The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 å resolution 1 1Edited by I. A. Wilson

Jain, Shri C.; Shinde, Ujwal; Li, Yuyun; Inouye, Masayori; Berman, Helen M.

doi:10.1006/jmbi.1998.2161

Cited by 148 publications

(169 citation statements)

References 35 publications

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“…N1 and N2 represent motifs that were identified using closely related subtilases and appear to be fairly well conserved (26). The sequence identity in Motif N1 and the C-terminal part of Motif N2 is significant, and these motifs are located in ␤1 and ␣2 Ϫ␤4, as seen from the x-ray crystallographic structure of ProWT (28). NMR structure analysis suggests that ␤1 and ␣2-␤4 display conformational rigidity and may represent potential folding nucleation sites in ProWT (27).…”

Section: Resultsmentioning

confidence: 89%

“…Equimolar concentrations of ProD and ProWT were added to S221C-subtilisin, and the samples were incubated overnight. The S221C substitution at the active site lowers the proteolytic activity by 10,000-fold and is insufficient to degrade the inhibitory propeptide (28). Far-UV CD spectroscopy establishes that unlike ProWT, which is completely unstructured (3), ProD adopts a well defined ␣-␤ conformation (Fig.…”

Section: Prod Is Structured As An Isolated Peptide and Forms A Complementioning

confidence: 98%

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Folding Pathway Mediated by an Intramolecular Chaperone

Yabuta

Subbian

Oiry

et al. 2003

Journal of Biological Chemistry

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Catalytic domains of several prokaryotic and eukaryotic protease families require dedicated N-terminal propeptide domains or ''intramolecular chaperones'' to facilitate correct folding. Amino acid sequence analysis of these families establishes three important characteristics: (i) propeptides are almost always less conserved than their cognate catalytic domains, (ii) they contain a large number of charged amino acids, and (iii) propeptides within different protease families display insignificant sequence similarity. The implications of these findings are, however, unclear. In this study, we have used subtilisin as our model to redesign a peptide chaperone using information databases. Our goal was to establish the minimum sequence requirements for a functional subtilisin propeptide, because such information could facilitate subsequent design of tailor-made chaperones. A decision-based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment was developed and utilized to design a novel peptide sequence (ProD). Interestingly, despite a difference of 5 pH units between their isoelectric points and despite displaying only 16% sequence identity with the wildtype propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor. The computed secondary structures and hydrophobic patterns within these two propeptides are similar. However, unlike ProWT, ProD adopts a well defined ␣؊␤ conformation as an isolated peptide and forms a stoichiometric complex with mature subtilisin. The CD spectra of this complex is similar to ProWT⅐subtilisin. Our results establish that despite low sequence identity and dramatically different charge distribution, both propeptides adopt similar structural scaffolds. Hence, conserved scaffolds and hydrophobic patterns, but not absolute charge, dictate propeptide function.Subtilisin E is an alkaline serine protease isolated from Bacillus subtilis (1). In vivo this protein is produced as pre-prosubtilisin (2), wherein the pre-region (signal peptide) facilitates protein secretion and the pro-region (propeptide) functions as an intramolecular chaperone that guides correct folding of subtilisin (2-6). Upon completion of folding, the propeptide is autoproteolytically removed (7). This is necessary because the propeptide is a potent inhibitor of subtilisin activity (4).Propeptide-mediated folding mechanisms exist in several unrelated protease families. However, there is no sequence conservation in propeptide domains within these families (8). This functional conservation across unrelated protease families suggests that propeptides have evolved in multiple parallel pathways and may share a common mechanism of action (9). Subtilisin (2-6) and ␣-lytic protease (10 -13) constitute the best-studied examples of propeptide-mediated protein folding. Bacterial subtilisins are models for the subtilase family that spans prokaryotes and eukaryotes. Amino acid sequence analysis of this family establishes that alth...

