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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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(A) Molecular architecture of the AGT:Pex5-pbd complex (PDB code: 3R9A), indicating the three regions in AGT involved in the interaction surface. The amount of polar and apolar surface buried is determined from this complex and used to estimate theoretical values of intrinsic enthalpy and heat capacity of binding using Equations (1) and (2) (B) and (C) Changes in accessible surface at the residue level for AGT (B) and Pex5p-pbd (C) upon complex formation.
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biomolecules-05-00121-f001: (A) Molecular architecture of the AGT:Pex5-pbd complex (PDB code: 3R9A), indicating the three regions in AGT involved in the interaction surface. The amount of polar and apolar surface buried is determined from this complex and used to estimate theoretical values of intrinsic enthalpy and heat capacity of binding using Equations (1) and (2) (B) and (C) Changes in accessible surface at the residue level for AGT (B) and Pex5p-pbd (C) upon complex formation.

Mentions: The interaction between AGT and the PTS1 binding domain of Pex5p (Pex5p-pbd) exclusively involves the C-terminal domain of AGT (residues 283–392) (PDB: 3R9A; Figure 1A). While the overall conformation and activity of AGT are not significantly affected by the interaction, a conformational change is observed in the C-terminal bundle domain of Pex5p-pbd [5]. The reported structure shows three topologically distinct regions of AGT interacting in the complex (Figure 1A): (i) residues 389–392, which constitute the minimal PTS1 sequence and interact with the central cavity of the TPR domains of Pex5p-pbd. This region forms an interface with the receptor of about 600 Å2 [5]; (ii) residues 381–388 (helix α13) and 327–330 (contained in the ancillary targeting sequence or PTS1A; [17]), and recently named the extended PTS1 [5]. Mutation of residues in this region show mild to moderate changes in affinity (about 2-fold lower affinity in Y330W and A328W, and about 5-fold in Y330A; [5]); (iii) residues 303–307. Regions II and III form an additional surface of about 400 Å2 [5]. Changes in molecular surface upon complex formation at the residue level are shown in Figure 1B,C.


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)

(A) Molecular architecture of the AGT:Pex5-pbd complex (PDB code: 3R9A), indicating the three regions in AGT involved in the interaction surface. The amount of polar and apolar surface buried is determined from this complex and used to estimate theoretical values of intrinsic enthalpy and heat capacity of binding using Equations (1) and (2) (B) and (C) Changes in accessible surface at the residue level for AGT (B) and Pex5p-pbd (C) upon complex formation.
© Copyright Policy
Related In: Results  -  Collection

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

biomolecules-05-00121-f001: (A) Molecular architecture of the AGT:Pex5-pbd complex (PDB code: 3R9A), indicating the three regions in AGT involved in the interaction surface. The amount of polar and apolar surface buried is determined from this complex and used to estimate theoretical values of intrinsic enthalpy and heat capacity of binding using Equations (1) and (2) (B) and (C) Changes in accessible surface at the residue level for AGT (B) and Pex5p-pbd (C) upon complex formation.
Mentions: The interaction between AGT and the PTS1 binding domain of Pex5p (Pex5p-pbd) exclusively involves the C-terminal domain of AGT (residues 283–392) (PDB: 3R9A; Figure 1A). While the overall conformation and activity of AGT are not significantly affected by the interaction, a conformational change is observed in the C-terminal bundle domain of Pex5p-pbd [5]. The reported structure shows three topologically distinct regions of AGT interacting in the complex (Figure 1A): (i) residues 389–392, which constitute the minimal PTS1 sequence and interact with the central cavity of the TPR domains of Pex5p-pbd. This region forms an interface with the receptor of about 600 Å2 [5]; (ii) residues 381–388 (helix α13) and 327–330 (contained in the ancillary targeting sequence or PTS1A; [17]), and recently named the extended PTS1 [5]. Mutation of residues in this region show mild to moderate changes in affinity (about 2-fold lower affinity in Y330W and A328W, and about 5-fold in Y330A; [5]); (iii) residues 303–307. Regions II and III form an additional surface of about 400 Å2 [5]. Changes in molecular surface upon complex formation at the residue level are shown in Figure 1B,C.

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