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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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Related in: MedlinePlus

Context dependence of binding thermodynamics to Pex5p-pbd. (A) correlation of binding thermodynamic parameters between AGT proteins and PTS1 nonapeptides; (B) Binding free energies show little or no correlation with the different contributions (conformational or intrinsic) to binding enthalpies. In panel b, closed symbols are for AGT proteins and open symbols for PTS1 nonapeptides.
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biomolecules-05-00121-f006: Context dependence of binding thermodynamics to Pex5p-pbd. (A) correlation of binding thermodynamic parameters between AGT proteins and PTS1 nonapeptides; (B) Binding free energies show little or no correlation with the different contributions (conformational or intrinsic) to binding enthalpies. In panel b, closed symbols are for AGT proteins and open symbols for PTS1 nonapeptides.

Mentions: Representative ITC binding analyses of nonapeptides (containing PTS1 octapeptides plus a N-terminal tyrosine residue) to Pex5p-pbd are shown in Figure 5. A comparison of the binding affinities of these PTS1 nonapeptides with their corresponding full-length AGT counterparts reveals about one order of magnitude lower affinity for the peptides (Table 4; with the exception of AGT-SKL, that is close to the detection limit of ITC for a direct titration), in reasonable agreement with previous reports for AGT and SCP2 proteins [5,6,7]. The lower affinity in the peptides likely reflects the contributions from ancillary regions in the full-length cargo protein to complex formation, even though other minor contributions (such as electrostatic effects due to the presence of a charged N-terminal amine group in the peptides) should not be ruled out. Nevertheless, the difference in affinity between AGT proteins and PTS1 nonapeptides shows a reasonable correlation in terms of binding enthalpies and entropies (Figure 6A), suggesting the conservation of this context dependent cargo recognition (full-length protein vs. peptide) to some extent. This supports the idea that, overall, the binding mode of all AGT proteins to Pex5p-pbd is similar and mainly encoded in the C-terminal octapeptide sequence, while other ancillary regions in the AGT protein roughly add one order of magnitude to the binding affinity.


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)

Context dependence of binding thermodynamics to Pex5p-pbd. (A) correlation of binding thermodynamic parameters between AGT proteins and PTS1 nonapeptides; (B) Binding free energies show little or no correlation with the different contributions (conformational or intrinsic) to binding enthalpies. In panel b, closed symbols are for AGT proteins and open symbols for PTS1 nonapeptides.
© Copyright Policy
Related In: Results  -  Collection

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

biomolecules-05-00121-f006: Context dependence of binding thermodynamics to Pex5p-pbd. (A) correlation of binding thermodynamic parameters between AGT proteins and PTS1 nonapeptides; (B) Binding free energies show little or no correlation with the different contributions (conformational or intrinsic) to binding enthalpies. In panel b, closed symbols are for AGT proteins and open symbols for PTS1 nonapeptides.
Mentions: Representative ITC binding analyses of nonapeptides (containing PTS1 octapeptides plus a N-terminal tyrosine residue) to Pex5p-pbd are shown in Figure 5. A comparison of the binding affinities of these PTS1 nonapeptides with their corresponding full-length AGT counterparts reveals about one order of magnitude lower affinity for the peptides (Table 4; with the exception of AGT-SKL, that is close to the detection limit of ITC for a direct titration), in reasonable agreement with previous reports for AGT and SCP2 proteins [5,6,7]. The lower affinity in the peptides likely reflects the contributions from ancillary regions in the full-length cargo protein to complex formation, even though other minor contributions (such as electrostatic effects due to the presence of a charged N-terminal amine group in the peptides) should not be ruled out. Nevertheless, the difference in affinity between AGT proteins and PTS1 nonapeptides shows a reasonable correlation in terms of binding enthalpies and entropies (Figure 6A), suggesting the conservation of this context dependent cargo recognition (full-length protein vs. peptide) to some extent. This supports the idea that, overall, the binding mode of all AGT proteins to Pex5p-pbd is similar and mainly encoded in the C-terminal octapeptide sequence, while other ancillary regions in the AGT protein roughly add one order of magnitude to the binding affinity.

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