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Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

Gawlik K, Shiryaev SA, Zhu W, Motamedchaboki K, Desjardins R, Day R, Remacle AG, Stec B, Strongin AY - PLoS ONE (2009)

Bottom Line: Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms.In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.

Methodology/principal findings: We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.

Conclusions/significance: Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

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Related in: MedlinePlus

Structural model of the WT and K117P furins.(A) Stereo view of the WT furin catalytic cleft before (a red ribbon) and after (a blue ribbon) the cleavage at the primary Arg- Thr-Lys-Arg107↓Asp108 site. The red curved arrow indicates the motion of the 108–117 sequence from its position in profurin to its final position in the mature furin enzyme. The open conformation of the mature furin enzyme is stabilized by strong electrostatic interactions of Asp115 and a calcium ion (a blue sphere), and, in addition, by two salt bridges (Glu112-Lys135 and Lys117-Glu362). (B) The 26–107 prodomain and the 108–574 catalytic domain-P domain of furin are shown in green and grey, respectively. The modeled structure of the 108–117 sequence in profurin is shown in red while the actual structure of the 108–117 sequence in the mature WT furin enzyme is in blue. Top panels - In the structure of the WT furin, the 76–107 peptide sequence, which is generated as a result of the secondary cleavage, is shown as an orange ribbon. The arrows 1 and 2 indicate the primary and the secondary cleavage sites, respectively. Left panel, profurin; middle panel, mature furin, right panel, close-up. Bottom panels – In the K117P mutant the formation of an additional helix is necessary to eject the emerging N-terminus from the active site but it is sterically forbidden because of the adjacent Pro116. As a result, the N-end region of the mature mutant enzyme continue to occupy the S′ sub-sites leading to the inactive enzyme. The arrow 1 indicates the cleavage at the primary cleavage site alone that, as a result, generates the liberated intact prodomain (green). Left panel, profurin; middle panel, mature furin, right panel, close-up.
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pone-0005031-g001: Structural model of the WT and K117P furins.(A) Stereo view of the WT furin catalytic cleft before (a red ribbon) and after (a blue ribbon) the cleavage at the primary Arg- Thr-Lys-Arg107↓Asp108 site. The red curved arrow indicates the motion of the 108–117 sequence from its position in profurin to its final position in the mature furin enzyme. The open conformation of the mature furin enzyme is stabilized by strong electrostatic interactions of Asp115 and a calcium ion (a blue sphere), and, in addition, by two salt bridges (Glu112-Lys135 and Lys117-Glu362). (B) The 26–107 prodomain and the 108–574 catalytic domain-P domain of furin are shown in green and grey, respectively. The modeled structure of the 108–117 sequence in profurin is shown in red while the actual structure of the 108–117 sequence in the mature WT furin enzyme is in blue. Top panels - In the structure of the WT furin, the 76–107 peptide sequence, which is generated as a result of the secondary cleavage, is shown as an orange ribbon. The arrows 1 and 2 indicate the primary and the secondary cleavage sites, respectively. Left panel, profurin; middle panel, mature furin, right panel, close-up. Bottom panels – In the K117P mutant the formation of an additional helix is necessary to eject the emerging N-terminus from the active site but it is sterically forbidden because of the adjacent Pro116. As a result, the N-end region of the mature mutant enzyme continue to occupy the S′ sub-sites leading to the inactive enzyme. The arrow 1 indicates the cleavage at the primary cleavage site alone that, as a result, generates the liberated intact prodomain (green). Left panel, profurin; middle panel, mature furin, right panel, close-up.

Mentions: In our modeled profurin structure, the relative position of the prodomain and the catalytic domain was similar to that reported for the subtilisin-propeptide complex (Protein Data Bank code 1SCJ). The identified helix sheet interaction of the propeptide with the catalytic domain is conserved in the subtilisin family [22], [23], [24] and also in PCs including furin (Figure 1A). The 101–113 sequence region that spans the primary Arg-Thr-Lys-Arg107↓Asp108 cleavage site was positioned in the active site of profurin in a manner similar to the dec-RVKR-cmk inhibitor in the 1P8J crystal structure of murine furin [25], [26].


Autocatalytic activation of the furin zymogen requires removal of the emerging enzyme's N-terminus from the active site.

