pH-Dependent Unfolding of Aspergillopepsin II Studied by Small-Angle X-ray Scattering

Kojima, Masaki; Tanokura, Masaru; Kimura, Kazumoto; Amemiya, Yoshiyuki; Kihara, Hiroshi; Takahashi, Kenji

doi:10.1021/bi991584o

Cited by 35 publications

(25 citation statements)

References 15 publications

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“…The critical role of charge repulsion in protein stability has been indicated in several cases. For example, whereas aspergillopepsin II, an aspartic proteinase composed of many (20% of total) negatively charged residues, is stable at acidic pH, it unfolds at alkaline pH because of the negative charge repulsion (39).…”

Section: Discussionmentioning

confidence: 99%

Partially Folded Structure of Flavin Adenine Dinucleotide-depleted Ferredoxin-NADP+ Reductase with Residual NADP+ Binding Domain

Hamada¹,

Hoshino

Onda³

et al. 2002

Journal of Biological Chemistry

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Protein folding is one of the most important issues in structural biology and many other related fields in molecular biology and biotechnology. The characterization of an intermediate state between the native and unfolded structures is important in understanding the folding mechanism of proteins (1, 2). Although many small proteins of Ͻ100 amino acids in length show highly cooperative folding reactions from the unfolded to the native state without accumulating observable intermediates, proteins with Ͼ100 amino acid residues (3) and some smaller proteins (4, 5) show populated intermediates with partially folded structures, e.g. the molten globule state, in the first millisecond of folding reactions (6). For a number of proteins, partially folded intermediates with conformational properties similar to those of kinetic folding intermediates have been found under mild denaturing conditions in equilibrium (7,8). These observations offer the opportunity to analyze the detailed conformational properties of the partially folded structures or the roles of individual subdomains.Some large proteins can be decomposed into folding units (9, 10). The existence of several domains in such proteins is assumed to reflect both the organization of the genes and the dynamics of the folding process (11, 12). It has been proposed that domains and subdomains fold independently and subsequently merge to produce the native molecule (13). Recent studies of protein folding have focused on small proteins or domains of larger proteins. This is not only because of the simplicity of the mechanisms and reversibility of folding reactions, but also because of the probability that these reactions reflect earlier events in the folding process of larger proteins. On the other hand, the direct characterization of folding and unfolding behavior with the entire molecule is necessary for complete understanding of the folding mechanisms of larger proteins (5,14). In the present study, we analyzed ferredoxin-NADP ϩ reductase (FNR) 1 (EC 1.18.1.2) as an example of such a large protein.FNR catalyzes reduction of NADP ϩ to NADPH during photosynthesis in higher plants (15). The crystal structure of maize FNR has recently been determined by x-ray crystallography ( Fig. 1) (16). The molecule was shown to be composed of welldefined FAD and NADP ϩ binding domains. The FAD binding domain (residues 1-153) is made up of a scaffold of six antiparallel ␤-strands arranged in two perpendicular ␤-sheets, the bottom of which is capped by an ␣-helix and a long loop. The NADP ϩ binding domain (residues 154 -314) consists of a core of five parallel ␤-strands surrounded by seven ␣-helices. The FAD cofactor is bound to the protein through hydrogen bonds, van der Waals contacts, and -stacking interactions. The isoalloxazine ring system, which constitutes the reactive part of FAD, is stacked on the aromatic rings of two tyrosine residues, i.e. Tyr 95 and Tyr 314 (Fig. 1A). We studied the structural properties of holo-FNR and two forms of apoenzyme, i.e. apo-FNR prepared by CaCl ...

