大家好，今天推送的文章是2022年11月发表在 Acta crystallographica. Section F, Structural biology communications 上的 The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a ‘spanner in the works’，通讯作者为新西兰坎特伯雷大学物理与化学科学学院生物分子相互作用中心的 Jodie M. Johnston。
Purine nucleoside phosphorylase (PNPase) is an enzyme found in the purine rescue pathway in some organisms that reversibly phospholyzes nucleosides to form ribose 1-phosphate and purine bases (Figure 1). PNPase has two main structural forms: a trimeric form, mainly found in eukaryotes, and a hexameric form ('trimer-of-dimers'), mainly found in bacteria. The trimeric and hexamer forms of PNPase have similar subunit structures, i.e., α/β folds, and their main difference is substrate specificity, most of the trimeric PNPase substrates studied are limited to 6-oxopurine ribonucleoside, while hexamer PNPase can use 6-oxopurine ribonucleoside and 6-aminopurine ribonucleoside as substrates. While the hexamer form is predominantly found in bacteria, many bacteria have both forms encoded in their genomes, including Geobacillus stearistophilus, which is a gram-positive thermophilic bacterium and a common cause of food spoilage.
PNPase is a potential antimicrobial drug target and an industrial catalyst for the production of antiviral nucleoside compounds. The substrate promiscibility of PNPase is relevant for the latter application, as the wider range of purine bases that PNPase can accommodate, the wider the range of antiviral nucleoside candidates it can use for production. Due to the limited solubility of many potential purine bases at ambient temperatures, PNPases from thermophilic organisms such as Bacillus thermophilus can be used at higher temperatures, improving substrate solubility, allowing for a wider range of production of these compounds. The authors resolved the structure of the N-terminal His-tagged PNPase (His-GsePNPase) of Bacillus thermophilus with a resolution of 1.72 Å.
Figure 1
1
Structure of His-GsePNPase
His-GsePNPase crystallized in space group P43212 in the absence of ligands with a diffraction resolution of 1.72 Å. Three strands (a-c) are present in the asymmetric unit, from which hexamer bioassemblies can be generated by applying crystallographic symmetry manipulations (Figure 2A). The hexamer structure shows triple symmetry by ring hexamers, and His-GsePNPase can be considered as trimers of dimers (a/b, a0/b0, and c/c0), like other hexamer PNPases. The observed electron density of each strand is continuous except for residues 209-210 in chain c and the part of the N-terminal tag.
Subunit folding analysis showed that HisGsePNPase was structurally similar to other hexamer PNPases, with the His-GsePNPase subunit structure having a conserved folding, and its structure was most similar to that of H. pylori PNPase and well-studied EcPNPase, with a root mean square standard deviation (r.m.s.d.) of 0.42–0.51 Å. The subunits of HisGsePNPase are similar to each other, with r.m.s.d. of 0.136–0.160 Å for pairwise comparison. Each subunit consists of seven β-helices (H1-H7) and two β-folds (Figure 2B). The first β-sheet (S1–S4) has four chains, where S3 is antiparallel, and the second sheet forms a sheet barrel roll (S5-S10), where S5 and S10 are antiparallel. According to the analysis in SCOP, HisGsePNPase is part of the phosphorylase/hydrolase-like folding superfamily, as are other PNPases.
Figure 2
2
His-GsePNPase 和 EcPNPase
High degree of conservation between active sites
The PNPase active site is formed near the interface between two subunits (e.g., subunits a and b; Figure 3a), and there are six active sites in the hexamer. The active site is mainly formed by the residues of one subunit, and the residues of the other subunit in each dimer pair also contribute (Figure 3B). There is a high degree of sequence and structural conservation between the active sites of EcPNPase and HisGsePNPase. In EcPNPase, residues that interact with purine bases are identified as Ala156, Phe159, Val178, Met180, Asp204, and Ile206, which are equivalent to Ala156, Phe159, Val177, Met179, Asp203, and Ile205 in His-GsePNPase (Figure 3b). The ribose binding site in EcPNPase is primarily formed by interaction with Glu181 of one subunit and His4 of adjacent subunits, which are equivalent to Glu180 and His4 in His-GsePNPase, respectively (Figure 3b). The phospho-binding site of EcPNPase consists of two arginine residues, Arg87 in one subunit and Arg43 in the adjacent subunit, which are also conserved in HisGsePNPase (Figure 3b).
