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Molecular recognition of PTS-1 cargo proteins by Pex5p: implications for protein mistargeting in primary hyperoxaluria.

Mesa-Torres N, Tomic N, Albert A, Salido E, Pey AL - Biomolecules (2015)

Bottom Line: Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation.Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities.Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, 18071 Granada, Spain. noelmesa@ugr.es.

ABSTRACT
Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3-4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

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Thermodynamic binding properties for the interaction of PTS1 nonapeptides with Pex5p-pbd. (A) Free energy (ΔG), enthalpy (ΔH) and entropy (−TΔS) contributions to the binding reaction; (B,C) Thermodynamic dissection of binding enthalpies (B) and heat capacity changes (ΔCp, C) into their intrinsic contribution (estimated from polar and apolar ΔASA using the crystal structure for the complex; Figure 1) and the contribution from conformational changes. In (A,B), data are means for two independent experiments at 25.
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biomolecules-05-00121-f007: Thermodynamic binding properties for the interaction of PTS1 nonapeptides with Pex5p-pbd. (A) Free energy (ΔG), enthalpy (ΔH) and entropy (−TΔS) contributions to the binding reaction; (B,C) Thermodynamic dissection of binding enthalpies (B) and heat capacity changes (ΔCp, C) into their intrinsic contribution (estimated from polar and apolar ΔASA using the crystal structure for the complex; Figure 1) and the contribution from conformational changes. In (A,B), data are means for two independent experiments at 25.

Mentions: A comparative analyses of the intrinsic and conformational contributions to the experimental ΔH and ΔCp value may provide some insight into this gain in affinity between full-length cargo protein and the nonapeptides (Figure 6 and Figure 7). In general, the peptides show a lower enthalpic penalization to binding, which indicates that a significant fraction of this penalization in the full-length protein arises from ancillary regions, and is somewhat entropically compensated. While the contributions to the enthalpy show similar nature in the full-length AGT and the peptides, the average favorable contribution from the intrinsic component is 17 kJ/mol lower in the peptide, while the conformational component reduces its penalization by about 33 kJ/mol, thus supporting the notion that the ancillary regions contribute favorably in terms of solvent exposure but unfavorably in terms of conformational changes (which is the main source of the enthalpic penalization to binding in the full-length AGT). As found for full-length proteins, the correlation between intrinsic and conformational components to the binding affinity is weak (Figure 6B). In the case of binding heat capacities, the behavior of the peptides is similar to that of the full-length protein, but as expected, the contribution of the intrinsic component is lower due to the smaller changes in solvent accessibility compared to the full-length protein.


Molecular recognition of PTS-1 cargo proteins by Pex5p: implications for protein mistargeting in primary hyperoxaluria.

Mesa-Torres N, Tomic N, Albert A, Salido E, Pey AL - Biomolecules (2015)

Thermodynamic binding properties for the interaction of PTS1 nonapeptides with Pex5p-pbd. (A) Free energy (ΔG), enthalpy (ΔH) and entropy (−TΔS) contributions to the binding reaction; (B,C) Thermodynamic dissection of binding enthalpies (B) and heat capacity changes (ΔCp, C) into their intrinsic contribution (estimated from polar and apolar ΔASA using the crystal structure for the complex; Figure 1) and the contribution from conformational changes. In (A,B), data are means for two independent experiments at 25.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384115&req=5

biomolecules-05-00121-f007: Thermodynamic binding properties for the interaction of PTS1 nonapeptides with Pex5p-pbd. (A) Free energy (ΔG), enthalpy (ΔH) and entropy (−TΔS) contributions to the binding reaction; (B,C) Thermodynamic dissection of binding enthalpies (B) and heat capacity changes (ΔCp, C) into their intrinsic contribution (estimated from polar and apolar ΔASA using the crystal structure for the complex; Figure 1) and the contribution from conformational changes. In (A,B), data are means for two independent experiments at 25.
Mentions: A comparative analyses of the intrinsic and conformational contributions to the experimental ΔH and ΔCp value may provide some insight into this gain in affinity between full-length cargo protein and the nonapeptides (Figure 6 and Figure 7). In general, the peptides show a lower enthalpic penalization to binding, which indicates that a significant fraction of this penalization in the full-length protein arises from ancillary regions, and is somewhat entropically compensated. While the contributions to the enthalpy show similar nature in the full-length AGT and the peptides, the average favorable contribution from the intrinsic component is 17 kJ/mol lower in the peptide, while the conformational component reduces its penalization by about 33 kJ/mol, thus supporting the notion that the ancillary regions contribute favorably in terms of solvent exposure but unfavorably in terms of conformational changes (which is the main source of the enthalpic penalization to binding in the full-length AGT). As found for full-length proteins, the correlation between intrinsic and conformational components to the binding affinity is weak (Figure 6B). In the case of binding heat capacities, the behavior of the peptides is similar to that of the full-length protein, but as expected, the contribution of the intrinsic component is lower due to the smaller changes in solvent accessibility compared to the full-length protein.

Bottom Line: Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation.Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities.Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, 18071 Granada, Spain. noelmesa@ugr.es.

ABSTRACT
Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3-4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

Show MeSH
Related in: MedlinePlus