Date Published: July 01, 2020
Publisher: International Union of Crystallography
Author(s): Joanna Smietanska, Joanna Sliwiak, Miroslaw Gilski, Zbigniew Dauter, Radoslaw Strzalka, Janusz Wolny, Mariusz Jaskolski.
When crystallized in complex with the fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) in the presence of melatonin, Hyp-1, a pathogenesis-related class 10 protein from Hypericum perforatum, produced tetartohedrally twinned C2 crystals with commensurate structure modulation, which was interpreted as a ninefold expansion of the unit cell in the c direction. The asymmetric unit of this supercell contains 36 protein molecules (differently populated by 156 ANS ligands) arranged into columns by a combination of ninefold translational noncrystallographic symmetry and pseudotetragonal rotational NCS.
Crystal structure modulation (also known as superstructure) is classified with other aperiodic phenomena as it consists of the violation of short-range unit-cell-to-unit-cell periodicity, which over the long range is regained by a wave of structural deformations, described by an atomic modulation function (AMF). In reciprocal space, this structural phenomenon is manifested by the presence of main reflections (usually stronger but also affected by the modulation) arising from the (approximately periodic) main lattice of the basic unit cells and of (usually weaker) satellite reflections arising from the periodic AMF. Since in real space the period of the AMF is longer than the corresponding period of the basic lattice, in reciprocal space the satellites subdivide the wider main-to-main distances. The situation is relatively simple, and the phenomenon is termed commensurate modulation, if the period of the AMF is an integral multiple of the corresponding period of the basic lattice. The structure may be then described, not quite elegantly but efficiently, in an expanded unit cell called a supercell. In the more general incommensurate case such a trick is not possible and the mathematical solution lies in the concept of superspace, as introduced by Janner, Janssen and de Wolff (Janner & Janssen, 1977 ▸; de Wolff, 1974 ▸), which makes explicit use of the AMF function. The phenomenon of superstructure modulation has been relatively well studied in small-molecule crystallography (Schönleber, 2011 ▸), but is very rarely mentioned in the context of macromolecular crystals. Superspace theory and its application by Sander van Smaalen (van Smaalen, 2004 ▸, 2007 ▸) inspired work on symmetry analysis of the diffraction from (3 + 1)-dimensionally incommensurately modulated crystals of profilin–actin (PA; Lovelace et al., 2004 ▸; Porta et al., 2011 ▸). The solution and refinement of 11 twinned and incommensurately modulated structures of engineered variants of the Escherichia coli enzyme N-acetylneuraminic acid lyase have been reported by Ivan Campeotto (Campeotto et al., 2018 ▸). Recent years brought promising results in assigning a (3 + 1)-dimensional superspace group to an incommensurately modulated protein crystal (Porta et al., 2017 ▸), as well as in solving and refining a model structure using a supercell approximation (Lovelace et al., 2013 ▸, 2019 ▸). Jeffrey Lovelace and Gloria Borgstahl have given an excellent overview of the available methods for solving problematic macromolecular crystal structures marked by order–disorder phenomena and positional modulation (Lovelace & Borgstahl, 2020 ▸).
Previous studies of Hyp-1, a PR-10 protein from H. perforatum, revealed that in complex with the fluorescent dye ANS (8-anilinonaphthalene-1-sulfonate) it forms tetartohedrally twinned crystals (7Hyp/ANS) with sevenfold commensurate modulation along the c direction of the C2 supercell. Pre-incubation of the Hyp-1 protein with an ANS–melatonin mixture and further crystallization trials using the final crystal-growth conditions for the 7Hyp/ANS complex resulted in a different tetartohedrally twinned C2 crystal form (9Hyp/ANS) with ninefold noncrystallographic repetition of the same structural motif (two Hyp-1 dimers) along c as well as noncrystallographic translational perturbations along the a and b directions. A physical manifestation of this superstructure modulation is the fluctuation of the diffraction-pattern intensity, with strong reflections for the l index of 9n and also in between at l = 9n ± 4. In structural terms, interpretation of this crystal structure as commensurately modulated requires as many as 36 protein molecules in the asymmetric part of an expanded unit cell (supercell) for its description. The present 9Hyp/ANS structure with ninefold modulation was solved by MR using a modified Phaser algorithm that takes account of the effects of translational and rotational noncrystallographic symmetry, using the previous 7Hyp/ANS model as a probe. The S(H) and L-tests suggested at least partial crystal twinning, despite the fact that the detection of twinning is obliterated by translational NCS (tNCS). The twinning was ultimately confirmed (with the four twinning fractions refined at ∼0.25) by successful refinement of the superstructure with REFMAC, which converged with an R and Rfree of 0.226 and 0.257, respectively, for the diffraction data extending to 2.3 Å resolution. Each Hyp-1 molecule harbors three internal ligand-binding sites, which are variously populated by 95 ANS ligands in the 36 copies of the protein. In addition, there are 61 ANS molecules bound on the surface of the Hyp-1 molecules, which are most likely to be the generator of superstructure modulation. Furthermore, other buffer-solution molecules (sulfate, HEPES, citrate and dimethyl sulfoxide) were found in the structure at selected ligand-binding sites that are typically reserved for ANS. This phenomenon of ligand substitution at what appears to be a random selection of binding sites has not been observed in Hyp-1 complexes before. Although the presence of the single melatonin molecule (which was the ligand of choice for this crystallization experiment) proved to be impossible to maintain after analysis using polder maps, this mysterious area of electron density, which was pragmatically modeled as an unidentified ligand marked by several water molecules, may correspond after all to a poorly ordered or partially degraded melatonin molecule, although its location in binding site 7 does not correspond to the melatonin binding sites (1, 2 and 3) known from a Hyp-1–MEL complex structure, but rather overlaps with a typical interstitial ANS site. Since in the present study slightly modified crystallization conditions (the addition of melatonin to the incubation mixture with ANS) triggered a new type of structure modulation, it is quite possible that other superstructure modulation variants of Hyp-1 complexes could be engineered using different melatonin (or other additive) concentrations.