The self-inhibited structure of full-length PCSK9 at 1.9 Å reveals structural homology with resistin within the C-terminal domain

Hampton, Eric; Knuth, Mark W.; Li, Jun; Harris, Jennifer L.; Lesley, Scott A.; Spraggon, Glen

doi:10.1073/pnas.0703402104

Cited by 138 publications

(117 citation statements)

References 44 publications

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“…The crystal structure of apo-PCSK9 revealed a tightly bound prodomain that is predicted to render the active site inaccessible to exogenous substrates (12)(13)(14). The structure of the PCSK9 prodomain and catalytic domain is similar to that of other subtilisin-like serine proteases.…”

mentioning

confidence: 81%

Molecular basis for LDL receptor recognition by PCSK9

Kwon¹,

Lagace²,

McNutt³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

375

355

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hypercholesterolemia ͉ proprotein convertase ͉ crystal structure P roprotein convertase (PC) subtilisin/kexin type 9 (PCSK9) is a serine protease of the PC family that has profound effects on plasma low-density lipoprotein (LDL)-cholesterol (LDL-C) levels through its ability to mediate LDL receptor (LDLR) protein degradation (1, 2). The link between PCSK9 and plasma LDL-C levels was first established by the discovery of missense mutations in PCSK9 that were present in patients with an autosomal dominant form of familial hypercholesterolemia (FH) (3). These mutations were speculated to result in a gain-offunction of PCSK9 owing to their mode of inheritance. Subsequently, loss-of-function mutations in PCSK9 were discovered that are associated with low plasma LDL-C levels and significantly reduced coronary heart disease (CHD) (4, 5).The overall domain structure of PCSK9 is similar to other PC family members. It includes a signal peptide, followed by a prodomain, a subtilisin-like catalytic domain, and a variable C-terminal domain (6). The prodomain serves a dual role as a chaperone for folding and as an inhibitor of catalytic activity (7-9). Autocatalysis between Gln-152 and Ser-153 (VFAQ:SIP) separates the prodomain from the catalytic domain, but the prodomain remains bound, occluding the catalytic site (10, 11). For other PC family members, a second catalytic cleavage is required to release the prodomain, which unmasks the catalytic site, resulting in an active protease (7). No site of secondary cleavage has been identified that activates PCSK9.The crystal structure of apo-PCSK9 revealed a tightly bound prodomain that is predicted to render the active site inaccessible to exogenous substrates (12-14). The structure of the PCSK9 prodomain and catalytic domain is similar to that of other subtilisin-like serine proteases. The C-terminal domain of PCSK9 contains three six-stranded ␤-sheet subdomains arranged with quasi-threefold symmetry. This domain shares structural homology to the adipokine resistin and has been speculated to mediate protein-protein interactions (12-14).Recent studies have provided insights into the site and mode of PCSK9's action. Addition of recombinant PCSK9 to the medium of cultured hepatocytes results in a dramatic reduction in LDLR number. PCSK9 binds directly to the LDLR on the cell surface and PCSK9-stimulated degradation of the LDLR requires autosomal recessive hypercholesterolemia (ARH), an adaptor protein needed for internalization of LDLRs (11). The affinity of PCSK9 binding to the LDLR is enhanced at acidic pH, suggesting that PCSK9 binds more avidly to LDLRs in the lysosomal/endosomal compartments (12,15,16). One gain-offunction mutant, Asp-374-Tyr, was Ϸ10-fold more active than wild-type PCSK9 in mediating degradation of LDLRs, owing to an Ϸ5-to 30-fold increased affinity of PCSK9 for the LDLR (11,12,15). Although autocatalysis of PCSK9 is required for proper folding and secretion, catalytic activity was not required for PCSK9-mediated LDLR degradation when added to cultured cells (...

