. When corrected for the estimated extinction coefficients of 11,000 M− 1·cm− 1 for the dimer and 5500 M− 1·cm− 1 for the intramolecular disulfide species, the ratios of dimer-to-intramolecular disulfide were 1:1 for the T2A variant, 2:1 for the F3A variant, 3:1 for the L9A variant, and 1:2 for the L10A variant. All dimers were determined to be antiparallel based on trypsin digestion (Figure S3). Based on these data, Leu6 and Leu10 appear to be major contributors to dimerization with additional contributions from Thr2, Phe3, and Leu9. Importantly, the minimal contribution of Trp7 contrasts sharply with the previously proposed model in which tryptophan residues interact at the dimer interface (Victorio & Sawyer, 2023).
Evaluation of dimers by CD spectroscopy indicated that dimers formed from each variant exhibited different degrees of α-helical folding. At 25°C, the MRE at 222 nm ranged from approximately − 22,000 deg·cm2/dmol (70% α-helical) for CV1 to approximately − 7300 deg·cm2/dmol for the T2A variant (20% α-helical). The nine dimers (CV1 and eight variants) can be grouped into four pairs with one outlier (Fig. 2):
The first pair is CV1 and L10A, which were almost 70% α-helical at 25°C (Fig. 2A, black and light blue; Figure S4). Nonetheless, folding stability was starkly different for these peptides. The CV1 dimer shows gradual unfolding with an MRE of -9000 deg·cm2/dmol at 95°C, corresponding to approximately 50% α-helicity at this temperature. In contrast, the dimer of the L10A variant displayed a cooperative loss of α-helical folding between 75°C and 85°C (Fig. 2F, light blue). Thus, consistent with HPLC analysis, Leu10 appears to play a significant role in dimer stability.
Six of the remaining variants can be paired based on their α-helical folding as dimers at 25°C (Fig. 2B-D and Figure S4). At 25 ℃, variants N5A and N13A were approximately 50% folded (-17,500 deg·cm2/dmol), variants W7Y and L9A variants were approximately 40% folded (-13,500 deg·cm2/dmol), and variants F3A and Q12A were approximately 30% folded (-9400 deg·cm2/dmol). The relative stability of these variants was maintained across the full temperature range up to 95°C, with retention of greater than 90% of their respective maximum folding up to 70°C.
Dimers of the final T2A variant displayed a highly unusual CD profile as a function of temperature (Fig. 2E-F, red; Figure S4). At 5–25°C, the MRE at 222 nm was relatively constant at approximately 20% α-helical (-7200 deg·cm2/dmol). As the temperature increases from 25°C to 55°C, the MRE at 222 nm decreased to approximately
-11,000 deg·cm2/dmol, indicating an unusual increase in α-helical folding to approximately 40% in this temperature range. Beyond 55°C, the MRE for the T2A variant increased to -6200 deg·cm2/dmol at 95°C, approximately 40% of the maximum α-helical folding for this temperature range.
One important caveat to this analysis of relative α-helical folding of dimers is that all but three peptides (T2A, F3A, and W7Y) showed an apparent “shift” in the characteristic 222 nm CD minimum for α-helices to approximately 227 nm (Fig. 2). This shift was not observed for any of the bisthiol peptides, suggesting that the shift is related to dimerization. Aromatic-aromatic interactions, especially those involving tryptophan, frequently give rise to CD minima/maxima within this wavelength range (Andersen et al., 2006; Cochran et al., 2001; Nazzaro et al., 2023; Sawyer & Arora, 2018). This fact contributed to the previously proposed model involving tryptophan interactions at the dimerization interface (Victorio & Sawyer, 2023). The fact that the W7Y variant does not display this CD shift supports the hypothesis that the CD shift arises from interaction between tryptophan residues. The absence of this shift in the T2A and F3A variants was not initially clear from CD analyses but was subsequently rationalized based on the three-dimensional structure of the CV1 dimer (vide infra).
Concurrent with alanine scanning and CD analyses, we also determined the three-dimensional structure of the CV1 dimer at 1.0 Å resolution. The asymmetric unit is composed of two interacting dimers (Fig. 3A). In contrast to the previously proposed dimer interface involving tryptophan residues (Victorio & Sawyer, 2023), the structure of each dimer in the crystal structure reveals a canonical knobs-into-holes packing between Leu6, Leu9, and Leu10 as expected for a dimer of α-helices (Hadley et al., 2008; Keating et al., 2001; Regan & DeGrado, 1988; Tripet et al., 2000). The helical portion of each peptide is capped at the N-terminus by Thr2 (Fig. 3B; (Aurora & Rose, 1998)). Cysteine residues extend beyond the helical segment of each peptide to form bridging disulfide bonds. Asn5 and Asn13 form long-range hydrogen bonding interactions within each dimer (Fig. 3C), which is consistent with the slightly decreased stability of the N5A and N13A dimers relative to CV1 in CD experiments. For each dimer, one of the Phe3 residues and both Trp7 residues are oriented away from the dimer interface, suggesting a limited role for these residues in dimerization/disulfide bond formation (Fig. 3D).
