Journal:Acta Cryst D:S2059798320006841
From Proteopedia
(Difference between revisions)
| Line 5: | Line 5: | ||
<b>Molecular Tour</b><br> | <b>Molecular Tour</b><br> | ||
Hyp-1 overall structure and function | Hyp-1 overall structure and function | ||
| - | Hyp-1 from St John’s wort (''Hypericum perforatum'') belongs to pathogenesis-related proteins of class 10 (PR-10). These ubiquitous plant proteins are expressed under stressful conditions, such as salinity, drought or pathogen invasion <ref name="Sliwiak">PMID:24531481</ref>. St. John’s wort is an important herb, widely used for millennia in folk medicine for the treatment of various ailments <ref name="Sliwiak1">PMID:25849394</ref>. PR-10 proteins are among the most intriguing plant proteins because of the difficulty of assigning to them a unique biological function, despite their abundance, coexistence of many isoforms in one plant, and comprehensive structural characterization. The characteristic PR-10 fold consists of a seven-stranded antiparallel β-sheets wrapped around a long C-terminal helix α3, which rests on a V-shaped fork of two additional short helices α1 and α2. A progression of β-hairpins and loops connect the β-strands, except strands β1 and β2 which form the opposite edges of the β-sheet <ref name="Michalska">PMID:19853038</ref>. The PR-10 fold creates a large hydrophobic cavity between the β-sheet and helix α3 that is suggestive of a ligand binding function. A number of hydrophobic mediators have been already confirmed as PR-10 partners, most notably phytohormones such as cytokinins. Such complexes are usually studied using ANS Displacement Assays (ADA), in which the fluorescent dye 8-anilino-1-naphthalene sulfonate (ANS) is displaced by the ligand of interest | + | Hyp-1 from St John’s wort (''Hypericum perforatum'') belongs to pathogenesis-related proteins of class 10 (PR-10). These ubiquitous plant proteins are expressed under stressful conditions, such as salinity, drought or pathogen invasion <ref name="Sliwiak">PMID:24531481</ref>. St. John’s wort is an important herb, widely used for millennia in folk medicine for the treatment of various ailments <ref name="Sliwiak1">PMID:25849394</ref>. PR-10 proteins are among the most intriguing plant proteins because of the difficulty of assigning to them a unique biological function, despite their abundance, coexistence of many isoforms in one plant, and comprehensive structural characterization. The characteristic PR-10 fold consists of a seven-stranded antiparallel β-sheets wrapped around a long C-terminal helix α3, which rests on a V-shaped fork of two additional short helices α1 and α2. A progression of β-hairpins and loops connect the β-strands, except strands β1 and β2 which form the opposite edges of the β-sheet <ref name="Michalska">PMID:19853038</ref>. The PR-10 fold creates a large hydrophobic cavity between the β-sheet and helix α3 that is suggestive of a ligand binding function. A number of hydrophobic mediators have been already confirmed as PR-10 partners, most notably phytohormones such as cytokinins. Such complexes are usually studied using ANS Displacement Assays (ADA), in which the fluorescent dye 8-anilino-1-naphthalene sulfonate (ANS) is displaced by the ligand of interest <ref name="Gasymov">PMID:17321809</ref>. In order to elucidate the molecular basis of this method we crystallized Hyp-1 in complex with ANS. Our first crystal turned out to be tetartohedrally twinned and have a modulated superstructure, which was interpreted in a sevenfold expanded unit cell containing 28 Hyp-1 molecules and 89 ANS ligands <ref name="Sliwiak">PMID:24531481</ref><ref name="Sliwiak1">PMID:25849394</ref>. In the present study, we co-crystallized Hyp-1 with a mixture of ANS and melatonin, which was recently found to be a PR-10 ligand for cross-talk with other phytohormones [5]. To our surprise, melatonin was unable to displace ANS and instead we obtained a crystal of Hyp-1/ANS complex with an even more daunting modulation: 36 Hyp-1 and 156 ANS molecules in a ninefold expanded unit cell. |
The phenomenon of superstructure modulation | The phenomenon of superstructure modulation | ||
