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, 3 (Pt 4), 282-93
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Structure of a Heterogeneous, Glycosylated, Lipid-Bound, in Vivo-Grown Protein Crystal at Atomic Resolution From the Viviparous Cockroach Diploptera Punctata

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Structure of a Heterogeneous, Glycosylated, Lipid-Bound, in Vivo-Grown Protein Crystal at Atomic Resolution From the Viviparous Cockroach Diploptera Punctata

Sanchari Banerjee et al. IUCrJ.

Abstract

Macromolecular crystals for X-ray diffraction studies are typically grown in vitro from pure and homogeneous samples; however, there are examples of protein crystals that have been identified in vivo. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown in cellulo. Here, an atomic resolution (1.2 Å) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These in vivo-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, Diploptera punctata. The milk proteins crystallized in space group P1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single in vivo-grown crystal of a natural protein in its native environment at atomic resolution.

Keywords: glycosylation; protein heterogeneity; sulfur-SAD; viviparity in cockroach.

Figures

Figure 1
Figure 1
In vivo-grown Lili-Mip crystals from D. punctata. Polarized microscopy reveals birefringent protein crystals enclosed inside the embryo midgut and an enlarged view of the extracted crystals (inset).
Figure 2
Figure 2
Crystal structure of Lili-Mip. (a) Cartoon diagram of the Lili-Mip structure consisting of one C-terminal α-helix (light blue) and nine β-strands (magenta) that form a barrel to loosely coordinate the lipid. The N-glycans (yellow) at the four glycosylation sites are modelled in 2F oF c electron density (white). (b) Surface view of Lili-Mip showing the 2F oF c electron-density map (blue) contoured at 1× r.m.s. for the N-glycans at Asn35, Asn79 and Asn145, and 0.5σ for that at Asn66. The wire mesh (green) in the middle of the structures in both panels is the difference map showing density for the lipid drawn at 3.0σ.
Figure 3
Figure 3
Lipid binding to Lili-Mip. (a) Close-up view of the interface between the lipids (linoleic acid, purple; oleic acid, yellow) and the hydrophobic cavity. Residues involved in the formation of the cavity are modelled and labelled. The F oF c electron-density map (drawn at 3.0σ) for the lipids in the binding cavity is shown in green. As mentioned in the text, the last few C atoms and the charged group are disordered and different in the different structures. The electron-density map depicted is using data from PDB entry 4nyq. (b) Two-dimensional projection of lipid coordination by Lili-Mip residues: LIGPLOT diagram (Wallace et al., 1995 ▸). Atoms of the lipid are labelled in black and Lili-Mip residues are shown in red. The direction of the hydrophobic interactions between each atom of the lipid and Lili-Mip is represented.
Figure 4
Figure 4
2F oF c (white) and F oF c (green) electron-density maps for three residues where heterogeneity is observed by crystallography and mass spectrometry. All 2F oF c maps are contoured at 1 × r.m.s. values. (a) Residue 12 is Pro in Lili-Mip 1 and Thr in Lili-Mip 2. The difference map (green) is at the 3σ level. (b) Residue 39 is Val in Lili-Mip 1 and Phe in Lili-Mip 2. The difference map (green) is at the 2σ level in the first panel to show the complete ring of Phe. (c) Residue 50 is Asn in Lili-Mip 1 and Thr in Lili-Mip 2. The difference map (green) is contoured at the 3σ level. In all three figures, panel 1 shows the additional densities after refining only the Lili-Mip 1 sequence (residues in yellow) and panel 2 after refining only the Lili-Mip 2 sequence (residues in orange). Panel 3 shows that after refining with both Lili-Mip 1 and 2 sequences, no additional densities are observed. The electron-density map depicted is using data from PDB entry 4nyq.
Figure 5
Figure 5
Crystal packing in Lili-Mip. (a) The arrangement of molecules in a plane. Each molecule is surrounded by six other molecules. (b) Overall crystal packing showing a sheath within a cylinder arrangement. (c) The π–π stacking interaction between Tyr153 and Tyr142 of two neighbouring molecules. (d) The C-­terminal helix interaction with a groove formed by the loops and β-strand in the opening of the ligand-binding site through a salt bridge between Lys131 and Glu61. (e) The third interacting region between neighbouring molecules through hydrogen bonds between Asn45 and Ser109 as well as Arg14 and Gln32.
Figure 6
Figure 6
Porcupine plots showing relative motions of the four regions in the deglycosylated models of (a) native, (b) oleic acid-bound and (c) linoleic acid-bound Lili-Mip. Regions I, II, III and IV are coloured blue, red, green and orange, respectively.

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