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mentioning

confidence: 81%

Molecular basis for LDL receptor recognition by PCSK9

Kwon¹,

Lagace²,

McNutt³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

375

355

View full text Add to dashboard Cite

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“…2B, lane 7) shows that the proteolysis of substrate PCSK9 is highly specific for the active protease PCSK9⌬Pro . Furthermore, because protease PCSK9⌬Pro is significantly more active than protease PCSK9 , these data show that the prodomain is a cis-acting inhibitor of intermolecular proteolysis, consistent with the known PCSK9 structure (22,24,31). We were particularly encouraged by our ability to amplify the detection of intermolecular PCSK9 proteolysis, especially given that the PCSK9 protease is thought to only undergo a single turnover event.…”

Section: The Pcsk9 Catalytic Domain Can Perform Intermolecularmentioning

confidence: 99%

“…Based on the occupancy of the active site by the prodomain C terminus (22,24,31) as well as our own results of the prodomain inhibiting intermolecular proteolysis in cis, we hypothesized that exogenous prodomain would serve as a model inhibitor and effectively block intermolecular proteolysis in trans. To test this we performed co-transfections in the intermolecular cleavage system (protease PCSK9⌬Pro acting upon substrate PCSK9 ) with an additional wild-type prodomain construct termed prodomain WT (Fig.…”

Section: The Pcsk9 Catalytic Domain Can Perform Intermolecularmentioning

confidence: 99%

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Active Site and Cleavage Sequence Differentially Regulate Protein Secretion from Proteolysis

Chorba

Shokat

2014

Journal of Biological Chemistry

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Background: PCSK9 inhibition shows promise in the treatment of hypercholesterolemia and prevention of atherosclerotic heart disease. Results: PCSK9 proteolysis and secretion have different structural requirements near the protein active site. Conclusion: PCSK9 proteolysis and secretion occur in two separate steps. Significance: The PCSK9 active site can serve as an allosteric modulator of protein secretion, suggesting a new strategy for inhibition.

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“…The structure of PCSK9 has been determined (7)(8)(9). The prodomain and catalytic domain (Pro-Cat domain) bind to EGF-A of the LDLR (6,10,11).…”

mentioning

confidence: 99%

A Two-step Binding Model of PCSK9 Interaction with the Low Density Lipoprotein Receptor

Yamamoto¹,

Lu²,

Ryan³

2011

Journal of Biological Chemistry

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PCSK9 (proprotein convertase subtilisin-like/kexin type 9) is an emerging target for pharmaceutical intervention. This multidomain protein interacts with the LDL receptor (LDLR), promoting receptor degradation. Insofar as PCSK9 inhibition induces a decrease in plasma cholesterol levels, understanding the nature of the binding interaction between PCSK9 and the LDLR is of critical importance. In this study, the ability of PCSK9 to compete with apoE3 N-terminal domain-containing reconstituted HDL for receptor binding was examined. Whereas full-length PCSK9 was an effective competitor, the N-terminal domain (composed of the prodomain and catalytic domain) was not. Surprisingly, the C-terminal domain (CT domain) of PCSK9 was able to compete. Using a direct binding interaction assay, we show that the PCSK9 CT domain bound to the LDLR in a calcium-dependent manner and that co-incubation with the prodomain and catalytic domain had no effect on this binding. To further characterize this interaction, two LDLR fragments, the classical ligand-binding domain (LBD) and the EGF precursor homology domain, were expressed in stably transfected HEK 293 cells and isolated. Binding assays showed that the PCSK9 CT domain bound to the LBD at pH 5.4. Thus, CT domain interaction with the LBD of the LDLR at endosomal pH constitutes a second step in the PCSK9-mediated LDLR binding that leads to receptor degradation.

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The self-inhibited structure of full-length PCSK9 at 1.9 Å reveals structural homology with resistin within the C-terminal domain

Cited by 138 publications

References 44 publications

Molecular basis for LDL receptor recognition by PCSK9

Molecular basis for LDL receptor recognition by PCSK9

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Active Site and Cleavage Sequence Differentially Regulate Protein Secretion from Proteolysis

A Two-step Binding Model of PCSK9 Interaction with the Low Density Lipoprotein Receptor

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