Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide–protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition☆

Kaur, Hardeep; Datt, Manish; Ekka, Mary Krishna; Mittal, Monica; Singh, Appu Kumar; Kumaran, S.

doi:10.1016/j.biochi.2010.09.008

Cited by 12 publications

(22 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although HGC tripeptide is resistant to proteolytic degradation by Dug1p, this tripeptide forms the enzyme−peptide complex, supporting our earlier proposal that hydrolysis might be the rate-limiting step in this case. 3 Therefore, our results demonstrate that Dug1p binds tripeptides but hydrolyzes selectively. To improve our understanding of the specificity of binding, we used a hexameric peptide with the sequence Gly-Cys-Gly-Cys-Gly-Cys and examined the binding.…”

Section: Comparison Of Peptide and Inhibitor Binding Posesmentioning

confidence: 60%

“…The stereochemical features of interactions between the peptide and active site residues explain how Dug1p binds a variety of small peptides in a sequence-independent manner. 3 The mode of peptide binding is similar to the "twoprong plug two-hole socket" model. 35 The N-terminus of the peptide fits into the catalytic S1 binding pocket, whereas the Cterminus of the peptide is firmly bound to the S1′ pocket.…”

Section: ■ Discussionmentioning

confidence: 90%

“…The same mechanism is also proposed for Dug1p-mediated hydrolysis of peptides. 3 To obtain evidence of active site remodeling, we compared active site features of Dug1p and CN2 structures. Positions and conformational features of metal ions and catalytic residues differ between these two structures, suggesting that peptide binding and inhibitor binding influence active sites differently.…”

Section: ■ Discussionmentioning

confidence: 99%

“…2 To improve our understanding of the peptide recognition properties of Dug1p, we studied the binding of a variety of peptides to this enzyme and found that the substrate specificity of Dug1p may be encoded at two different levels. 3 We showed that Dug1p binds a broad range of small peptides but cleaves Cys-Gly peptide with specificity, suggesting that catalytic specificity is achieved after binding. We proposed that Dug1p may employ an induced fitlike mechanism for discriminating substrates from nonsubstrate peptides.…”

mentioning

confidence: 86%

See 3 more Smart Citations

Molecular Basis of Peptide Recognition in Metallopeptidase Dug1p from Saccharomyces cerevisiae

Singh

Pandya

et al. 2014

Biochemistry

Self Cite

View full text Add to dashboard Cite

Dug1p, a M20 family metallopeptidase and human orthologue of carnosinase, hydrolyzes Cys-Gly dipeptide, the last step of glutathione (GSH) degradation in Saccharomyces cerevisiae. Molecular bases of peptide recognition by Dug1p and other M20 family peptidases remain unclear in the absence of structural information about enzyme-peptide complexes. We report the crystal structure of Dug1p at 2.55 Å resolution in complex with a Gly-Cys dipeptide and two Zn(2+) ions. The dipeptide is trapped in the tunnel-like active site; its C-terminus is held by residues at the S1' binding pocket, whereas the S1 pocket coordinates Zn(2+) ions and the N-terminus of the peptide. Superposition with the carnosinase structure shows that peptide mimics the inhibitor bestatin, but active site features are altered upon peptide binding. The space occupied by the N-terminus of bestatin is left unoccupied in the Dug1p structure, suggesting that tripeptides could bind. Modeling of tripeptides into the Dug1p active site showed tripeptides fit well. Guided by the structure and modeling, we examined the ability of Dug1p to hydrolyze tripeptides, and results show that Dug1p hydrolyzes tripeptides selectively. Point mutations of catalytic residues do not abolish the peptide binding but abolish the hydrolytic activity, suggesting a noncooperative mode in peptide recognition. In summary, results reveal that peptides are recognized primarily through their amino and carboxyl termini, but hydrolysis depends on the properties of peptide substrates, dictated by their respective sequences. Structural similarity between the Dug1p-peptide complex and the bestatin-bound complex of CN2 suggests that the Dug1p-peptide structure can be used as a template for designing natural peptide inhibitors.

show abstract

Section: Comparison Of Peptide and Inhibitor Binding Posesmentioning

confidence: 60%

Section: ■ Discussionmentioning

confidence: 90%

Section: ■ Discussionmentioning

confidence: 99%

mentioning

confidence: 86%

See 2 more Smart Citations

Molecular Basis of Peptide Recognition in Metallopeptidase Dug1p from Saccharomyces cerevisiae

Singh

Pandya

et al. 2014

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The formation of enzyme-substrate complex is best described by induced fit model which suggests that binding of the substrate induces specific conformational changes within the active site and the productive enzyme-substrate complex formation is determined by these conformational dynamics [7]–[10]. A structurally related “non-substrate” molecule can also bind the active site, but binding may not result in the formation of catalytically productive enzyme-substrate complex due to different conformational dynamics induced by the “non-substrate” molecule [8], [11], [12]. Structurally related “non-substrate” molecules pose great challenges when enzyme has to selectively catalyze the substrate from a mixture of compounds, a condition often encountered under physiological conditions [11].…”

Section: Introductionmentioning

confidence: 99%

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

et al. 2012

Self Cite

View full text Add to dashboard Cite

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (Kd∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of Kd values with Km values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.

show abstract

Specific Electrostatic Molecular Recognition in Water

Hoeck

Schoffelen

et al. 2016

Chemistry A European J

View full text Add to dashboard Cite

The identification of pairs of small peptides that recognize each other in water exclusively through electrostatic interactions is reported. The target peptide and a structure-biased combinatorial ligand library consisting of ≈78 125 compounds were synthesized on different sized beads. Peptide-peptide interactions could conveniently be observed by clustering of the small, fluorescently labeled target beads on the surface of larger ligand-containing beads. Sequences of isolated hits were determined by MS/MS. The interactions of the complex showing the highest affinity were investigated by a novel single-bead binding assay and by 2D NMR spectroscopy. Molecular dynamics (MD) studies revealed a putative mode of interaction for this unusual electrostatic binding event. High binding specificity occurred through a combination of topological matching and electrostatic and hydrogen-bond complementarities. From MD simulations binding also seemed to involve three tightly bound water molecules in the interface between the binding partners. Binding constants in the submicromolar range, useful for biomolecular adhesion and in nanostructure design, were measured.

show abstract

Cys-Gly specific dipeptidase Dug1p from S. cerevisiae binds promiscuously to di-, tri-, and tetra-peptides: Peptide–protein interaction, homology modeling, and activity studies reveal a latent promiscuity in substrate recognition☆

Cited by 12 publications

References 44 publications

Molecular Basis of Peptide Recognition in Metallopeptidase Dug1p from Saccharomyces cerevisiae

Molecular Basis of Peptide Recognition in Metallopeptidase Dug1p from Saccharomyces cerevisiae

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

Specific Electrostatic Molecular Recognition in Water

Contact Info

Product

Resources

About