Studies on the active site of Escherichia coli adenylosuccinate synthetase by chemical modification and site-directed mutagenesis

Reaction of E. coli adenylosuccinate synthetase with thiol reagents leaded to modification of one cysteine residue per subunit without significant loss of enzyme activity. Modification of a second cysteine residue occurred under 3.4 M urea and resulted in complete loss of enzyme activity. The remaining two cystein residues were modified only after treatment with 8 M urea. The reactive cystein had been identified as Cys[superscript]291, and the thiol exposed with 3.5 M urea is Cys[superscript]344. When Cys[superscript]344 was replaced by either serine or alanine, the mutant enzymes were as active as the wild type enzyme. These findings point to the nonessential roles of sulfhydryl groups in the enzyme;Incubation of the enzyme with low concentrations of pyridoxal 5[superscript]\u27-phosphate (PLP) resulted in a rapid loss of activity. Stoichiometry and peptide isolation studies showed that two lysine residues were modified. Complete protection of the enzyme against inactivation can be observed only in the presence of GTP. One lysine residue was protected under these conditions. In contrast to GTP, addition of the other two substrates either alone or together to reaction mixtures did not render protection. Peptide mapping revealed that the lysine shielded by GTP is Lys[superscript]140. Replacing the Lys[superscript]140 with Ile[superscript]140 resulted in total loss of the activity. These results suggest that Lys[superscript]140 may play an important role in enzymatic activity;Chemical modification of the enzyme with phenylglyoxal resulted in an inhibition of enzyme acitivity. The substrates, GTP or IMP, can partially protect the enzyme against inactivation. The other substrate, aspartate, had no such effect. In the presence of both IMP and GTP, nearly complete protection of the enzyme against inactivation can be observed. Stoichiometry studies showed that only 1 reactive arginine residue was modified and this arginine residue could be shielded by GTP and IMP. Sequence analysis indicated that Arg[superscript]147 is the site of phenylglyoxal modification. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild type system, whereas the K[subscript] m for aspartate exhibits no significant change compared with the native enzyme. In addition, k[subscript] cat of the R147L mutant decreased by a factor of 1.3 x 10[superscript]4. These results indicate that Arg[superscript]147 is critical for enzyme catalysis

Electrostatics in the Stability and Misfolding of the Prion Protein: Salt Bridges, Self-Energy, and Solvation

Using a recently developed mesoscopic theory of protein dielectrics, we have calculated the salt bridge energies, total residue electrostatic potential energies, and transfer energies into a low dielectric amyloid-like phase for 12 species and mutants of the prion protein. Salt bridges and self energies play key roles in stabilizing secondary and tertiary structural elements of the prion protein. The total electrostatic potential energy of each residue was found to be invariably stabilizing. Residues frequently found to be mutated in familial prion disease were among those with the largest electrostatic energies. The large barrier to charged group desolvation imposes regional constraints on involvement of the prion protein in an amyloid aggregate, resulting in an electrostatic amyloid recruitment profile that favours regions of sequence between alpha helix 1 and beta strand 2, the middles of helices 2 and 3, and the region N-terminal to alpha helix 1. We found that the stabilization due to salt bridges is minimal among the proteins studied for disease-susceptible human mutants of prion protein

Crystal structure of a murine α-class glutathione S-transferase involved in cellular defense against oxidative stress

Glutathione S-transferases (GSTs) are ubiquitous multifunctional enzymes which play a key role in cellular detoxification. The enzymes protect the cells against toxicants by conjugating them to glutathione. Recently, a novel subgroup of α-class GSTs has been identified with altered substrate specificity which is particularly important for cellular defense against oxidative stress. Here, we report the crystal structure of murine GSTA4-4, which is the first structure of a prototypical member of this subgroup. The structure was solved by molecular replacement and refined to 2.9 Å resolution. It resembles the structure of other members of the GST superfamily, but reveals a distinct substrate binding site.

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein-RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein-double-stranded DNA and protein-single-stranded DNA complexes. The interface properties of the protein-RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein-RNA and protein-DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein-RNA complexes, backbone contacts were more dominant in the protein-DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level

Whey-derived peptides interactions with ACE by molecular docking as a potential predictive tool of natural ACE inhibitors

Several milk/whey derived peptides possess high in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. However, in some cases, poor correlation between the in vitro ACE inhibitory activity and the in vivo antihypertensive activity has been observed. The aim of this study is to gain insight into the structure-activity relationship of peptide sequences present in whey/milk protein hydrolysates with high ACE inhibitory activity, which could lead to a better understanding and prediction of their in vivo antihypertensive activity. The potential interactions between peptides produced from whey proteins, previously reported as high ACE inhibitors such as IPP, LIVTQ, IIAE, LVYPFP, and human ACE were assessed using a molecular docking approach. The results show that peptides IIAE, LIVTQ, and LVYPFP formed strong H bonds with the amino acids Gln 259, His 331, and Thr 358 in the active site of the human ACE. Interestingly, the same residues were found to form strong hydrogen bonds with the ACE inhibitory drug Sampatrilat. Furthermore, peptides IIAE and LVYPFP interacted with the amino acid residues Gln 259 and His 331, respectively, also in common with other ACE-inhibitory drugs such as Captopril, Lisinopril and Elanapril. Additionally, IIAE interacted with the amino acid residue Asp 140 in common with Lisinopril, and LIVTQ interacted with Ala 332 in common with both Lisinopril and Elanapril. The peptides produced naturally from whey by enzymatic hydrolysis interacted with residues of the human ACE in common with potent ACE-inhibitory drugs which suggests that these natural peptides may be potent ACE inhibitors

Engineering of Cyclodextrin Product Specificity and pH Optima of the Thermostable Cyclodextrin Glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 β-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-Å resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced α-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (β- and γ-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of α-CD. Glu258 is involved in catalysis in CGTases as well as α-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.

Deduced Primary Structure of the β Subunit of Brain Type II Ca2+/calmodulin-dependent Protein Kinase Determined by Molecular Cloning

cDNA clones coding for the β subunit of rat brain type II Ca2+/calmodulin-dependent protein kinase were isolated and sequenced. The clones, including one containing the entire coding region, hybridize at high stringency to a single band of poly(A)+ RNA of length 4.8 kilobases. The subunit coded for by the clones was identified by in vitro transcription of the cDNA followed by translation of the resulting RNA. The DNA sequence of the clones contains a single long open reading frame (1626 nucleotides) coding for a protein of 542 amino acids with a molecular weight of 60,333, the amino-terminal half of which is homologous to several other protein kinases. Potential ATP- and calmodulin-binding domains were identified. Two independent clones contain an identical 45-nucleotide deletion, relative to the clones described above, resulting in a shorter open reading frame coding for a protein of molecular weight 58,000. This suggests that the minor, 58-kDa β' subunit of the type II Ca2+/calmodulin-dependent kinase may be synthesized on a separate message

Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway

Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway’s ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ~700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ~70 residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with Kd of ~1 microM, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides, but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of (cognate) dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically, and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1’s type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding