Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 1

Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.     

Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1

The mammalian AP-endonuclease (APE1) repairs apurinic/apyrimidinic (AP) sites and strand breaks with 3′ blocks in the genome that are formed both endogenously and as intermediates during base excision repair. APE1 has an unrelated activity as a redox activator (and named Ref-1) for several trans-acting factors. In order to identify whether any of the seven cysteine residues in human APE1 affects its enzymatic function, we substituted these singly or multiply with serine. The repair activity is not affected in any of the mutants except those with C99S mutation. The Ser99-containing mutant lost affinity for DNA and its activity was inhibited by 10 mM Mg²⁺. However, the Ser99 mutant has normal activity in 2 mM Mg²⁺. Using crystallographic data and molecular dynamics simulation, we have provided a mechanistic basis for the altered properties of the C99S mutant. We earlier predicted that Mg²⁺, with potential binding sites A and B, binds at the B site of wild-type APE1-substrate complex and moves to the A site after cleavage occurs, as observed in the crystal structure. The APE1-substrate complex is stabilized by a H bond between His309 and the AP site. We now show that this bond is broken to destabilize the complex in the absence of the Mg²⁺. This effect due to the mutation of Cys99, ∼ 16 Å from the active site, on the DNA binding and activity is surprising. Mg²⁺ at the B site promotes stabilization of the C99S mutant complex. At higher Mg²⁺ concentration the A site is also filled, causing the B-site Mg²⁺ to shift together with the AP site. At the same time, the H bond between His309 and the AP site shifts toward the 5′ site of DNA. These shifts could explain the lower activity of the C99S mutant at higher [Mg²⁺]. The unexpected involvement of Cys99 in APE1's substrate binding and catalysis provides an example of involvement of a residue far from the active site.     

Multi-drug resistance profile of PR20 HIV-1 protease is attributed to distorted conformational and drug binding landscape: molecular dynamics insights

The PR20 HIV-1 protease, a variant with 20 mutations, exhibits high levels of multi-drug resistance; however, to date, there has been no report detailing the impact of these 20 mutations on the conformational and drug binding landscape at a molecular level. In this report, we demonstrate the first account of a comprehensive study designed to elaborate on the impact of these mutations on the dynamic features as well as drug binding and resistance profile, using extensive molecular dynamics analyses. Comparative MD simulations for the wild-type and PR20 HIV proteases, starting from bound and unbound conformations in each case, were performed. Results showed that the apo conformation of the PR20 variant of the HIV protease displayed a tendency to remain in the open conformation for a longer period of time when compared to the wild type. This led to a phenomena in which the inhibitor seated at the active site of PR20 tends to diffuse away from the binding site leading to a significant change in inhibitor–protein association. Calculating the per-residue fluctuation (RMSF) and radius of gyration, further validated these findings. MM/GBSA showed that the occurrence of 20 mutations led to a drop in the calculated binding free energies (ΔG_bind) by ~25.17 kcal/mol and ~5 kcal/mol for p2-NC, a natural peptide substrate, and darunavir, respectively, when compared to wild type. Furthermore, the residue interaction network showed a diminished inter-residue hydrogen bond network and changes in inter-residue connections as a result of these mutations. The increased conformational flexibility in PR20 as a result of loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces led to a loss of protease grip on ligand. It is interesting to note that the difference in conformational flexibility between PR20 and WT conformations was much higher in the case of substrate-bound conformation as compared to DRV. Thus, developing analogues of DRV by retaining its key pharmacophore features will be the way forward in the search for novel protease inhibitors against multi-drug resistant strains.     

Insights on the structural perturbations in human MTHFR Ala222Val mutant by protein modeling and molecular dynamics

Methylenetetrahydrofolate reductase (MTHFR) protein catalyzes the only biochemical reaction which produces methyltetrahydrofolate, the active form of folic acid essential for several molecular functions. The Ala222Val polymorphism of human MTHFR encodes a thermolabile protein associated with increased risk of neural tube defects and cardiovascular disease. Experimental studies have shown that the mutation does not affect the kinetic properties of MTHFR, but inactivates the protein by increasing flavin adenine dinucleotide (FAD) loss. The lack of completely solved crystal structure of MTHFR is an impediment in understanding the structural perturbations caused by the Ala222Val mutation; computational modeling provides a suitable alternative. The three-dimensional structure of human MTHFR protein was obtained through homology modeling, by taking the MTHFR structures from Escherichia coli and Thermus thermophilus as templates. Subsequently, the modeled structure was docked with FAD using Glide, which revealed a very good binding affinity, authenticated by a Glide XP score of ?10.3983 (kcal mol^?1). The MTHFR was mutated by changing Alanine 222 to Valine. The wild-type MTHFR-FAD complex and the Ala222Val mutant MTHFR-FAD complex were subjected to molecular dynamics simulation over 50 ns period. The average difference in backbone root mean square deviation (RMSD) between wild and mutant variant was found to be ~.11 Å. The greater degree of fluctuations in the mutant protein translates to increased conformational stability as a result of mutation. The FAD-binding ability of the mutant MTHFR was also found to be significantly lowered as a result of decreased protein grip caused by increased conformational flexibility. The study provides insights into the Ala222Val mutation of human MTHFR that induces major conformational changes in the tertiary structure, causing a significant reduction in the FAD-binding affinity.     

The mutK Gene of Vibrio cholerae: a New Gene Involved in DNA Mismatch Repair

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).     

Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-1

Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.