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Section: Discussionmentioning

confidence: 99%

Partially Folded Structure of Flavin Adenine Dinucleotide-depleted Ferredoxin-NADP+ Reductase with Residual NADP+ Binding Domain

Hamada¹,

Hoshino

Onda³

et al. 2002

Journal of Biological Chemistry

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“…Refolding of Aspergillopepsinogen II-Aspergillopepsin II is known to be unfolded above neutral pH mainly because of the electrostatic repulsion inside the molecule (31). The pro-peptide of aspergillopepsinogen II is rich in basic residues whereas the mature enzyme moiety is abundant in acidic residues.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Glutamic Acid and an Aspartic Acid Residue Essential for Catalytic Activity of Aspergillopepsin II, a Non-pepsin Type Acid Proteinase

Huang

Kagami

Inoue

et al. 2000

Journal of Biological Chemistry

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Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase. To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions. The circular dichroism spectra of the mutant pro-and mature enzymes were essentially the same as those of the wildtype pro-and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.The regular aspartic proteinase family (1-5) includes such enzymes as pepsin, chymosin, cathepsins D and E, renin, and fungal and retroviral pepsins. The family is homologous both in primary and tertiary structures so they are thought to share a common ancestor in evolution. Generally, members of the aspartic proteinase family are composed of two homologous domains, each containing a catalytic aspartyl residue in a consensus sequence of Asp-(Thr/Ser)-Gly, in close proximity to enable catalytic function (6). The proteinases are often characterized by susceptibility to specific inhibitors such as pepstatin A (7), 1,2-epoxy-3-(p-nitrophenoxy)propane (8), and the diazoacetyl-DL-norleucine methyl ester in the presence of cupric ions (9).In addition to the aspartic proteinases, there exist some distinct families of aspartic or acid proteinases that are not homologous, having neither apparent internal sequence homology nor the consensus Asp-(Thr/Ser)-Gly sequence present in the pepsin-type aspartic proteinases. Aspergillopepsin II is unique in the primary structure of known proteins with the exception of scytalidopepsin B (20) and the EapB and EapC proteins from C. parasitica (14). Aspergillopepsin II consists of two polypeptide chains, a 39-residue light chain and a 173-residue heavy chain (11), which are derived from a precursor polypeptide of 282 residues (21). Although this two-chain structure is different from the one-chain structures of scytalidopepsin B and the EapB and EapC proteins, a 44 -65% identity in amino acid sequence indicates that these proteins have been derived in evolution from the same ancestral protein and presumably share common catalytic residues and mechanisms. Recently Barrett et al. (22) have classified...

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“…This is consistent with the secondary structure prediction made previously from the circular dichroism spectroscopy. 16) There are two seven-stranded anti-parallel β-sheets (shown in green and red). These two sheets overlap with each other, thus forming a β-sandwich structure.…”

Section: Introductionmentioning

confidence: 99%

The three-dimensional structure of aspergilloglutamic peptidase from Aspergillus niger

Sasaki

Nakagawa

Muramatsu

et al. 2004

Proceedings of the Japan Academy. Ser. B: Physical and Biologic

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Aspergilloglutamic peptidase from Aspergillus niger is a novel pepstatin-insensitive acid endopeptidase distinct from the well-studied aspartic peptidases, and thus is an interesting target for protein structure/function studies. In the present study, we have determined the three-dimensional structure of the enzyme by X-ray crystallography to a 1.4-Å resolution. The results revealed that the enzyme has a unique structure, composed of two seven-stranded anti-parallel β-sheets which form a β-sandwich structure and appear to have a partial two-fold symmetry, suggesting its possible evolution by gene duplication and that the glutamic acid-110 and glutamine-24 in the heavy chain form a catalytic dyad, consistent with our results obtained by site-directed mutagenesis.

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pH-Dependent Unfolding of Aspergillopepsin II Studied by Small-Angle X-ray Scattering

Cited by 35 publications

References 15 publications

Partially Folded Structure of Flavin Adenine Dinucleotide-depleted Ferredoxin-NADP+ Reductase with Residual NADP+ Binding Domain

Partially Folded Structure of Flavin Adenine Dinucleotide-depleted Ferredoxin-NADP+ Reductase with Residual NADP+ Binding Domain

Identification of a Glutamic Acid and an Aspartic Acid Residue Essential for Catalytic Activity of Aspergillopepsin II, a Non-pepsin Type Acid Proteinase

The three-dimensional structure of aspergilloglutamic peptidase from Aspergillus niger

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