Crystal and NMR structures of a Trp-cage mini-protein benchmark for computational fold prediction

Abstract

To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior. A hexameric oligomer of the cyclic peptide is found in both crystal forms and indicates that under appropriate conditions, this minimized system may also serve as a model for protein-protein interactions.protein-protein interaction | folding simulation target | protein cyclization | peptide oligomerization | H-bonding motifs T rp-cage miniproteins (at 18-20 residue length) are among the smallest peptides to fold into a stable protein-like structure (1, 2); as a result, the Trp-cage fold has emerged as a protein folding paradigm (3, 4). Trp-cage species have been extensively studied experimentally (4-11) and computationally (12-19), but the conclusions regarding the folding process and results drawn from these studies are not in accord at the level that should be attainable for such a small system.The structural model used for testing computational folding methods has, in almost all cases, been that derived by NMR for the initially reported Trp-cage (TC5b), which displays a rather low fold stability, T m ¼ 43°C (1). Additional Trp-cage structures based on NMR measurements are now available (2,6,10,(20)(21)(22). All of these appear to possess a common fold topology that is supported by an extensive set of structuring-induced shifts (CSDs, chemical shift deviations from random coil norms) that are largely the result of ring current effects. The uniformity of these CSDs suggests a well-defined common structure. However, other spectroscopic measurements of Trp-cage molecules have been interpreted as being consistent with a folded structure undergoing a greater degree of dynamic fluctuation (7,8). In addition, "folded" structures from molecular dynamics simulations, with a few notable exceptions (18,19), fail to predict the ring current effects observed by NMR (2). The failure of numerous calculations to reproduce the NMR structures and contrary conclusions from other experimental methods is problematic and points to outstanding issues concerning the continuing* use of a Trp-cage as a benchmark for molecular dynamics folding simulations. Further structural studies of Trp-cage species are required to validate the target and to ascertain the key interatomic interactions leading to the folded state.Herein we report X-ray structure determinations for two crystal forms of a cyclized Trp-cage (23), cyclo-TC1 (-GDAYAQW-LADGGPSSGRPPPSG-). Raising additional questions about the oligomeric state of the molec...

Folding Dynamics and Pathways of the Trp-Cage Miniproteins

“…In contrast, the large upfield shifts at the P18Hα (CSD = −2.33) and P18Hβ3 (CSD = −2.14 ppm, at 280 K), based on the shifts observed in a hyperstable cyclic construct 14,23 appeared to be superior measures of full cage formation. These appear only on complete cage formation; the P18Hα signal is usually overlapped with other peaks, but the P18Hβ3 signal appears far upfield with no other signals nearby.…”

Structure of the Extracellular Domain of Matrix Protein 2 of Influenza A Virus in Complex with a Protective Monoclonal Antibody

“…Hydrophobic interactions are formed between the indole ring of M2e-Trp15 and the aliphatic parts of M2e-Glu6 and M2e-Arg12. The central position of M2e-Trp15 in M2e resembles the positioning of tryptophan in so-called Trp cages, which fold into stable miniproteins (40). As in Trp cages, the indole ring nitrogen of M2e-Trp15 hydrogen bonds with the carbonyl oxygen of M2e-Thr9, and a network of other hydrogen bonds between main-chain carbonyl oxygens and nitrogens of M2e confines M2e-Trp15 in a central position (Fig.…”

Aryl–aryl interactions in designed peptide folds: Spectroscopic characteristics and optimal placement for structure stabilization

“…The common, rigid fold of sequence-optimized, e.g. DAYAQ WLADX GPASX RPPPS, X = Gly (TC13b) or D-Ala (TC16b), Trp-cage species has been established by numerous NMR structures and two crystal structures 41 and is stable enough to allow substitutions at L7, P12 and P18, as well as swapping Y3 for a Phe or Trp residue. This allowed us to examine the effects of potential W/W as well as Y/W cluster geometries upon the CD spectrum of the Trp-cage.…”