Superstructure modulation is a crystal abnormality, where exact periodicity of the unit cells is violated, and the wave of distortion has a long period (running over many unit cells), which can be commensurate or incommensurate with the basic lattice periodicity. In reciprocal space, this phenomenon is manifested by the presence of weaker satellites dividing the spaces between the main Bragg reflections, according to a modulation vector q [6]. Superstructure modulation is well known in small-molecule crystallography but in macromolecular crystallography it is practically unheard of [7]. The first successful structure solution and refinement of a modulated protein crystal structure was presented for the Hyp-1/ANS with protein with sevenfold expansion of the ''C''2 unit cell <ref name="Sliwiak">PMID:24531481</ref><ref name="Sliwiak1">PMID:25849394</ref>. Here, we present the crystal structure of the same complex but with ninefold commensurate modulation. The asymmetric unit contains 36 independent Hyp-1 molecules and 156 ANS ligands. 95 ANS molecules occupy (with variable frequency) three binding sites of the Hyp-1 molecule, two of which are located within the familiar hydrophobic cavity, while the third one is formed as a deep surface invagination. The remaining 61 ANS ligands are bound on protein surfaces, gluing together neighboring protein molecules and generating the bizarre pattern of crystal packing. | Superstructure modulation is a crystal abnormality, where exact periodicity of the unit cells is violated, and the wave of distortion has a long period (running over many unit cells), which can be commensurate or incommensurate with the basic lattice periodicity. In reciprocal space, this phenomenon is manifested by the presence of weaker satellites dividing the spaces between the main Bragg reflections, according to a modulation vector q [6]. Superstructure modulation is well known in small-molecule crystallography but in macromolecular crystallography it is practically unheard of [7]. The first successful structure solution and refinement of a modulated protein crystal structure was presented for the Hyp-1/ANS with protein with sevenfold expansion of the ''C''2 unit cell <ref name="Sliwiak">PMID:24531481</ref><ref name="Sliwiak1">PMID:25849394</ref>. Here, we present the crystal structure of the same complex but with ninefold commensurate modulation. The asymmetric unit contains 36 independent Hyp-1 molecules and 156 ANS ligands. 95 ANS molecules occupy (with variable frequency) three binding sites of the Hyp-1 molecule, two of which are located within the familiar hydrophobic cavity, while the third one is formed as a deep surface invagination. The remaining 61 ANS ligands are bound on protein surfaces, gluing together neighboring protein molecules and generating the bizarre pattern of crystal packing. | ||
| Line 14: | Line 14: | ||
There are 61 ANS molecules bound on the surface of the Hyp-1 molecules, which are most likely the generator of superstructure modulation. | There are 61 ANS molecules bound on the surface of the Hyp-1 molecules, which are most likely the generator of superstructure modulation. | ||
| - | [4] Gasymov, O. K. & Glasgow, B. J., ANS fluorescence: potential to augment the identification of the external binding sites of proteins, Biochim. Biophys. Acta. (2007) 1774, 403-411. | ||
[5] Sliwiak, J., Sikorski, M. & Jaskolski, M., PR-10 proteins as potential mediators of melatonin-cytokinin cross-talk in plants: crystallographic studies of LlPR-10.2B isoform from yellow lupine, FEBS J. 10 (2018), 1907-1922. | [5] Sliwiak, J., Sikorski, M. & Jaskolski, M., PR-10 proteins as potential mediators of melatonin-cytokinin cross-talk in plants: crystallographic studies of LlPR-10.2B isoform from yellow lupine, FEBS J. 10 (2018), 1907-1922. | ||
[6] Smaalen, S. van (2007). Incommensurate Crystallography, Oxford University Press. | [6] Smaalen, S. van (2007). Incommensurate Crystallography, Oxford University Press. | ||
Revision as of 14:02, 31 May 2020
| |||||||||||
This page complements a publication in scientific journals and is one of the Proteopedia's Interactive 3D Complement pages. For aditional details please see I3DC.
