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The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1.

Evnouchidou I, Momburg F, Papakyriakou A, Chroni A, Leondiadis L, Chang SC, Goldberg AL, Stratikos E - PLoS ONE (2008)

Bottom Line: Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini.Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus.It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Scientific Research Demokritos, IRRP, Aghia Paraskevi, Greece.

ABSTRACT

Background: Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.

Methodology/principal findings: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains.

Conclusions/significance: To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

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Homology model of ERAP1 based on the TIFF3 structure.Top panel: The model is displayed in surface representation and coloured by electrostatic potential (red indicates negative electrostatic potential, blue indicates positive electrostatic potential). Notice the highly negatively charged (red) wide groove in the middle of the molecule adjacent to the catalytic Zn(II). The model peptide (of the sequence LMAAFAKAF in yellow) designed based on the library results to represent a near optimal substrate was docked in the active site so that the scissile amino-terminal peptide bond is positioned adjacent to the catalytic site Zn(II). Bottom panel, detail view of docked peptide in postulated binding site; the obstructing part of the protein has been removed to allow easier visualization of the peptide. The predicted location of the catalytic Zn(II) is indicated as a cyan sphere. Electrostatic potentials were calculated by the PME electrostatics add-on of VMD 1.8.5 and picture created using Pymol 0.99.
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pone-0003658-g007: Homology model of ERAP1 based on the TIFF3 structure.Top panel: The model is displayed in surface representation and coloured by electrostatic potential (red indicates negative electrostatic potential, blue indicates positive electrostatic potential). Notice the highly negatively charged (red) wide groove in the middle of the molecule adjacent to the catalytic Zn(II). The model peptide (of the sequence LMAAFAKAF in yellow) designed based on the library results to represent a near optimal substrate was docked in the active site so that the scissile amino-terminal peptide bond is positioned adjacent to the catalytic site Zn(II). Bottom panel, detail view of docked peptide in postulated binding site; the obstructing part of the protein has been removed to allow easier visualization of the peptide. The predicted location of the catalytic Zn(II) is indicated as a cyan sphere. Electrostatic potentials were calculated by the PME electrostatics add-on of VMD 1.8.5 and picture created using Pymol 0.99.

Mentions: The ERAP1 models we generated exhibit structural characteristics that appear highly relevant to the peptide library screen results. A central wide and deep cavity leads to the active site Zn(II) and is an obvious candidate to accommodate the peptide substrate (Figure 7, top panel). The cavity is large enough to accommodate a 9mer or even larger peptide (Figure 7, bottom panel). The cavity carries a strongly negative electrostatic potential but at the same time contains several hydrophobic pockets. The equivalent region of TIFF3 displays no such electrostatic potential distribution (data not shown). Based on the crystallographic analysis of TIFF3 there are 3 different molecular models that slightly vary in their inter-domain configuration. Depending on the TIFF3 model used for the construction of the ERAP1 homology model the opening to the aforementioned central cavity varies slightly in size; however the gross features (electrostatic potential and size) were found to be virtually unchanged (data not shown).


The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1.

Evnouchidou I, Momburg F, Papakyriakou A, Chroni A, Leondiadis L, Chang SC, Goldberg AL, Stratikos E - PLoS ONE (2008)

Homology model of ERAP1 based on the TIFF3 structure.Top panel: The model is displayed in surface representation and coloured by electrostatic potential (red indicates negative electrostatic potential, blue indicates positive electrostatic potential). Notice the highly negatively charged (red) wide groove in the middle of the molecule adjacent to the catalytic Zn(II). The model peptide (of the sequence LMAAFAKAF in yellow) designed based on the library results to represent a near optimal substrate was docked in the active site so that the scissile amino-terminal peptide bond is positioned adjacent to the catalytic site Zn(II). Bottom panel, detail view of docked peptide in postulated binding site; the obstructing part of the protein has been removed to allow easier visualization of the peptide. The predicted location of the catalytic Zn(II) is indicated as a cyan sphere. Electrostatic potentials were calculated by the PME electrostatics add-on of VMD 1.8.5 and picture created using Pymol 0.99.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2573961&req=5

pone-0003658-g007: Homology model of ERAP1 based on the TIFF3 structure.Top panel: The model is displayed in surface representation and coloured by electrostatic potential (red indicates negative electrostatic potential, blue indicates positive electrostatic potential). Notice the highly negatively charged (red) wide groove in the middle of the molecule adjacent to the catalytic Zn(II). The model peptide (of the sequence LMAAFAKAF in yellow) designed based on the library results to represent a near optimal substrate was docked in the active site so that the scissile amino-terminal peptide bond is positioned adjacent to the catalytic site Zn(II). Bottom panel, detail view of docked peptide in postulated binding site; the obstructing part of the protein has been removed to allow easier visualization of the peptide. The predicted location of the catalytic Zn(II) is indicated as a cyan sphere. Electrostatic potentials were calculated by the PME electrostatics add-on of VMD 1.8.5 and picture created using Pymol 0.99.
Mentions: The ERAP1 models we generated exhibit structural characteristics that appear highly relevant to the peptide library screen results. A central wide and deep cavity leads to the active site Zn(II) and is an obvious candidate to accommodate the peptide substrate (Figure 7, top panel). The cavity is large enough to accommodate a 9mer or even larger peptide (Figure 7, bottom panel). The cavity carries a strongly negative electrostatic potential but at the same time contains several hydrophobic pockets. The equivalent region of TIFF3 displays no such electrostatic potential distribution (data not shown). Based on the crystallographic analysis of TIFF3 there are 3 different molecular models that slightly vary in their inter-domain configuration. Depending on the TIFF3 model used for the construction of the ERAP1 homology model the opening to the aforementioned central cavity varies slightly in size; however the gross features (electrostatic potential and size) were found to be virtually unchanged (data not shown).

Bottom Line: Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini.Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus.It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Scientific Research Demokritos, IRRP, Aghia Paraskevi, Greece.

ABSTRACT

Background: Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.

Methodology/principal findings: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains.

Conclusions/significance: To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

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