Crystal Structure of Hyp-1, a Hypericum perforatum PR-10 Protein, in Complex with Melatonin

Abstract

Hyp-1, a PR-10-fold protein from Hypericum perforatum, was crystallized in complex with melatonin (MEL). The structure confirms the conserved protein fold and the presence of three unusual ligand binding sites, two of which are internal chambers (1,2), while the third one (3) is formed as an invagination of the protein surface. The MEL ligand in site 1 is well defined while that in site 3 seems to be rotating between the side chains of Lys33 and Tyr150 that act as a molecular vise. The patch of electron density in site 2 does not allow unambiguous modeling of a melatonin molecule but suggests a possible presence of its degradation product. This pattern of ligand occupation is reproducible in repeated crystallization/structure determination experiments. Although the binding of melatonin by Hyp-1 does not appear to be very strong (for example, MEL cannot displace the artificial fluorescence probe ANS), it is strong enough to suggest a physiological role of this interaction. For example, trans-zeatin, which is a common ligand of PR-10 proteins, does not overcompete melatonin for binding to Hyp-1 as it does not affect the crystallization process of the Hyp-1/MEL complex, and among a number of potential natural mediators tested, melatonin was the only one to form a crystalline complex with Hyp-1 with the use of standard crystallization screens. Hyp-1 is the second protein in the Protein Data Bank for which melatonin binding has been demonstrated crystallographically, the first one being human quinone reductase.

PR‐10 proteins as potential mediators of melatonin‐cytokinin cross‐talk in plants: crystallographic studies of LlPR‐10.2B isoform from yellow lupine

“…We also tested the behavior of samples of pure melatonin subjected to high doses of X‐ray radiation. As reported previously , the 1 H NMR spectra of melatonin irradiated with X‐ray doses up to 10 times higher than those absorbed during a diffraction experiment, showed no indication of any chemical change.…”

“…However, it was not possible to model the latter ligand with reasonable occupancy at this site, even though the UNL electron density overlaps the ZEA2 and ZEA4 sites of the Z complex . The UNL electron density in structure M is bigger and better defined than the patch of unidentified electron density reported in the Hyp‐1/MEL complex (PDB ID , ), which in addition has a different location in the protein cavity, as described in the section “Comparison of melatonin complexes of LlPR‐10.2B and Hyp‐1”.…”

“…It is of special note that when screening for complex formation of two other yellow lupine PR‐10 isoforms representing subclass 1, namely LlPR‐10.1A and LlPR‐10.1B, with the same repertoire of ligands (except 2HM, SER, MeJa, and VIT), we could only obtain a crystalline complex of LlPR‐10.1A with ZEA and only the ligand‐free form of LlPR‐10.1B. On the other hand, another PR‐10 member, the Hyp‐1 protein from H. perforatum screened against the same repertoire of ligands (except DPU, CCPU, 2HM, VIT, and NAR, but extended by emodin and hypericin) produced a crystalline complex with MEL only, although it did form a crystalline complex with the artificial fluorescent dye ANS (8‐anilino‐naphthalene‐1‐sulfonate) .…”

“…The PR‐10 cavity can be shallow (type I), capable of specific binding of usually one ligand molecule, as found in PR‐10‐fold proteins with regulatory function (e.g., PhBP, major latex proteins, abscisic acid receptors, or MtN13), or it can be extended (type II) and capable of unspecific binding of more than two ligand molecules (thus suggesting transport/storage functions), as found in classic PR‐10 proteins . Moreover, in the case of Hyp‐1, a PR‐10 protein from St John's wort ( Hypericum perforatum ), it has been shown that the cavity (type III) is in fact divided into three separated sites capable of binding chemically diversified ligands in well conserved positions .…”

Beyond a neurotransmitter: The role of serotonin in plants

“…To date a single plant protein has been identified in complex with melatonin, Hyp-1, a putative pathogenesis related PR-10 type protein from H. perforatum [91] . Though serotonin was not examined in this study, this receptor subtype does represent a potential candidate class.…”