| 1. |
- Gianni, Stefano, et al.
(författare)
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Sequence-specific Long Range networks in PSD-95/Discs Large/ZO-1 (PDZ) Domains Tune Their Binding Selectivity
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:31, s. 27167-27175
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Tidskriftsartikel (refereegranskat)abstract
- Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.
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| 2. |
- Haq, S Raza, et al.
(författare)
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Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society. - 0002-7863 .- 1520-5126. ; 134:1, s. 599-605
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Tidskriftsartikel (refereegranskat)abstract
- Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K-d correlate with changes in k(off).
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| 3. |
- Savino, Carmelinda, et al.
(författare)
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Investigating the structural plasticity of a cytochrome P450 : three-dimensional structures of P450 EryK and binding to its physiological substrate
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:42, s. 29170-29179
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism.
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