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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 protein variants on the AGT-LRM background 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 mean ± s.d. for three independent experiments at 25 °C.
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biomolecules-05-00121-f004: Thermodynamic binding properties for the interaction of protein variants on the AGT-LRM background 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 mean ± s.d. for three independent experiments at 25 °C.

Mentions: As noted above, residues upstream from the C-terminal tripeptide of the PTS1 sequence may strongly modulate binding affinity [18,19]. Therefore, we have introduced PTS1 octapeptides previously described by [7] and displaying widely different binding affinities for Pex5p-pbd (Table 1). As expected from this previous report, we observed an increase in binding affinity compared to the natural PTS1 sequence of AGT (Figure 4A and Table 4). On the AGT-LRM background, several PTS1 sequences (AGT-SKL, CRA and HMG, Table 1) increased the affinity with a significant reduction of the enthalpic penalties (about 16 kJ/mol), while the BFE sequence (showing only a 2-fold increase in affinity) shows much favorable enthalpic contribution (about 35 kJ/mol) and much less favorable entropic contribution to binding (Figure 4A). These changes in the enthalpic contributions to binding could be at least partly explained by structural optimization of the PTS1 sequence at the Pex5p-pbd binding site similar to that proposed for the AGT–SKL variant (Table 3) and found for the K390A AGT mutant [6]. We must also note that the changes in binding affinity found are relatively modest in most of the cases due to significant enthalpy/entropy compensations (Figure 4A and [6]), highlighting the complex sequence/energetic dependence of the molecular recognition of PTS1 cargo proteins by Pex5p-pbd, which goes beyond the –SKL consensus tripeptide.


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 protein variants on the AGT-LRM background 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 mean ± s.d. for three independent experiments at 25 °C.
© Copyright Policy
Related In: Results  -  Collection

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

biomolecules-05-00121-f004: Thermodynamic binding properties for the interaction of protein variants on the AGT-LRM background 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 mean ± s.d. for three independent experiments at 25 °C.
Mentions: As noted above, residues upstream from the C-terminal tripeptide of the PTS1 sequence may strongly modulate binding affinity [18,19]. Therefore, we have introduced PTS1 octapeptides previously described by [7] and displaying widely different binding affinities for Pex5p-pbd (Table 1). As expected from this previous report, we observed an increase in binding affinity compared to the natural PTS1 sequence of AGT (Figure 4A and Table 4). On the AGT-LRM background, several PTS1 sequences (AGT-SKL, CRA and HMG, Table 1) increased the affinity with a significant reduction of the enthalpic penalties (about 16 kJ/mol), while the BFE sequence (showing only a 2-fold increase in affinity) shows much favorable enthalpic contribution (about 35 kJ/mol) and much less favorable entropic contribution to binding (Figure 4A). These changes in the enthalpic contributions to binding could be at least partly explained by structural optimization of the PTS1 sequence at the Pex5p-pbd binding site similar to that proposed for the AGT–SKL variant (Table 3) and found for the K390A AGT mutant [6]. We must also note that the changes in binding affinity found are relatively modest in most of the cases due to significant enthalpy/entropy compensations (Figure 4A and [6]), highlighting the complex sequence/energetic dependence of the molecular recognition of PTS1 cargo proteins by Pex5p-pbd, which goes beyond the –SKL consensus tripeptide.

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