Gawlik K, Shiryaev SA, Zhu W, Motamedchaboki K, Desjardins R, Day R, Remacle AG, Stec B, Strongin AY - PLoS ONE (2009)

Structural model of the WT and K117P furins.(A) Stereo view of the WT furin catalytic cleft before (a red ribbon) and after (a blue ribbon) the cleavage at the primary Arg- Thr-Lys-Arg107↓Asp108 site. The red curved arrow indicates the motion of the 108–117 sequence from its position in profurin to its final position in the mature furin enzyme. The open conformation of the mature furin enzyme is stabilized by strong electrostatic interactions of Asp115 and a calcium ion (a blue sphere), and, in addition, by two salt bridges (Glu112-Lys135 and Lys117-Glu362). (B) The 26–107 prodomain and the 108–574 catalytic domain-P domain of furin are shown in green and grey, respectively. The modeled structure of the 108–117 sequence in profurin is shown in red while the actual structure of the 108–117 sequence in the mature WT furin enzyme is in blue. Top panels - In the structure of the WT furin, the 76–107 peptide sequence, which is generated as a result of the secondary cleavage, is shown as an orange ribbon. The arrows 1 and 2 indicate the primary and the secondary cleavage sites, respectively. Left panel, profurin; middle panel, mature furin, right panel, close-up. Bottom panels – In the K117P mutant the formation of an additional helix is necessary to eject the emerging N-terminus from the active site but it is sterically forbidden because of the adjacent Pro116. As a result, the N-end region of the mature mutant enzyme continue to occupy the S′ sub-sites leading to the inactive enzyme. The arrow 1 indicates the cleavage at the primary cleavage site alone that, as a result, generates the liberated intact prodomain (green). Left panel, profurin; middle panel, mature furin, right panel, close-up.
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Related In: Results  -  Collection

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pone-0005031-g001: Structural model of the WT and K117P furins.(A) Stereo view of the WT furin catalytic cleft before (a red ribbon) and after (a blue ribbon) the cleavage at the primary Arg- Thr-Lys-Arg107↓Asp108 site. The red curved arrow indicates the motion of the 108–117 sequence from its position in profurin to its final position in the mature furin enzyme. The open conformation of the mature furin enzyme is stabilized by strong electrostatic interactions of Asp115 and a calcium ion (a blue sphere), and, in addition, by two salt bridges (Glu112-Lys135 and Lys117-Glu362). (B) The 26–107 prodomain and the 108–574 catalytic domain-P domain of furin are shown in green and grey, respectively. The modeled structure of the 108–117 sequence in profurin is shown in red while the actual structure of the 108–117 sequence in the mature WT furin enzyme is in blue. Top panels - In the structure of the WT furin, the 76–107 peptide sequence, which is generated as a result of the secondary cleavage, is shown as an orange ribbon. The arrows 1 and 2 indicate the primary and the secondary cleavage sites, respectively. Left panel, profurin; middle panel, mature furin, right panel, close-up. Bottom panels – In the K117P mutant the formation of an additional helix is necessary to eject the emerging N-terminus from the active site but it is sterically forbidden because of the adjacent Pro116. As a result, the N-end region of the mature mutant enzyme continue to occupy the S′ sub-sites leading to the inactive enzyme. The arrow 1 indicates the cleavage at the primary cleavage site alone that, as a result, generates the liberated intact prodomain (green). Left panel, profurin; middle panel, mature furin, right panel, close-up.
Mentions: In our modeled profurin structure, the relative position of the prodomain and the catalytic domain was similar to that reported for the subtilisin-propeptide complex (Protein Data Bank code 1SCJ). The identified helix sheet interaction of the propeptide with the catalytic domain is conserved in the subtilisin family [22], [23], [24] and also in PCs including furin (Figure 1A). The 101–113 sequence region that spans the primary Arg-Thr-Lys-Arg107↓Asp108 cleavage site was positioned in the active site of profurin in a manner similar to the dec-RVKR-cmk inhibitor in the 1P8J crystal structure of murine furin [25], [26].

Bottom Line: Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms.In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

View Article: PubMed Central - PubMed

Affiliation: Burnham Institute for Medical Research, La Jolla, California, United States of America.

ABSTRACT

Background: Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.

Methodology/principal findings: We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.

Conclusions/significance: Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.

Show MeSH
Related in: MedlinePlus