While not contributing to individual dimers, the Phe3 and Trp7 residues appear to stabilize a dimer of dimers in the asymmetric unit and help to rationalize several observations from previous and current CD data (Victorio & Sawyer, 2023). The individual dimer copies within the dimer of dimers overlay well (Fig. 4A, RMSD = 0.28 Å for all backbone atoms). The arrangement of dimers is antiparallel and highly asymmetrical (Fig. 4B). The interface between dimers resembles a hydrophobic protein core and is composed of four copies each of Phe3, Leu6, Trp7, and Leu10. The Phe3 residues form interacting pairs on either end of the hydrophobic core with a specific packing arrangement in which Hβ atoms from Phe3 of chains B and C form apparent CH-π interactions (Nishio et al., 2014; Zondlo, 2013) with the ring faces of Phe3 from chains D and A, respectively (Fig. 4C). The Trp7 residues form two types of interactions: the Trp7 residues from chains B and C form a parallel displaced stacking arrangement at a distance of approximately 3.2 Å (Figure
4D, left) while the Trp7 residues from chains A and D are parallel to each other but interacting with other hydrophobic core residues instead of each other (Fig. 4D, right).
In particular, the parallel displaced stacking of Trp residues at the interface between dimers provides a rationale for the apparent shift of the 222 nm CD minimum to 227 nm, which is observed in the spectra of CV1 and many other variants (Grishina & Woody, 1994). As mentioned previously, aromatic-aromatic interactions, especially those involving tryptophan, frequently give rise to CD minima/maxima within the 225–235 nm wavelength range (Andersen et al., 2006; Cochran et al., 2001; Nazzaro et al., 2023; Sawyer & Arora, 2018). The absence of the CD shift for the W7Y dimer supports the idea that the CD shift is related to Trp-Trp interaction. The absence of the 227 nm CD shift for the F3A dimer can be rationalized by the apparent role of the Phe3 residues in stabilizing the dimer of dimers that results in Trp-Trp stacking. The reason for the absence of a CD shift for the T2A dimer is less clear but may be related to the capping function of
Thr2, which presumably stabilizes individual helices to promote the formation of the dimer of dimers.
In addition to revealing the interactions driving dimerization of CV1 and its dimer of dimers assembly, the X-ray structure also revealed interactions that drive crystal packing. Residues Trp7 and Leu10 from chains A and D form a relatively flat face that forms hydrophobic and π-π interactions with the same residues from an adjacent dimer of dimers to form an octameric (or dimer of dimer of dimers) structure (Fig. 4E). This hierarchical assembly leads to a porous crystal structure with polar residues (Asn5, Arg8, Gln12, and Asn13) facing the pores, reminiscent of functionalized peptide-derived crystalline frameworks based on π-stacking strategies (Heinz-Kunert et al., 2022; Vijayakanth et al., 2024).
Overall, we report biophysical and crystallographic characterization of spontaneous peptide dimers based on the N-terminus of the p53 protein. CD spectral and structural data highlight major roles for the Leu6 and Leu10 residues in mediating interaction between peptide chains within each dimer. Phe3 and Trp7 residues promote higher-order assemblies of four and eight peptide chains, though such assemblies are not required for efficient dimerization. The four-chain dimer of dimers possesses a protein-like hydrophobic core, and its existence in dilute aqueous solution is supported by the 227 nm minimum observed for almost all Trp-containing variants.
Moreover, structural data suggests a designable molecular architecture beyond the p53 sequence. The all-leucine core at the dimer interface forms a LxxLL pattern, which is a consensus motif through which transcriptional coactivators bind to nuclear receptors in an α-helical conformation (Galande et al., 2005; Heery et al., 1997; Plevin et al., 2005). Beyond its role in transcription, the LxxLL motif is also found in the ubiquitin ligase UBE3A, also known as E6AP, which interacts with the E6 protein of human papillomavirus (HPV) to mediate p53 degradation as a hallmark of the HPV-related carcinogenicity (Ye et al., 2023; Zanier et al., 2013). Thus, it seems highly likely that other LxxLL motif-containing peptides could be dimerized in the same way as the CV1 peptide for potential protein targeting and/or intracellular delivery.