1.2 Å Crystal Structure of the Serine Carboxyl Proteinase Pro-Kumamolisin

Comellas-Bigler, M.; Maskos, K.; Huber, Robert; Oyama, Hiroshi; Oda, Kōhei; Bode, Wolfram

doi:10.1016/j.str.2004.04.013

Cited by 57 publications

(83 citation statements)

References 43 publications

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“…Given that mixed inhibition can lead to an uncompetitive kinetics (32), it may be difficult to unequivocally distinguish these two modes of inhibition (33). The high resolution structure of the intact prokumamolisin, as the first prosedolisin, has been resolved recently (38), and it has been shown that the prosegment exhibits a half-␤ sandwich core docking to the catalytic domain, similarly to the equivalent subtilases and PCs. Given the overall structural similarity of sedolisins (5), a similar arrangement of the TPP I prosegment and catalytic domain of TPP I might be expected.…”

Section: Discussionmentioning

confidence: 99%

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Golabek

Dolzhanskaya

Walus

et al. 2008

Journal of Biological Chemistry

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Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment. Tripeptidyl peptidase I (TPP I)2 is an acidic lysosomal hydrolase sequentially removing tripeptides en bloc from the N terminus of small, unstructured polypeptides (for a review, see Ref.1). The natural substrate(s) of the enzyme still remains elusive. TPP I is widely distributed in the human body, with a high expression level in brain tissue (2, 3). Naturally occurring mutations in TPP I underlie a devastating neurodegenerative disorder of early childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2) (4).On the basis of significant sequence homology and inhibitory studies, TPP I was assigned to a family of pepstatin-insensitive serine-carboxyl proteases recently renamed sedolisins (for a Like most proteolytic enzymes, TPP I is synthesized as an inactive precursor, zymogen (8, 9). The prepro-TPP I consists of a 19-amino acid (aa) signal peptide cleaved off when the newly forming polypeptide chain is inserted into the endoplasmic reticulum lumen, the 176-aa prosegment (or prodomain), and a 368-aa catalytic domain. As we demonstrated earlier, the removal of the prosegment from a purified TPP I proenzyme (pro-TPP I) occurs via an autocatalytic, intramolecular mechanism, whereby the mature enzyme does not significantly participate in its own generation (10). Efficient in vitro processing and autoactivation of the proenzyme is triggered by lowering the pH of the proenzyme solution to below 4.5. When pro-TPP I is activated at pH 4.5 and above, it is processed slowly and generates additional 6-and 14-aa N-terminal extensions in the newly formed polypeptide, rendering it enzymatically inactive (10). However, as we showed earlier, the presence of polyanionic compounds, such...

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Section: Discussionmentioning

confidence: 99%

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Golabek

Dolzhanskaya

Walus

et al. 2008

Journal of Biological Chemistry

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“…The positioning of Pro8 is critical as it points away from the body of the protein so moving the scissile peptide bond out of reach of the catalytic serine. This is in contrast to the mode of binding of the classical prodomains from ESPs (26)(27)(28) and the subtilisin-like proteins kumamolisin (29,30) and fervidolysin (31) over the active site, which retain the target peptide bond in a position to allow autocatalytic processing. Interactions between subtilisins and classical proteinacous inhibitors (32,33) such as CI2 (34) are normally restricted to the active site and substrate binding region whereas the N-terminal extension of ISP forms distinct interactions with regions of the protein beyond the substrate binding pocket.…”

Section: Discussionmentioning

confidence: 89%

“…Comparison of the structure of proISP with that of NΔ 18 -ISP S250A determined here provides an explanation and reveals that the N-terminal extension regulates protease activity via an additional mechanism: disruption of the catalytic triad and S1 binding pocket. Leu6 and Tyr9 within the N-terminal extension point toward the loop carrying the catalytic serine mutation resulting in displacement of the main-chain atoms of the loop containing Ala250 by ∼1 Å compared to other subtilisins, including those with the active site serine residue mutated to alanine (26,30). This results in Ala250 being displaced by ∼1.6 Å from the position required for the formation of a catalytic triad.…”

