Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non–Specificity, and Inhibition

The single–strand–specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1–P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X–ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1–P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes–shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1–P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.


Thermal unfolding using differential scanning fluorimetry
Thermal stability of fully glycosylated S1 nuclease and a sample treated with Endoglycosidase F1 (see deglycosylation details in the main article) was analyzed by differential scanning fluorimetry using a Prometheus NT.48 apparatus and Prometheus NT.48 Series nanoDSF Grade Standard Capillaries (NanoTemper Technologies GmbH). Samples were in the storage buffer (25 mM Bis-Tris pH 6.0 with addition of 50 mM NaCl). Concentration of both samples was about 0.5 mg/ml. Thermal unfolding was performed in the range from 20 °C to 95 °C at a scan rate of 2.5 °C per minute.

Surface electrostatic potential distribution
Surface electrostatic potential distribution was calculated for protonation states at pH 4, 6, and 8.5; pH 4 is close to the pH optimum for nuclease activity, pH 6 is close to the pH optimum for 3'-mononucleotidase activity, and pH 8.5 was chosen as a point of minimal catalytic activity of S1 nuclease. The calculations were done using APBS [1]. Parameter files were created by PDB2PQR 1.8 using the AMBER force field [2]. Protonation states were assigned by PropKa [3].  Values in parentheses are for the highest resolution shell. ASU stands for asymmetric unit, Pi for phosphate ion, PEG MME for a fragment of polyethylene glycol monomethyl ether, 5'AMP for adenosine 5'-monophosphate, 5'dAMP(S) for 2'-deoxyadenosine 5'-thio-monophosphate, 5'dCMP for 2'-deoxycytidine 5'-monophosphate, dGua for 2'-deoxyguanosine, and dCyt for 2'-deoxycytidine. Both deglycosylated versions behave similar to S1wt in the storage buffer and they are monomeric. The measured hydrodynamic radius is 2.49 ± 0.70 nm for S1wt, 2.47 ± 0.89 nm for S1-α-mann, and 2.23 ± 0.69 nm for S1-Endo F1. The apparent trend of decrease of hydrodynamic radius by deglycosylation cannot be reliably interpreted due to the observed experimental errors. (d) The activity of S1wt and S1 treated with Endoglycosidase F1 against ssDNA and RNA. Activity is reported as a change of absorbance at 260 nm over time normalized to the amount of enzyme used. S1wt and S1-Endo F1 display similar activity taking into account the decrease of enzyme mass by about 18% by deglycosylation and also the experimental error.   The presence of a sodium ion (magenta sphere) is supported by the coordination distances and behavior in the refinement. 5'AMP interacts with the sodium ion through the phosphate group and through this sodium ion also with Asp83 of NBS1. The sodium ion has no direct contact with symmetry-related protein molecules. The phosphate moiety of 5'AMP also interacts with Glu42 (shown in sticks, carbon -dark grey, marked by *) from a symmetry-related protein chain. This is the only direct contact of the ligand with a symmetry-related molecule. Molecular graphics were created using PyMOL (Schrödinger, LLC) and chain A of the structure 5FBB.      Homologs of S1 nuclease were identified using an NCBI BLAST search [5]. Due to the high number of found homologs the list presented here was manually edited based on the intended demonstration of the selected features. Most of the bacterial homologs were selected based on Pimkin et al. [6]. The sequences were aligned using ClustalW2 [7]. Names of the enzymes with known structure are in bold characters. The column following the name shows sequence identity to S1 nuclease. The figure was created using ESPript [8] and edited. (Glyc112) Glycosylation site at position 112 along with its interacting partner, an aromatic amino acid at position 75.

SUPPLEMENTARY TABLES
(NBS1) Alignment of selected residues involved in the formation of NBS1. The side chain of the residue at position 81 is involved in the stacking interaction with a nucleobase. The side chain of the amino acid at position 83 provides hydrogen bonding to a nucleobase. Asp or Asn is conserved at position 83 in fungi and plants. In trypanozomatidae and bacteria this pattern is often broken. However, without the structure of such nuclease it is hard to estimate whether the function of the hydrogen bond partner is substituted by another amino acid, or nucleobase binding is facilitated in a different way. The second π-system donor is the peptide bond between the residue 151 and Gly152. Asn154 is always conserved. (Tyr site and Half-Tyr site) Residues involved in the formation of the P1 nuclease Tyr site are in the light blue box. Residues involved in the formation of the S1 nuclease Half-Tyr site are in the green box. These two sites are conserved only in fungi. Moreover, several S1-P1 like nucleases from fungi apparently do not possess either site. Residues with a possible role in the Half-Tyr site in plants are in the light green box. However, it is not possible to confirm the role of these residues in plants without the corresponding structures.
Figure K. Electrostatic potential distribution on the solvent accessible surface of the S1-P1 nuclease family members with known structure. Electrostatic potential distribution for S1 nuclease was calculated for protonation states at pH 4.0, pH 6.0, and pH 8.5 using the structure 5FBF -nuclease products. In the cases of P1 (PDB ID: 1AK0 [9]), TBN1 (PDB ID: 3SNG [10]), and AtBFN2 (PDB ID: 4CXO [11]) the electrostatic potential distribution was calculated only at pH 6.0 (close to their pH optimum of activity). All structures are shown in the same orientation with respect to the zinc cluster. Molecular graphics were created using PyMOL (Schrödinger, LLC).

Figure L.
Thermal stability of fully glycosylated S1 nuclease (red) and S1 nuclease treated with Endoglycosidase F1 (blue) measured by DSF. Measurements were performed in 25 mM Bis-Tris pH 6.0 with the addition of 50 mM NaCl, with protein concentration 0.5 mg/ml, and in temperature range 20 -95 °C.