Figure 3
3
The N-terminal tag of the adjacent subunit occupies the active site
A surprising aspect regarding the structure of His-GsePNPase is the presence of several residue (NLYFQ) N-terminal tags from the nine residue rTEV site (ENLYFQGAM) in the active site. This portion of the rTEV site was observed to bind to the active site of the adjacent subunit in each dimer (Figure 3a). The interaction between the active site and the N-terminal tag is dominated by the Tyr residue in the rTEV recognition site. This residue is oriented in a manner similar to the purine-base portion of the substrate (Figure 3C). The similarity of this binding posture suggests that the hydrophobic stacking interaction of this residue with the active site residue may help to keep the N-terminal tag at the position of binding to the active site, and therefore may also prevent cleavage between Gln and Gly in the protease pair sequence.
N-terminal tag occupancy prevents the His-GsePNPase active site from shutting down, which is characterized by having a α helix (H7, using the α helix number of EcPNPase) that can be split into two parts to close the active site. In His-GsePNPase H7, it consists of the residue V al200-Gln217. Two distinct active site conformations were observed in EcPNPase, expressed as "open" (non-segmented) and "closed" (segmented). In the closed conformation, the ring eliminates access to the active site. In the structure of His-GsePNPase, the conformation of the ring in all three subunits is most consistent with the "open" conformation of EcPNPase (Figure 3d), with the tag occupying the active site, excluding helical segmentation and the ability to adopt a "closed" conformation.
4
Interaction of the N-terminal tag with the active site is possible
Affects enzyme function
Before considering the functional effect of the N-terminal tag binding at the active site, it is first necessary to investigate whether this is just a crystallization artifact driven by proximity effects and entropy. Therefore, the authors' team attempted to cleave the tag and purify the unlabeled form in order to perform joint structural and functional characterization of the labeled and unlabeled forms. However, regardless of experimental conditions, only mixed populations of cleaved and uncut subunits were obtained, of which the predominantly uncleaved ones. This result suggests that the tag may be difficult to remove because His-GsePNPase exists in solution as tag-binding, preventing the protease from reaching the cleavage site. Crystallization screening experiments performed in the presence of high concentrations of the PNPase inhibitor acyclovir and the substrate 7-methylguanine support the strong affinity of the tag for the active site of the crystalline form, suggesting that neither substrate can replace the tag from the active site. However, UV-Vis activity assays have shown that N-terminal labeled His-GsePNPase has catalytic activity in solution (data not shown). Enzymatic activity means that the active site must be at least somewhat accessible to the substrate under standard assay conditions. While the substrate can bind to the active site in the tag-competing solution, the vice versa, i.e., the tag may compete with the substrate and may affect the activity of the enzyme.
Taken together, it is shown that His-GsePNPase is most likely to exist in both the solution and crystalline phases in label-bound form, but has a lower affinity enthalpy in solution due to the entropy favorability of the disordered unbound form that partially counteracts the binding. This circumstance, combined with the dynamic properties of the protein in solution, may explain why substrate binding is observed in the activity assay but not in the crystallization screen.
The authors' team resolved the purine nucleoside phosphorylase structure from Geobacillus thermophilus at a resolution of 1.72 Å. Several residues of the N-terminal tag cleavage site of the recombinant tobacco etching virus protease (rTEV) are located in the active sites of adjacent subunits in the dimer, and the key residue for this interaction is the tyrosine residue, which is located where the substrate nucleoside ring is normally located. Tag binding appears to be driven by a combination of enthalpy, entropy, and proximity effects, and attempts to cleavage the tag in solution yielded only a small fraction of the untagged protein, suggesting that the enzyme is predominantly present in solution in tag-bound form, thus preventing rTEV from entering the cleavage site. However, the tagged protein retains some activity in solution, suggesting that the tagged does not completely block the active site, but may act as a competitive inhibitor.
This article inspires us to be careful to determine how affinity tags affect protein structure and function, especially for cases where tag removal is difficult.
Article Information:
https://doi.org/10.1107/S2053230X22011025