Section: Discussionmentioning

confidence: 99%

Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism

Gamble

Künze

Dodson

et al. 2011

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

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A distinct class of the biologically important subtilisin family of serine proteases functions exclusively within the cell and forms a major component of the bacilli degradome. However, the mode and mechanism of posttranslational regulation of intracellular protease activity are unknown. Here we describe the role played by a short N-terminal extension prosequence novel amongst the subtilisins that regulates intracellular subtilisin protease (ISP) activity through two distinct modes: active site blocking and catalytic triad rearrangement. The full-length proenzyme (proISP) is inactive until specific proteolytic processing removes the first 18 amino acids that comprise the N-terminal extension, with processing appearing to be performed by ISP itself. A synthetic peptide corresponding to the N-terminal extension behaves as a mixed noncompetitive inhibitor of active ISP with a K i of 1 μM. The structure of the processed form has been determined at 2.6 Å resolution and compared with that of the full-length protein, in which the N-terminal extension binds back over the active site. Unique to ISP, a conserved proline introduces a backbone kink that shifts the scissile bond beyond reach of the catalytic serine and in addition the catalytic triad is disrupted. In the processed form, access to the active site is unblocked by removal of the N-terminal extension and the catalytic triad rearranges to a functional conformation. These studies provide a new molecular insight concerning the mechanisms by which subtilisins and protease activity as a whole, especially within the confines of a cell, can be regulated.posttranslational modification | protease regulation | proprotein activation | protease structure S ubtilisins are serine endopeptidases ubiquitous in nature, spread across the eubacteria, archaebacteria, eukaryotes, and viruses (1). They play a variety of important biological roles that include specific posttranslational processing of hormones (2, 3), virulence and infection factors in various pathogens (4, 5), and as nonspecific digestive proteases (1). The distinctive primary structure features common to the majority of subtilisins, and typified by the bacilli extracellular subtilisins (ESP), comprise a N-terminal signal sequence with an adjacent prodomain required for correct folding of the mature, catalytic domain (6-8) (Fig. 1A). Both the signal sequence and the prodomain are posttranslationally removed, the latter autocatalytically. The bacilli ESPs have a broad substrate specificity that suites their role as scavenging proteases, and their robustness to harsh environments coupled with extensive protein engineering has resulted in their exploitation by industry for a variety of applications, in particular as an active ingredient in laundry detergents (6, 7, 9, 10). The ESPs have proved excellent paradigms for understanding key molecular features of proteins, including enzyme catalysis (11) and protein folding (6-8).The intracellular subtilisins (ISPs) are a distinctive class found in many different bacilli and re...

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“…As pointed out by Guhaniyogi et al (28), there is a good agreement between the structures of the glycosylated TPP1 precursor and the Endo H-deglycosylated one (PDB ID 3EDY). Furthermore, the crystal structure of pro-kumamolisin, the most homologous bacterial member of the sedolisin family, showed that the connecting linker runs through the entire active site cleft (17). Superposition of pro-kumamolisin with the active kumamolisin revealed that the catalytic domain of the proenzyme exhibits an almost identical structure and is already properly prefolded within the proenzyme (18).…”

Section: Discussionmentioning

confidence: 99%

“…The TPP1 precursor shares only 18.7% of its amino acid sequence with pro-kumamolisin that is currently the only reported structure of a full-length S53 peptidase. However, superposition of the reported structure of pro-kuma- molisin (17) with this full-length TPP1 structure reveals a 1.64 Å root mean square deviation of the 376 matching C-␣ atoms (supplemental Fig. S2).…”

Section: Homology Of Tpp1 To Members Of the Sedolisin Family Of Serinementioning

confidence: 99%

Structure of Tripeptidyl-peptidase I Provides Insight into the Molecular Basis of Late Infantile Neuronal Ceroid Lipofuscinosis

Pal

Kraetzner

Gruene

et al. 2009

Journal of Biological Chemistry

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Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 Å resolution reveals a globular structure with a subtilisin-like fold, a Ser 475 -Glu 272 -Asp 360 catalytic triad, and an octahedrally coordinated Ca 2؉ -binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp 276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

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1.2 Å Crystal Structure of the Serine Carboxyl Proteinase Pro-Kumamolisin

Cited by 57 publications

References 43 publications

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Regulation of an intracellular subtilisin protease activity by a short propeptide sequence through an original combined dual mechanism

Structure of Tripeptidyl-peptidase I Provides Insight into the Molecular Basis of Late Infantile Neuronal Ceroid Lipofuscinosis

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