High-Resolution X-ray Structure of the Unexpectedly Stable Dimer of the [Lys^(-2)-Arg^(-1)-des(17−21)]Endothelin-1 Peptide

Abstract

Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable. The KR-CSH-ET peptide has now been crystallized at pH 5.0 and its high-resolution structure determined ab initio at 1.13 A using direct methods. Unexpectedly, KR-CSH-ET was shown to be a head-to-tail symmetric dimer, and the overall interface involves two intermolecular R(-1)-D(8) salt bridges, a two-stranded antiparallel beta-sheet, and hydrophobic contacts. Molecular dynamics simulations carried out on this dimer clearly showed that the two intermolecular salt bridges were in this case very stable. Sedimentation equilibrium experiments unambiguously confirmed that KR-ET-1 and KR-CSH-ET also exist as dimers in solution at pH 5.0. On the basis of the new dimeric structure, previous NMR data were reinterpreted. Structure calculations were performed using 484 intramolecular and 38 intermolecular NMR-derived constraints. The solution and the X-ray structures of the dimer are very similar (mean rmsd of 0.85 A). Since the KR dipeptide at the N-terminus of KR-CSH-ET is present in the prosequence, it can be hypothesized that similar intermolecular salt bridges could be involved in the in vivo formation of the native disulfide bonds of ET-1. Therefore, it appears to be likely that the prosequence does assist the ET-1 folding in a chaperone-like manner before successive cleavages that yield the bioactive ET-1 hormone.

A virtual-system coupled multicanonical molecular dynamics simulation: Principles and applications to free-energy landscape of protein–protein interaction with an all-atom model in explicit solvent

“…In the obtained free-energy landscape, the most stable complex at 300 K sampled from V-McMD was the same as the X-ray complex structure. 44 We also found some minor complex forms. V-McMD has shown faster convergence than McMD does.…”

“…Therefore, we were able to quit the simulation up to the tenth V-McMD. The N cnt for the native complex 44 is 128. Later we demonstrate that the lowest free-energy region of the free-energy landscape at 300 K converged to the native complex.…”

“…Two intramolecular disulfide bonds ( 3 Cys- 17 Cys and 5 Cys-13 Cys) stabilize the CSH motif. 41,42 The X-ray structure 44 shows that the homod- imer complex of KR-CSH-ET1 is stabilized by intermolecular interactions of three types: two salt bridges, phenylalanine stacking (i.e., hydrophobic core) at the interface, and a β-sheet including the strands in the CSH motif.…”

“…43 X-ray crystallography has shown that this molecule forms a homodimer, where the orientations of the two molecules are well packed with an antisymmetry mutually (PDBID; 1t7h). 44 In this X-ray crystallography, five amino-acid residues at the C-terminal of KR-ET1 were removed because these residues are presumably disordered and exposed in solution. This truncated peptide, denoted as KR-CSH-ET1, is an appropriate target to assess the current sampling method V-McMD because this peptide is small and because the complex formation is well monitored using a degree of the antisymmetry.…”

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

“…In the study of Hoh et al (2004), the target for TTP-V-McMD was a bioactive peptide, Endothelin-1 derivative (KR-CSH-ET1), which forms a stable antisymmetric homodimer (Fig. 5a).…”