NBCZone: Universal three-dimensional construction of eleven amino acids near the catalytic nucleophile and base in the superfamily of (chymo)trypsin-like serine fold proteases

Abstract

(Chymo)trypsin-like serine fold proteases belong to the serine/cysteine proteases found in eukaryotes, prokaryotes, and viruses. Their catalytic activity is carried out using a triad of amino acids, a nucleophile, a base, and an acid. For this superfamily of proteases, we propose the existence of a universal 3D structure comprising 11 amino acids near the catalytic nucleophile and base - Nucleophile-Base Catalytic Zone (NBCZone). The comparison of NBCZones among 169 eukaryotic, prokaryotic, and viral (chymo)trypsin-like proteases suggested the existence of 15 distinct groups determined by the combination of amino acids located at two "key" structure-functional positions 54_T and 55_T near the catalytic base His57_T. Most eukaryotic and prokaryotic proteases fell into two major groups, [ST]A and TN. Usually, proteases of [ST]A group contain a disulfide bond between cysteines Cys42_T and Cys58_T of the NBCZone. In contrast, viral proteases were distributed among seven groups, and lack this disulfide bond. Furthermore, only the [ST]A group of eukaryotic proteases contains glycine at position 43_T, which is instrumental for activation of these enzymes. In contrast, due to the side chains of residues at position 43_T prokaryotic and viral proteases do not have the ability to carry out the structural transition of the eukaryotic zymogen-zyme type.

Keywords: (Chymo)trypsin-like proteases; Catalytic triad; Structural framework; Structural motif.

Fig. 1

Structure of the active site in (chymo)trypsin-like serine fold proteases. Amino acid numbers are taken as in Trypsin (PDB ID: 4I8H_A). The catalytic triad includes Asp102 (the catalytic acid), His57 (the catalytic base) and Ser195 (the catalytic nucleophile). The PROSITE “TRYPSIN_SER” pattern (PS00135; G–[DE]–S–G–[GS]) includes Gly193-Asp194-Ser195(cat. nucleophile)-Gly196-Gly197. The PROSITE “TRYPSIN_HIS” pattern (PS00134; [LIVM]-[ST]-A-[STAG]-H-C) includes Val53-Ser54-Ala55-Ala56-His57(cat. base)-Cys58. Two main-chain nitrogens, N/Gly193 and N/Ser195, are the two canonical oxyanions “N(oxyI)” and “N(oxyII)”. Gly43 and Val213 simultaneously interact with both the TRYPSIN_SER and TRYPSIN_HIS pattern, and thus constitute the “43/213 Nucleophile-Base Catalytic Zone” (43/213-NBCZone) of (chymo)trypsin-like serine fold proteases. The disulfide bond Cys42-Cys58 joins the elements of the “42/43 Base Catalytic Zone”, which includes the TRYPSIN_HIS pattern and the Cys42-Gly43 dipeptide. Two conserved structural water molecules in positions X and Y, HOH X and HOH Y, interact with the TRYPSIN_SER pattern and form the “Nucleophile-Base Catalytic Zone Conserved Extension” in eukaryotic serine (chymo)trypsin-like fold proteases. Structural data were visualized and analyzed using Discovery Studio [76] and Bodil [77]. Figures were drawn with MolScript [78] and Raster 3D [79].

Fig. 2

(A) shows the “43/213 Nucleophile-Base Catalytic Zone” (43/213-NBCZone) of the “[ST]A Group” of (chymo)trypsin-like serine fold proteases (see Table 1). The 43/213-NBCZone, shown in (A), together with the “42/43 Base Catalytic Zone” shown in (B) constitute the entire Nucleophile-Base Catalytic Zone (NBCZone) of trypsin, which is the representative structure of the “[ST]A Group” (see Table 1). Unlike the “[ST]A Group”, shown in (A), where the 55_T position of the 43/213-NBCZone is occupied by an alanine (Ala55 in panel A), in the “TN Group” enzymes, shown in (C), (D), (E) and (F), the 55_T position is occupied by an asparagine (Asn196 in panel C; Asn218 in panel D; Asn171 in panel E and Asn38 in panel F), whose conformation is different in four different groups, named Sets I to IV, of the TN Group enzymes. In Sets I and II, respectively shown in (C) and (D), the ND2 atom of Asn55_T takes part in the formation of the 43/213-NBCZone, while the OD1 atom of Asn55_T does either form an Asx-turn with the catalytic histidine (as in C) or interacts with the main-chain oxygen atom of the catalytic acid (as in D). In Sets III and IV, respectively shown in (E) and (F), the OD1 atom of Asn55_T takes part in the formation of the 43/213-NBCZone, while the ND2 atom of Asn55_T interacts with either the catalytic acid (as in E) or base (as in F).

Fig. 3

(A) shows the 43/213-NBCZone of the “43&[STG]V Group” of (chymo)trypsin-like serine fold proteases, which is not found in eukaryotes (example of immunoglobulin A1 protease; see Tables 1 and S1). In these prokaryotic and viral proteins, the change in the course of the polypeptide chain at the position 43_T leads to the replacement of the “key” canonical NH...O hydrogen bond (N/Gly43-O/cat.nucleophile in Fig. 2A) with a weak CH...O hydrogen bond (CG2/Ile86-O/cat.nucleophile in panel A), and the impossibility of forming a Cys42_T-Cys58_T disulfide bridge within the 42/43 Base Catalytic Zone. In (B), the viral “[KR]P group” is shown, where at the position 54_T, a lysine or an arginine is found instead of a threonine or a serine. In (C) and (D), extension of the NBCZone in trypsin and trypsinogen, respectively, is shown due to either inclusion of two conserved structural water molecules at the positions X and Y of trypsin (as in C), or a side-chain oxygen atom (OD1 atom of Asp194 in trypsinogen) and one water molecule at the same spatial positions X and Y (as in D). (E) shows the extension of the NBCZone in the “TN Group” of (chymo)trypsin-like serine fold proteases (example of the chloroplastic protease Do-like 2; PDB ID: 5ILB), where the OG atom of Ser43_T is located at position X instead of the structural water molecule found in the NBCZone extension of trypsin. (F) shows the NBCZone extension in the five “inactive” proteases (example of Heparin binding protein; PDB ID: 1A7S; see Tables S1 and S2), where instead of a glycine at position 197_T there is a serine, threonine, or aspartate (Thr177 in F), whose side-chain OG1 atom substitutes the HOH Y water molecule at the NBCZone extension.