Atomic structure of Lanreotide nanotubes revealed by cryo-EM

Abstract

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Keywords: helical polymers; peptide assemblies; three-dimensional reconstruction.

Fig. 1.

Crystal structure of the Lanreotide derivative Lan-dap5. Schematic representation of the crystalline stack formed by successive monolayers of peptides rotated by 60° with respect to each other: (A) top and (B) side views. Details of the peptide monolayers: (C and E) top views and (D) side view. (C and D) Superposition of three monolayers of peptides. The peptides in the middle layer are shown in a stick representation, while just the peptide backbones are shown for the surrounding layers. (E) Detailed structure of one peptide layer. Molecules a_x (magenta) and b_x (green) are stacked head-to-tail in the asymmetric unit of the crystal. The blue ellipsoid highlights the close proximity of diaminopropionic acid side chains (replacing Lys in Lan-dap5) between adjacent β-sheets. (F) The molecular packing proposed in 2003 for the external peptide layer of the lanreotide nanotube wall. The putative Lys-Lys close contacts between adjacent β-sheets are highlighted by the blue ellipsoids (adapted from ref. 10). (G) superposition of the two conformations a_x (magenta) and b_x (green) of Lan-dap5 present in the asymmetric unit.

Fig. 2.

Cryo-EM: analysis, density map and hand determination. (A) Cryo-EM image of Lanreotide nanotubes. (Scale bar, 50 nm.) (B) One representative 2D class average. (Scale bar, 10 nm.) (C) Close-up of the density map to show its quality. (D) Density maps for the two main conformations of Lanreotide within the helical asymmetric unit that differ by the position of the DNah side chains; that is, the capped molecule (orange carbons) and the away molecule (cyan carbons) (see SI Appendix, Fig. S3 for the density map of the asymmetric unit). (E) Determination of the correct hand for the structure of the nanotubes. Part of the Lanreotide model [Cys₂-Tyr₃-(D)-Trp₄ of a single molecule] is displayed after fitting into the correct map hand (Left) and into the inverse map hand (Right). Note the poor fit around the D-Trp₄ C_α in the inverse hand case, particularly for the carbonyl, C_β and side chain. In D and E, the map is displayed only around the selected atoms for clarity. (F) A further determination of the correct hand of the cryo-EM map can be made from examining the pattern of backbone hydrogen bonds. A single peptide is displayed after real-space refinement into the correct map hand (Upper) and into the mirrored map with the wrong hand (Lower). All of the expected antiparallel β-sheet hydrogen bonds only exist in the map with the correct hand.

Fig. 3.

Structure of Lanreotide nanotubes: (A) The density map for the tube, with a region outlined in the center. (B and C) Top (B) and side (C) views of the outlined region in A showing the density map together with the atomic model for the helical asymmetric unit. The eight molecules of the asymmetric unit are clustered into two main conformations: molecules a_c, b_c, g_c, and h_c (in orange-yellow) are capped and molecules c_a, d_a, e_a, and f_a (in cyan-green) are away. (D) Superposition of the eight conformations of Lanreotide found in the capped (orange) and the away (cyan) families of molecules present in the asymmetric unit. (E) The different environments for the eight molecules within the asymmetric unit. For clarity, the centers of the two tetramers of an asymmetric unit are highlighted with light gray circles. (F–H) Main-chain H-bond networks as displayed by Pymol polar contacts with default parameters. H-bonds in antiparallel β-sheets are shown as blue dotted lines and in parallel β-sheets as magenta dotted lines. (F) Networks as seen from outside the tube in the same orientation as A. One asymmetric unit is displayed as spheres and the molecules around it only by their backbones. The two black dotted lines indicate the two β-sheets shown in side views in G and H. (G) Side view of the β-sheet formed by molecules a_c, d_a, g_c, and f_a. (H) Side view of the β-sheet formed by molecules c_a, b_c, e_a, and h_c. (I) superposition of molecule a_c (orange) and c_a (cyan) of the asymmetric unit of the nanotubes with one of the two Lan-dap5 conformations a_x found in the crystal (magenta). The DTrp and the Tyr of the molecules forming the nanotubes are at 180° (DTrp) and 120° (Tyr) with respect to the same residues of Lanreotide in the crystal. For Nah, whereas the two conformers of Lanreotide in the nanotubes mostly differ are the level of the C_α-CH₂, (i.e., side chain conformation), the difference with the crystal is at the level of the backbone between the Carbonyl-C_α.

Fig. 4.

Role of the side chains in the nanotube structure. (A) Three side views of the nanotube with either all the residues (Top), the aromatic residues only (Middle), or the aliphatic residues only (Bottom) shown. (B) Close-up of the nanotube wall: from the top to the bottom are highlighted Lys (gray blue), D-Trp (royal blue), Nah (yellow), Thr (spring green), Cys (green), Tyr (salmon), and Val (orange) residues. (C) Close-up of the D-Trp-Lys (Top), the Val cluster (Middle), and the aromatic stacking (Bottom).