Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

BACKGROUND: It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures. PRINCIPAL FINDINGS: We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates. CONCLUSIONS/SIGNIFICANCE: The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated

Potential inhibitors against papain-like protease of novel coronavirus (SARS-CoV-2) from FDA approved drugs

The cases of 2019 novel coronavirus (SARS-CoV-2) infection have been continuously increasing ever since its outbreak in China last December. Currently, there are no approved drugs to treat the infection. In this scenario, there is a need to utilize the existing repertoire of FDA approved drugs to treat the disease. The rational selection of these drugs could be made by testing their ability to inhibit any SARS-CoV-2 proteins essential for viral life-cycle. We chose one such crucial viral protein, the papain-like protease (PLpro), to screen the FDA approved drugs in silico. The homology model of the protease was built based on the SARS-coronavirus PLpro structure, and the drugs were docked in S3/S4 pockets of the active site of the enzyme. In our docking studies, sixteen FDA approved drugs, including chloroquine and formoterol, was found to bind the target enzyme with significant affinity and good geometry, suggesting their potential to be utilized against the virus.</p

Comparison of interactions of catalytic aspartates in the structures at pH 2.0 and pH7.0.

<p>Comparison of interactions of catalytic aspartates in the structures at pH 2.0 and pH7.0.</p

Structural comparison of present complex with tetrahedral intermediate complex [21] and product peptide complex [22]:

<p>Stereo diagram showing the ligand atoms at the catalytic centre along with catalytic aspartates. Protein Cα atoms are used in the structural superposition. WAT1 is within 1 Å from an oxygen atom in the newly generated gem-diol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007860#pone.0007860-Kumar1" target="_blank">[21]</a> or carboxyl group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007860#pone.0007860-Das1" target="_blank">[22]</a>.</p

Fit of carboxy terminal product peptide and active site water molecules into 2Fo-Fc electron density.

<p>The electron density map is contoured at 1.0σ level. The carboxyl product peptide ( violetpurple) and the active site water molecules are shown in the two orientations.</p

Relative positions of WAT1 and the modelled substrate in the active site:

<p>Diagram showing superposition of three structures: 1) present structure (yellow carbon), 2) unliganded HIV-1 protease (magenta carbon, PDB Id 1LV1) and 3) inactive HIV-1 protease/substrate complex (green carbon, PDB Id 1KJH). Water molecule observed in unliganded HIV-1 protease is also shown (magenta). The distances to the scissile carbon atom are indicated. SA OMIT density contoured at 3σ level is also shown for WAT1.</p

Data collection and refinement statistics.

<p>*Data for highest resolution shell are given in the parenthesis.</p

Comparison of conformation of catalytic aspartates in the structures at pH 2.0 and pH7.0.

<p>Comparison of conformation of catalytic aspartates in the structures at pH 2.0 and pH7.0.</p

X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases.

The alkaline phosphatase (AP) is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP). The crystal structure reveals many differences from other APs: 1) the catalytic residue is a threonine instead of serine, 2) there is no third metal ion binding pocket, and 3) the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily