Crystal Structure of a Dodecameric Tetrahedral-shaped Aminopeptidase

Abstract

Protein turnover is an essential process in living cells. The degradation of cytosolic polypeptides is mainly carried out by the proteasome, resulting in 7–9-amino acid long peptides. Further degradation is usually carried out by energy-independent proteases like the tricorn protease from Thermoplasma acidophilum. Recently, a novel tetrahedral-shaped dodecameric 480-kDa aminopeptidase complex (TET) has been described in Haloarcula marismortui that differs from the known ring- or barrel-shaped self-compartmentalizing proteases. This complex is capable of degrading most peptides down to amino acids. We present here the crystal structure of the tetrahedral aminopeptidase homolog FrvX from Pyrococcus horikoshii. The monomer has a typical clan MH fold, as found for example in Aeromonas proteolytica aminopeptidase, containing a dinuclear zinc active center. The quaternary structure is built by dimers with a length of 100 Å that form the edges of the tetrahedron. All 12 active sites are located on the inside of the tetrahedron. Substrate access is granted by pores with a maximal diameter of 10 Å, allowing only small peptides and unfolded proteins access to the active site. Protein turnover is an essential process in living cells. The degradation of cytosolic polypeptides is mainly carried out by the proteasome, resulting in 7–9-amino acid long peptides. Further degradation is usually carried out by energy-independent proteases like the tricorn protease from Thermoplasma acidophilum. Recently, a novel tetrahedral-shaped dodecameric 480-kDa aminopeptidase complex (TET) has been described in Haloarcula marismortui that differs from the known ring- or barrel-shaped self-compartmentalizing proteases. This complex is capable of degrading most peptides down to amino acids. We present here the crystal structure of the tetrahedral aminopeptidase homolog FrvX from Pyrococcus horikoshii. The monomer has a typical clan MH fold, as found for example in Aeromonas proteolytica aminopeptidase, containing a dinuclear zinc active center. The quaternary structure is built by dimers with a length of 100 Å that form the edges of the tetrahedron. All 12 active sites are located on the inside of the tetrahedron. Substrate access is granted by pores with a maximal diameter of 10 Å, allowing only small peptides and unfolded proteins access to the active site. The protein content of all living cells is constantly renewed through synthesis of new proteins and degradation of unneeded or misfolded proteins. This catabolism of proteins is a key cellular function and must be under spatial and temporal control to avert damage to the cell. Because prokaryotes lack membrane-bound compartments, degradation mostly takes place in large macromolecular self-compartmentalizing assemblies whose active sites are arranged in an inner cavity in order to only allow proteolysis of unfolded substrates.In all three kingdoms the degradation of cytosolic proteins is carried out predominantly by the ATP-dependent proteasome or similar energy-dependent proteases that generate oligopeptides 7–9 amino long are to be by assemblies like the energy-independent tricorn protease found in Thermoplasma large energy-independent protease that in the degradation of oligopeptides by the proteasome are the protease the from and All are and are of or with a and only a novel protease complex tetrahedral aminopeptidase (TET) tetrahedral Aeromonas proteolytica of tetrahedral Aeromonas proteolytica of has been from the Haloarcula marismortui of Å that is a complex with a novel tetrahedral that is by of to all self-compartmentalizing proteases described the cavity of complex is through and through to aminopeptidase of with a for and amino The complex most oligopeptides of amino peptides and for peptides to with dinuclear zinc from and is FrvX from the with is a of amino with a of and a of The protein mainly of a that to the clan to the This sites for zinc in the active by and dinuclear by a for the for and the of the of the with a active are to be zinc be or with like and be present with only in the active a of dinuclear is for aminopeptidase aminopeptidase and proteolytica aminopeptidase three of dinuclear All of similar of the active that FrvX with all proteases containing a to the the crystal structure of the energy-independent tetrahedral-shaped aminopeptidase complex FrvX from horikoshii. This dodecameric protein has the macromolecular as the described from The structure of the and the active the proteolytica and FrvX by from and a a The protein in cells in to an of by of cells in and by for the for and for The to a and with a from The containing FrvX a and The protein on a and by a FrvX by a in 100 The by found to be essential for the of the that the an Protein by acid only of the of protein as by of FrvX by the by of protein with of and 100 of an of The as with of a Å, Å, with in the of from protein by acid as the and of the of protein with of and 100 of and to an of 100 100 to with of a Å, the for structure to by the to and in a on the in a the zinc carried out the the in a from to for All and with are in and a Å, Å a Å, Å a 12 in of and to the as is the of the of the and is length The in of and to the as is the of the of the and is in a new and the the resulting of and of the to the of the and by the of aminopeptidase from as a by and by The resulting carried out by The in the large in a and from the as in the the zinc a of a in the active of three zinc in all are in of the structure with of the aminopeptidase of FrvX as The carried out in 100 with and or 10 The of by the of to present in the of FrvX and and and and as as of the all are of a in the the of amino acid and zinc are in as by and monomer found in MH clan from proteolytica is similar to the clan aminopeptidase from a of and The are only and similar are the from and of FrvX monomer is of and has of Å The the and the amino The is and The of is a that is by The on of the are and the are and The is of The of the the is located on the of the and a is located the of the is built of a by The are by the from a of to the of the of the from the of the of to the small of The of active zinc active is located in the the that is mainly by a by crystal All are of to the the active in the FrvX a zinc as by the zinc The in the and in and to all of the active of FrvX is in zinc are and are by and a The as is by and and by and is that the and a to zinc a with the The the is the active of FrvX is similar to the of the The dinuclear has the and the amino acid of the of FrvX and Aeromonas only in the of the and of the active site. acid are as are as and the are as or The in is the of the of the active sites of FrvX and that the of FrvX is similar to the for is mainly on and the of the to by of the to This of the to to the of the and the of the to in is to as the of the the to the of a tetrahedral The and is the of the a from to the new of the is known and that dinuclear are active with only in the active site. the Aeromonas that the zinc for and a on only the is the protein of has been that in the of a in the the is by in an of the zinc the of in all in the the the This that to in FrvX the a zinc and is for the site. This that the of and in FrvX are FrvX is active with only is the zinc is for the to that FrvX is a in from and and quaternary The function of FrvX in the and crystal the of tetrahedral by the structure of the on in the in the the tetrahedral-shaped by of the located of the has a length of 100 Å and is built by a and in the and form of an edges are by the by the The of the from of the and the and are in The are of and dodecameric tetrahedron. zinc are as down the a The and and are in and The and of the and are and protein protease of of amino are by a on the The and the and of Å and the and Å in and are dodecameric tetrahedral-shaped is the to as the to be dodecameric in the crystal the is by the of The resulting is similar to the in the FrvX structure with a of the with to in the is as Because of protein are is to the is a in to the to the FrvX quaternary structure and the crystal the with in that the is a under as from with to be a monomer in the crystal This is most of the to be the dodecameric an or and the a and the has a the for the the and the dodecameric to in the and is in the FrvX structure This is located the inside of the and to the and in the dodecameric is and has a with the amino in the is in the and with the with the dodecameric quaternary 12 active sites are located inside the tetrahedron. is by located the of the tetrahedron. The on the of the has a diameter of 10 are built by the and described for from through the of the and is by three the of is by the is small to allow proteins to only small peptides or unfolded protein as of FrvX in a The active zinc are as The is of the of large and small to the are active sites is Å and the three located the of the tetrahedron. The active sites is Å and the of FrvX and an The are in to be by FrvX All amino are in with for and of the amino is most similar to is the The of the is only a of This be as a by the for The is a for be by the of the active The is mainly by and to be for like the and acid be in the the by with that like with The with the of of as described under the amino of has been in in protein of active and protein like the tricorn protease with three aminopeptidase is in the degradation of peptides by the the of tricorn protease to be and for of The self-compartmentalizing aminopeptidase from Haloarcula the for most peptides down to amino acids. to all large self-compartmentalizing ring- or barrel-shaped protease like the proteasome, a tetrahedral-shaped dodecameric We present here for the the crystal structure of the similar dodecameric aminopeptidase FrvX from horikoshii. This protease to the aminopeptidase and a similar and active as proteolytica The is built by dimers the edges in a that all 12 active are located on the inner of the tetrahedron. Substrate access is located the of the of the tetrahedron. only small peptides and unfolded proteins as to the of FrvX has a for is a the the amino like or are with of for a dodecameric of the from and to as and on The crystal of the the a and dimers are self-compartmentalizing quaternary and for the of must be by the cell. the the of the or is known the for has to that the dodecameric are located the as has been for The protein content of all living cells is constantly renewed through synthesis of new proteins and degradation of unneeded or misfolded proteins. This catabolism of proteins is a key cellular function and must be under spatial and temporal control to avert damage to the cell. Because prokaryotes lack membrane-bound compartments, degradation mostly takes place in large macromolecular self-compartmentalizing assemblies whose active sites are arranged in an inner cavity in order to only allow proteolysis of unfolded all three kingdoms the degradation of cytosolic proteins is carried out predominantly by the ATP-dependent proteasome or similar energy-dependent proteases that generate oligopeptides 7–9 amino long are to be by assemblies like the energy-independent tricorn protease found in Thermoplasma large energy-independent protease that in the degradation of oligopeptides by the proteasome are the protease the from and All are and are of or with a and only Recently, a novel protease complex tetrahedral aminopeptidase (TET) tetrahedral Aeromonas proteolytica of tetrahedral Aeromonas proteolytica of has been from the Haloarcula marismortui of Å that is a complex with a novel tetrahedral that is by of to all self-compartmentalizing proteases described the cavity of complex is through and through to aminopeptidase of with a for and amino The complex most oligopeptides of amino peptides and for peptides to with dinuclear zinc from and is FrvX from the with FrvX is a of amino with a of and a of The protein mainly of a that to the clan to the This sites for zinc in the active by and dinuclear by a for the for and the of the of the with a active are to be zinc be or with like and be present with only in the active site. a of dinuclear is for aminopeptidase aminopeptidase and proteolytica aminopeptidase three of dinuclear All of similar of the active that FrvX with all proteases containing a to the the crystal structure of the energy-independent tetrahedral-shaped aminopeptidase complex FrvX from horikoshii. This dodecameric protein has the macromolecular as the described from The structure of the and the active the proteolytica and FrvX by from and a a The protein in cells in to an of by of cells in and by for the for and for The to a and with a from The containing FrvX a and The protein on a and by a FrvX by a in 100 The by found to be essential for the of the that the an Protein by acid only of the of protein as by of FrvX by the by of protein with of and 100 of an of The as with of a Å, Å, with in the of from protein by acid as the and of the of protein with of and 100 of and to an of 100 100 to with of a Å, the for structure to by the to and in a on the in a the zinc carried out the the in a from to for All and with are in and a Å, Å a Å, Å a 12 in of and to the as is the of the of the and is length The in of and to the as is the of the of the and is in a new and the the resulting of and of the to the of the and by the of aminopeptidase from as a by and by The resulting carried out by The in the large in a and from the as in the the zinc a of a in the active of three zinc in all are in of the structure with of the aminopeptidase of FrvX as The carried out in 100 with and or 10 The of by the of to Protein and FrvX by from and a a The protein in cells in to an of by of cells in and by for the for and for The to a and with a from The containing FrvX a and The protein on a and by a FrvX by a in 100 The by found to be essential for the of the that the an Protein by acid only of the of protein as by of FrvX by the by of protein with of and 100 of an of The as with of a Å, Å, with in the of from protein by acid as the and of the of protein with of and 100 of and to an of 100 100 to with of a Å, the for structure to by the to and in a on the in a the zinc carried out the the in a from to for All and with are in and the the resulting of and of the to the of the and by the of aminopeptidase from as a by and by The resulting carried out by The in the large in a and from the as in the the zinc a of a in the active of three zinc in all are in The of the structure with of the aminopeptidase of FrvX as The carried out in 100 with and or 10 The of by the of to present in the of FrvX and and and and as as of the all are of a in the the of amino acid and zinc are in as by and monomer found in MH clan from proteolytica is similar to the clan aminopeptidase from a of and The are only and similar are the from and of FrvX monomer is of and has of Å The the and the amino The is and The of is a that is by The on of the are and the are and The is of The of the the is located on the of the and a is located the of the is built of a by The are by the from a of to the of the of the from the of the of to the small of The of active zinc active is located in the the that is mainly by a by crystal All are of to the the active in the FrvX a zinc as by the zinc The in the and in and to all of the active of FrvX is in zinc are and are by and a The as is by and and by and is that the and a to zinc a with the The the is the active of FrvX is similar to the of the The dinuclear has the and the amino acid of the of FrvX and Aeromonas only in the of the and of the of the active sites of FrvX and that the of FrvX is similar to the for is mainly on and the of the to by of the to This of the to to the of the and the of the to in is to as the of the the to the of a tetrahedral The and is the of the a from to the new of the is known and that dinuclear are active with only in the active site. the Aeromonas that the zinc for and a on only the is the protein of has been that in the of a in the the is by in an of the zinc the of in all in the the the This that to in FrvX the a zinc and is for the site. This that the of and in FrvX are FrvX is active with only is the zinc is for the to that FrvX is a in from and and quaternary The function of FrvX in the and crystal the of tetrahedral by the structure of the on in the in the the tetrahedral-shaped by of the located of the has a length of 100 Å and is built by a and in the and form of an edges are by the by the The of the from of the and the and are in The are of and dodecameric tetrahedron. zinc are as down the a The and and are in and The and of the and are and protein protease of of amino are by a on the The and the and of Å and the and Å in and are dodecameric tetrahedral-shaped is the to as the to be dodecameric in the crystal the is by the of The resulting is similar to the in the FrvX structure with a of the with to in the is as Because of protein are is to the is a in to the to the FrvX quaternary structure and the crystal the with in that the is a under as from with to be a monomer in the crystal This is most of the to be the dodecameric an or and the a and the has a the for the the and the dodecameric to in the and is in the FrvX structure This is located the inside of the and to the and in the dodecameric is and has a with the amino in the is in the and with the with the dodecameric quaternary 12 active sites are located inside the tetrahedron. is by located the of the tetrahedron. The on the of the has a diameter of 10 are built by the and described for from through the of the and is by three the of is by the is small to allow proteins to only small peptides or unfolded protein as of FrvX in a The active zinc are as The is of the of large and small to the are active sites is Å and the three located the of the tetrahedron. The active sites is Å and the of FrvX and an The are in to be by FrvX All amino are in with for and of the amino is most similar to is the The of the is only a of This be as a by the for The is a for be by the of the active The is mainly by and to be for like the and acid be in the the by with that like with The with the of of as described under the amino present in the of FrvX and and and and as as of the all are of a in the the of amino acid and zinc are in as by and monomer found in MH clan from proteolytica is similar to the clan aminopeptidase from a of and The are only and similar are the from and of The FrvX monomer is of and has of Å The the and the amino The is and The of is a that is by The on of the are and the are and The is of The of the the is located on the of the and a is located the of the is built of a by The are by the from a of to the of the of the from the of the of to the small of The of active zinc The active is located in the the that is mainly by a by crystal All are of to the the active in the FrvX a zinc as by the zinc The in the and in and to all of the active of FrvX is in zinc are and are by and a The as is by and and by