Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria

Kobayashi, Hidetomo; Yoshida, Toru; Miyakawa, Takuya; Tashiro, Mitsuru; Okamoto, Keinosuke; Yamanaka, Hidetoshi; Tanokura, Masaru; Tsuge, Hideaki

doi:10.1074/jbc.m114.622852

Cited by 7 publications

(15 citation statements)

References 41 publications

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“…Since our previous studies demonstrated that ASP shows sufficient proteolytic action in phosphate buffer solution (pH ranging from 7.2 to 7.5) [17–19], in the present study we mixed 2 μl of the substrate solution (10 mM) with 148 μl of ASP or ASP[R566A] (1 nM) in 20 mM sodium phosphate buffer (pH 7.4) in order to examine the cleavage of each substrate by ASP or ASP[R566A]. The mixture was incubated at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, the chaperone protein has been designated as an ORF2 (open reading frame 2) [16]. In our PSI-BLAST search using the ORF2 sequence, we found that operons consisting of genes homologous to asp and orf2 are present in four genera other than Aeromonas : Vibrio , Shewanella , Chromobacterium , and Pseudoalteromonas [17]. However, it is not yet known how the chaperone protein works on a specific protein at the molecular level.…”

Section: Introductionmentioning

confidence: 99%

“…However, it is not yet known how the chaperone protein works on a specific protein at the molecular level. Our recent analysis of the complex of ASP with ORF2 showed that the carboxyl terminal region of ORF2 tightly interacts with the amino acid residues around the catalytic site of ASP [17]. It seems that the interaction prevents the nascent ASP from the proteolysis by itself (autolysis) during the folding process.…”

Section: Introductionmentioning

confidence: 99%

“…Further examination showed that the Gln residue at position C3 of ORF2 is close to the Arg-566 residue of ASP (Fig 1A). The Arg-566 residue of ASP is one of the amino acid residues comprising the extra occluding region of ASP, and the position of Arg-566 in the stereo structure is close to the catalytic site of ASP [17,18]. From these observations, we speculate that Arg-566 residue may have a role to interact with external molecules such as the chaperone protein ORF2.…”

Section: Introductionmentioning

confidence: 99%

“…After incubation with ORF2, the generated complex of ASP(S336A) with ORF2 was purified and its complex structure was analyzed [17]. The structures of ASP and ORF2 are depicted by a ribbon model and a stick model, respectively.…”

Section: Introductionmentioning

confidence: 99%

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Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity

Kobayashi

Otsubo²,

Teraoka³

et al. 2017

PLoS ONE

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Aeromonas sobria serine protease (ASP) is an extracellular serine protease secreted by the organism. Here, we identified the amino acid residue of ASP that contributes to substrate specificity by using both synthetic peptides and biological protein components. The results showed that the arginine residue at position 566 (Arg-566) of ASP, which is located in the extra occluding region of ASP close to an entrance of the catalytic cavity, is involved in the substrate specificity. A substitutional point mutation of the Arg-566 residue of ASP to Ala residue (ASP[R566A]) caused a decrease of the proteolytic efficiency for a certain substrate. In addition, ASP lost the ability to recognize the primary substrate by such a point mutation, and ASP[R566A] reacted to a wide range of synthetic substrates. It is likely that Arg-566 causes an interaction with the amino acid residue at position P3 of the substrate, which is the third amino acid residue upstream from the cleavage site. Another study using ORF2 protein, a chaperone protein of ASP, further suggested that Arg-566 could also play an important role in interaction with ORF2. We therefore conclude that the Arg-566 residue of ASP is likely responsible for the selection of substrates.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity

Kobayashi

Otsubo²,

Teraoka³

et al. 2017

PLoS ONE

Self Cite

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Membrane-Bound Transcription Factor Site-1 Protease in PF429242 Bound State: Computational Kinetics and Dynamics of Reversible Binding

et al. 2019

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Membrane-bound transcription factor site-1 protease (S1P) is an emerging clinical target due to its roles in lipogenesis, lysosomal biogenesis, unfolded protein response and viral glycoprotein processing. In this study, homology model of S1P was created in order to understand the structural basis for S1P inhibition by PF429242 using molecular docking, molecular dynamics simulation and in silico kinetics studies. PF429242 was docked (GlideScorePF429242=−5.20 kcal/mol) into the catalytic triad (D218, H249 and S414) and validated (R2=0.5686). The reversible binding kinetic parameter (Koff/Kon) was estimated at=7.28E-03 M with fully bound and apo-states interspersed by 3 transient ligand-bound states with unique binding signatures; water plays a major role in PF429242 dissociation from the catalytic site. Communication between key catalytic triad residues is altered in the presence of PF429242. In apo-S1P state, S414–S307/V216-D218 is the preferred route but in PF429242-bound state, S414-S417/V216-D218 is preferred. Communication between S414 and H249 is also shortened in PF429242 bound state; here, only L410 is required unlike apo-state, which requires P418, V256 and F252. Ligand binding did not alter the communication route between S414 and H249 as both recruited D244 and G220. In conclusion, PF429242 binds tightly but reversible to S1P and the details of this interaction has been presented to guide future efforts at developing novel inhibitors. Site-1-protease; PF429242; Kon/Koff; Network analysis

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A Tripeptidyl peptidase 1 is a binding partner of GPHR (Golgi pH regulator) in Dictyostelium

Stumpf

Müller

Gassen

et al. 2017

Disease Models &Amp; Mechanisms

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Mutations in tripeptidyl peptidase 1 (TPP1) have been associated with late infantile neuronal ceroid lipofuscinosis (NCL), a neurodegenerative disorder. TPP1 is a lysosomal serine protease, which removes tripeptides from the N-terminus of proteins and is composed of an N-terminal prodomain and a catalytic domain. It is conserved in mammals, amphibians, fish and the amoeba Dictyostelium discoideum. D. discoideum harbors at least six genes encoding TPP1, tpp1A to tpp1F. We identified TPP1F as binding partner of Dictyostelium GPHR (Golgi pH regulator), which is an evolutionarily highly conserved intracellular transmembrane protein. A region encompassing the DUF3735 (GPHR_N) domain of GPHR was responsible for the interaction. In TPP1F, the binding site is located in the prodomain of the protein. The tpp1F gene is transcribed throughout development and translated into a polypeptide of ∼65 kDa. TPP1 activity was demonstrated for TPP1F-GFP immunoprecipitated from D. discoideum cells. Its activity could be inhibited by addition of the recombinant DUF3735 domain of GPHR. Knockout tpp1F mutants did not display any particular phenotype, and TPP1 activity was not abrogated, presumably because tpp1B compensates as it has the highest expression level of all the TPP1 genes during growth. The GPHR interaction was not restricted to TPP1F but occurred also with TPP1B. As previous reports show that the majority of the TPP1 mutations in NCL resulted in reduction or loss of enzyme activity, we suggest that Dicyostelium could be used as a model system in which to test new reagents that could affect the activity of the protein and ameliorate the disease.

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Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria

Cited by 7 publications

References 41 publications

Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity

Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity

Membrane-Bound Transcription Factor Site-1 Protease in PF429242 Bound State: Computational Kinetics and Dynamics of Reversible Binding

A Tripeptidyl peptidase 1 is a binding partner of GPHR (Golgi pH regulator) in Dictyostelium

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