Structure and Mechanism of the Pepsin-Like Family of Aspartic Peptidases

Dunn, Ben M.

doi:10.1021/cr010167q

Cited by 336 publications

(335 citation statements)

References 230 publications

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“…Whereas other aspartic proteases, like pepsin, renin, and cathepsin D exist as soluble monomers, cathepsin E is the only known aspartic protease that exists as a homodimer consisting of two fully catalytically active monomers (42). Dimerization of cathepsin E results in an increased pH and temperature stability, which is consistent with its biological function in endosomal vesicles (43)(44)(45).…”

Section: In Brains Ofmentioning

confidence: 75%

Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

Westmeyer

Willem

Lichtenthaler

et al. 2004

Journal of Biological Chemistry

104

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Cleavage of the ␤-amyloid precursor protein (APP) by the aspartyl protease ␤-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid ␤-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by ␥-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

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Section: In Brains Ofmentioning

confidence: 75%

Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

Westmeyer

Willem

Lichtenthaler

et al. 2004

Journal of Biological Chemistry

104

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“…The shorter than van der Waal separation between scissile carbon and nitrogen atoms is an indication of the reaction having been stalled before completion. Complete separation and release of products P and Q require loss of hydrogen bonding and other interactions between the products and the enzyme (14). The residues from the flap and the catalytic aspartates are involved in a majority of these interactions, and opening of the flexible flaps, therefore, could disrupt these hydrogen bonds.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of HIV-1 protease in situ product complex and observation of a low-barrier hydrogen bond between catalytic aspartates

Das

Prashar

Mahale

et al. 2006

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

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HIV-1 protease is an effective target for designing drugs against AIDS, and structural information about the true transition state and the correct mechanism can provide important inputs. We present here the three-dimensional structure of a bi-product complex between HIV-1 protease and the two cleavage product peptides AETF and YVDGAA. The structure, refined against synchrotron data to 1.65 Å resolution, shows the occurrence of the cleavage reaction in the crystal, with the product peptides still held in the enzyme active site. The separation between the scissile carbon and nitrogen atoms is 2.67 Å, which is shorter than a normal van der Waal separation, but it is much longer than a peptide bond length. The substrate is thus in a stage just past the G'Z intermediate described in Northrop's mechanism [Northrop DB (2001) Acc Chem Res 34:790 -797]. Because the products are generated in situ, the structure, by extrapolation, can give insight into the mechanism of the cleavage reaction. Both oxygens of the generated carboxyl group form hydrogen bonds with atoms at the catalytic center: one to the OD2 atom of a catalytic aspartate and the other to the scissile nitrogen atom. The latter hydrogen bond may have mediated protonation of scissile nitrogen, triggering peptide bond cleavage. The inner oxygen atoms of the catalytic aspartates in the complex are 2.30 Å apart, indicating a low-barrier hydrogen bond between them at this stage of the reaction, an observation not included in Northrop's proposal. This structure forms a template for designing mechanism-based inhibitors.AIDS ͉ catalysis ͉ reaction intermediate ͉ x-ray crystallography H IV-1 protease (PR) is a homodimeric, aspartyl PR containing the signature sequence Asp-Thr-Gly in each monomer. The enzyme, which cleaves the viral polyprotein at eight different sites during the maturation process of the virus, is an important target for structure-based drug design (1-4). Emergence of drug-resistant mutations presents a new challenge, and additional inputs, such as the mechanism and interactions of the active enzyme with plain peptide substrates rather than with analogs, are required to tackle drug resistance. To address this question, we have undertaken to solve the crystal structures of HIV-1 PR oligopeptide substrate complexes. The enzyme used in the present study is a single polypeptide chain where the C terminus of the first monomer is linked with the N terminus of the second monomer through a pentapeptide (-GGSSG-) linker. The enzyme activity of such a tethered dimer (TD) construct is comparable with that of the native homodimer (5). Previously (6, 7), we have reported structures of TD and its complex with the undecapeptide substrate that corresponds in sequence to the reverse transcriptase-integrase junction in the viral polyprotein. Here we report the crystal structure of TD complexed with a decapeptide of amino acid sequence matching the reverse transcriptase-RNase H junction of the polyprotein. The structure has been refined to 1.65 Å resolution against diffra...

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“…Water 1 is in contact with the catalytic water 2 (in Fig. 3C), which is held in place by the carboxyls of the two catalytic Asp residues (Asp-32 and Asp-215) in all aspartic proteinases (1,4,11). The carboxyl group of D22 also contacts the side chain of Tyr-75 and water molecules 3 and 4 (in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Aspartic proteinases are widely distributed in nature and are involved in a variety of physiological processes and pathological conditions such as Alzheimer disease, cancer, hypertension, and AIDS (1). In stark contrast, naturally occurring protein inhibitors of aspartic proteinases are rare and are found only in a few specialized locations (1)(2)(3).…”

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confidence: 99%

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Key Features Determining the Specificity of Aspartic Proteinase Inhibition by the Helix-forming IA3 Polypeptide

Winterburn

Wyatt

Phylip

et al. 2007

Journal of Biological Chemistry

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The 68-residue IA 3 polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2-32 of the polypeptide adopt an amphipathic ␣-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (K i < 0.1 nM) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA 3 from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA 3 . The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained.

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Structure and Mechanism of the Pepsin-Like Family of Aspartic Peptidases

Cited by 336 publications

References 230 publications

Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

Crystal structure of HIV-1 protease in situ product complex and observation of a low-barrier hydrogen bond between catalytic aspartates

Key Features Determining the Specificity of Aspartic Proteinase Inhibition by the Helix-forming IA3 Polypeptide

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