and is that the and a to zinc a with the The the is the active of FrvX is similar to the of the The dinuclear has the and the amino acid of the of FrvX and Aeromonas only in the of the and Because of the of the active sites of FrvX and that the of FrvX is similar to the for is mainly on and the of the to by of the to This of the to to the of the and the of the to in is to as the of the the to the of a tetrahedral The and is the of the a from to the new of the is known and that dinuclear are active with only in the active site. the Aeromonas that the zinc for and a on only the is the protein of has been that in the of a in the the is by in an of the zinc the of in all in the the the This that to in FrvX the a zinc and is for the site. This that the of and in FrvX are FrvX is active with only is the zinc is for the to that FrvX is a in from and and quaternary The function of FrvX in the and crystal the of tetrahedral by the structure of the on in the The in the the tetrahedral-shaped by of the located of the has a length of 100 Å and is built by a and in the and form of an edges are by the by the The of the from of the and the and are in The are of and the to as the to be dodecameric in the crystal the is by the of The resulting is similar to the in the FrvX structure with a of the with to in the is as Because of protein are is to the is a in to the to the FrvX quaternary structure and the crystal the with in that the is a under as The from with to be a monomer in the crystal This is most of the to be the dodecameric an or and the a and the has a the for the the and the dodecameric to in the and is in the FrvX structure This is located the inside of the and to the and in the dodecameric is and has a with the amino in the is in the and with the with the dodecameric quaternary All 12 active sites are located inside the tetrahedron. is by located the of the tetrahedron. The on the of the has a diameter of 10 are built by the and described for from through the of the and is by three the of is by the is small to allow proteins to only small peptides or unfolded protein as The active sites is Å and the three located the of the tetrahedron. The active sites is Å and the of FrvX and an The are in to be by FrvX All amino are in with for and of the amino is most similar to is the The of the is only a of This be as a by the for The is a for be by the of the active The is mainly by and to be for like the and acid be in the the by with that like with The with the of of has been in in protein of active and protein like the tricorn protease with three aminopeptidase is in the degradation of peptides by the the of tricorn protease to be and for of The self-compartmentalizing aminopeptidase from Haloarcula the for most peptides down to amino acids. to all large self-compartmentalizing ring- or barrel-shaped protease like the proteasome, a tetrahedral-shaped dodecameric We present here for the the crystal structure of the similar dodecameric aminopeptidase FrvX from horikoshii. This protease to the aminopeptidase and a similar and active as proteolytica The is built by dimers the edges in a that all 12 active are located on the inner of the tetrahedron. Substrate access is located the of the of the tetrahedron. only small peptides and unfolded proteins as to the of FrvX has a for is a the the amino like or are with of for a dodecameric of the from and to as and on The crystal of the the a and dimers are self-compartmentalizing quaternary and for the of must be by the cell. the the of the or is known the for has to that the dodecameric are located the as has been for The of has been in in protein of active and protein like the tricorn protease with three aminopeptidase is in the degradation of peptides by the the of tricorn protease to be and for of The self-compartmentalizing aminopeptidase from Haloarcula the for most peptides down to amino acids. to all large self-compartmentalizing ring- or barrel-shaped protease like the proteasome, a tetrahedral-shaped dodecameric We present here for the the crystal structure of the similar dodecameric aminopeptidase FrvX from horikoshii. This protease to the aminopeptidase and a similar and active as proteolytica The is built by dimers the edges in a that all 12 active are located on the inner of the tetrahedron. Substrate access is located the of the of the tetrahedron. only small peptides and unfolded proteins as to the of FrvX has a for is a the the amino like or are with of for a dodecameric of the from and to as and on The crystal of the the a and dimers are self-compartmentalizing quaternary and for the of must be by the cell. the the of the or is known the for has to that the dodecameric are located the as has been for We from the in and from and and from the for with