Structural Characterization of Disulfide-Linked p53-Derived Peptide Dimers

Abstract

Disulfide bonds provide a convenient method for chemoselective alteration of peptide and protein structure and function. We previously reported that mild oxidation of a p53-derived bisthiol peptide (CTFANLWRLLAQNC) under dilute non-denaturing conditions led to unexpected disulfide-linked dimers as the exclusive product. The dimers were antiparallel, significantly α-helical, resistant to protease degradation, and easily reduced back to the original bisthiol peptide. Here we examine the intrinsic factors influencing peptide dimerization using a combination of amino acid substitution, circular dichroism (CD) spectroscopy, and X-ray crystallography. CD analysis of peptide variants suggests critical roles for Leu6 and Leu10 in the formation of stable disulfide-linked dimers. The 1.0 Å resolution crystal structure of the peptide dimer supports these data, revealing a leucine-rich LxxLL dimer interface with canonical knobs-into-holes packing. Two levels of higher-order oligomerization are also observed in the crystal: an antiparallel "dimer of dimers" mediated by Phe3 and Trp7 residues in the asymmetric unit and a tetramer of dimers mediated by Trp7 and Leu10. In CD spectra of Trp-containing peptide variants, minima at 227 nm provide evidence for the dimer of dimers in dilute aqueous solution. Importantly, and in contrast to the original dimer model, the canonical leucine-rich core and robust dimerization of most peptide variants suggests a tunable molecular architecture to target various proteins and evaluate how folding and oligomerization impact various properties, such as cell permeability.

Keywords: Dimerization; Disulfide; Folding; Structure.

Figure 1

Circular dichroism spectra for the CV1 bisthiol peptide and variants. Spectra were acquired using a Jasco J-1500 CD spectrometer at a concentration of 30 μM in 10 mM sodium phosphate, pH 7.5 in a 0.1 cm pathlength cell at 25 °C

Figure 2

Circular dichroism spectra for the disulfide-linked CV1 peptide dimer and disulfide-linked dimers of CV1 alanine variants. Spectra were acquired using a Jasco J-1500 CD spectrometer at a concentration of 30 μM in 10 mM sodium phosphate, pH 7.5 in a 0.1 cm pathlength cell. a-e) Spectra at 25 °C. f) Mean residue ellipticity at 222 nm as a function of temperature (5 °C to 95 °C)

Figure 3

Crystal structure of disulfide-linked CV1 peptide dimer (PDB 9C5S). In all panels, each helix is represented with the same color (chain A = green, chain B = blue,) and disulfide bonds are represented as yellow sticks. N- and C-termini are labeled as N and C, respectively, with subscripts to indicate the corresponding chain. a) The dimer interface involves interdigitation of the side-chains of Leu6, Leu9, and Leu10 (spheres). b) The oxygen atom of each Thr2 side-chain serves as a hydrogen bond acceptor for Asn5 NH to cap the helix at the N-terminus. c) Asn5 and Asn13 side-chains form long range side-chain-to-side-chain hydrogen bonds between helices. d) Phe3 and Trp7 side-chains (sticks) are peripheral to the helical interface

Figure 4

Analysis of crystallographic symmetry. In all panels, each helix is represented with the same color (chain A = green, chain B = blue, chain C = orange, chain D = pink) and disulfide bonds are represented as yellow sticks. N- and C-termini are labeled as N and C, respectively, with subscripts to indicate the corresponding chain. a) An overlay of the two copies of the dimer observed in the asymmetric unit shows high similarity (RMSD = 0.28 Å for all backbone atoms). b) The dimer of dimers is antiparallel with close contact between chains B (blue) and C (orange) and a wider interface between chains A (green) and D (pink). c) Phe3 residues (brown) cap an extended hydrophobic core formed primarily by Phe3, Leu6, Trp7, and Leu10 (spheres). d) Trp7 residues form two different arrangements. Between chains B (blue) and C (orange), Trp7 residues are separated by approximately 3.2 Å in a parallel displaced stacking arrangement. Between chains A (green) and D (pink), Trp7 residues are not interacting but pack in a parallel fashion against other hydrophobic core residues. e) The crystallographic octamer (dimer of dimer of dimers) is formed by extensive hydrophobic and π-π interactions between Trp7 and Leu10 from chains A (green) and D (pink) in adjacent dimer of dimers