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Section: Resultsmentioning

confidence: 89%

Section: Prod Is Structured As An Isolated Peptide and Forms A Complementioning

confidence: 98%

Folding Pathway Mediated by an Intramolecular Chaperone

Yabuta

Subbian

Oiry

et al. 2003

Journal of Biological Chemistry

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“…The core of the structure conforms to a standard subtilisin-like serine protease domain, an ␣/␤ protein consisting of a seven-stranded parallel ␤-strand sandwiches between sets of helices, inhibited by its prodomain (12). A C-terminal, accessory cysteine-rich domain (CRD) is situated next to the catalytic domain forming a cloverleaf of subdomains displaying a clear pseudothreefold axis (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Structures of bacterial subtilisin-like proteases complexed with their prodomain inhibitors have been published (12) constituting the first 70 or so residues of the N terminus. This prodomain appears to be essential for proper folding of the intact molecule.…”

Section: Resultsmentioning

confidence: 99%

“…1A)]; the first two correspond to an inhibitory prodomain (amino acids ) and a serine protease domain (amino acids 153-452) of the proteinase K subfamily member of the subtilisin-like serine proteases (1,11), respectively. The overall fold of the catalytic domain has been well characterized because of the early x-ray crystallography work done on bacterial subtilisins (11), followed by structures of the catalytic domain in complex with its prodomain (12). Recently, representative structures from eukaryotes have also been solved.…”

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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

Hampton

Knuth

et al. 2007

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

138

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Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) are strongly associated with levels of low-density lipoprotein cholesterol in the blood plasma and, thereby, occurrence or resistance to atherosclerosis and coronary heart disease. Despite this importance, relatively little is known about the biology of PCSK9. Here, the crystal structure of a full-length construct of PCSK9 solved to 1.9-Å resolution is presented. The structure contains a fully folded C-terminal cysteine-rich domain (CRD), showing a distinct structural similarity to the resistin homotrimer, a small cytokine associated with obesity and diabetes. This structural relationship between the CRD of PCSK9 and the resistin family is not observed in primary sequence comparisons and strongly suggests a distant evolutionary link between the two molecules. This three-dimensional homology provides insight into the function of PCSK9 at the molecular level and will help to dissect the link between PCSK9 and CHD.hypercholesterolemia ͉ low-density lipoprotein receptor ͉ proprotein convertase ͉ x-ray crystallography ͉ adipocytokine P CSK9 (also known as neural apoptosis-regulated convertase, NARC-1) is a 692-residue extracellular protein expressed primarily in the kidneys, liver and intestines (1) representing the 9th member of the secretory subtilase family. Various genetic observations subsequently mapped PCSK9 as the third gene (along with LDLR and APOB) to cause autosomal dominant hypercholesterolemia (ADH). These studies suggested that gainof-function mutations increase plasma levels of LDL-c (2-6), whereas nonsense or missense (loss-of-function) mutations, which interfere with folding or secretion of PCSK9, lead to a reduction of plasma levels of LDL-c and an 88% decrease in the risk of coronary heart disease (CHD) (5). In mice, adenoviral overexpression of PCSK9 results in increased plasma LDL-c level in normal mice but not in LDLR-deficient mice (7). Deletion of PCSK9 causes an increase in level of LDLR protein but not mRNA (8). These findings lead to a hypothesis that PCSK9 exerts its role in cholesterol metabolism through posttranslational down-regulation of LDLR, the receptor responsible for clearing LDL-c from plasma. In support of this hypothesis, a recent mouse parabiosis study revealed a nearly complete loss of liver LDLR proteins in wild-type recipients of human PCSK9 parabiosed from a transgenic littermate (9).Like other secretory subtilisin-like serine proteases, PCSK9 is synthesized in the endoplasmic reticulum (ER) as a precursor that undergoes autocatalytic processing, intracellular trafficking, and, finally, release into the extracellular medium with the capability of decreasing the cellular LDLR level and LDL-c uptake (10). Despite the rapid progress in understanding the genetics and biology of PCSK9, little is known of its structure, substrate specificity, or mode of action of PCSK9 in the regulation of LDLR. The full-length, mature sequence consists of three domains, [the first 30 residues of the N terminus contain signal se...

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Structural Calcium (Trypsin, Subtilisin)

Gilliland

Teplyakov

2004

Handbook of Metalloproteins

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The presence of structural calcium in trypsin and subtilisin does not play a role in catalysis, but instead, stabilizes the structures. Trypsin and subtilisin are two large families of serine proteases with different folds that take advantage of the stabilizing effects of binding calcium ions. Representative structures of the different calcium binding sites found in each protein family are described along with the implications for the presence of the ions. This article reviews the details, the energetics, and the roles that calcium plays in the structures of both these families of enzymes.

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The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 å resolution 1 1Edited by I. A. Wilson

Cited by 148 publications

References 35 publications

Folding Pathway Mediated by an Intramolecular Chaperone

Folding Pathway Mediated by an Intramolecular Chaperone

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

Structural Calcium (Trypsin, Subtilisin)

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