Vishal Prashar

X-ray snapshot of HIV-1 protease in action: observation of tetrahedral intermediate and short ionic hydrogen bond SIHB with catalytic aspartate.

Structural snapshots of each step in the catalytic cycle would help development of inhibitors of human immunodeficiency virus type 1 protease (HIV-1 PR) as effective drugs against HIV/AIDS. We report here one snapshot obtained by determining the structure of enzyme-substrate complex under conditions where the catalytic activity of the enzyme is greatly reduced. The 1.76 A crystal structure shows the oligopeptide substrate, AETFYVDGAA, converted in situ into a gem-diol tetrahedral intermediate (TI). The gem-diol intermediate is neutral and one of the hydroxyl oxygens forms a very short hydrogen bond (2.2 A) with the anionic aspartate of the catalytic dyad, which is monoprotonated. Further, there is no hydrogen atom on the outer oxygen of the neutral aspartate. These two observations provide direct evidence that, in the reaction mechanism, hydrogen bonding between catalytic aspartate and scissile carbonyl oxygen facilitates water attack on the scissile carbon atom. Comparison with the structural snapshot of the biproduct complex involving the same substrate reveals the reorganization of the hydrogen bonds at the catalytic center as the enzymatic reaction progresses toward completion. Accumulation of TI in the crystals provides direct evidence that collapse of TI is the rate-limiting step of hydrolysis.

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.

X-ray structure of HIV-1 protease in situ product complex.

HIV‐1 protease is an effective target for design of different types of drugs against AIDS. HIV‐1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV‐1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0‐Å resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen‐bonded with the N‐terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem‐hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid. Proteins 2009.

Resistance mechanism revealed by crystal structures of unliganded nelfinavir-resistant HIV-1 protease non-active site mutants N88D and N88S.

Nelfinavir is an inhibitor of HIV-1 protease, and is used for treatment of patients suffering from HIV/AIDS. However, treatment results in drug resistant mutations in HIV-1 protease. N88D and N88S are two such mutations which occur in the non-active site region of the enzyme. We have determined crystal structures of unliganded N88D and N88S mutants of HIV-1 protease to resolution of 1.65A and 1.8A, respectively. These structures refined against synchrotron data lead to R-factors of 0.1859 and 0.1780, respectively. While structural effects of N88D are very subtle, the mutation N88S has caused a significant conformational change in D30, an active site residue crucial for substrate and inhibitor binding.

Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex

The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

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.

Crystallization and preliminary x-ray crystallographic analysis of PhoK, an extracellular alkaline phosphatase from Sphingomonas sp. BSAR-1

Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87.37, c = 168.16 A, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95 A resolution at the ESRF.

Structural Basis of Why Nelfinavir‐Resistant D30N Mutant of HIV‐1 Protease Remains Susceptible to Saquinavir

Although anti‐HIV‐1 protease drugs nelfinavir (NFV) and saquinavir (SQV) share common functional groups, D30N is a major resistance mutation against NFV but remains susceptible to SQV. We have determined the crystal structure of D30N mutant‐tethered HIV‐1 protease in complex with SQV to 1.79 Å resolution. Structural analysis showed that SQV forms two direct hydrogen bonds with the main chain atoms of the residues Asp29 and Asp30 that are not observed in the D30N–NFV complex. Apart from maintaining these two main chain hydrogen bonds, the P2‐asparagine of SQV forms an additional hydrogen bond to the mutated side chain of the residue 30. These could be the reasons why D30N is not a drug resistance mutation against SQV. This structure supports the previous studies showing that the interactions between a potential inhibitor and backbone atoms of the enzyme are important to maintain potency against drug‐resistant HIV‐1 protease.

Crystallization and preliminary X-ray diffraction analysis of human seminal plasma protein PSP94.

The human seminal plasma protein PSP94 is a small protein of 94 residues that contains ten cysteines. Since its discovery about 25 years ago, several potential biological functions have been reported for this protein. Many PSP94 homologues have also been identified since then from various species, but no crystal structure has been determined to date. PSP94 has been purified from human seminal plasma and crystallized. These crystals diffracted to approximately 2.3 A resolution and belonged to space group P4(1)2(1)2, with unit-cell parameters a = 107.9, b = 107.9, c = 92.1 A. There are four molecules in the asymmetric unit. Structure solution by the heavy-atom method is currently in progress.