P3–P3′ Residues Flanking Scissile Bonds in Factor VIII Modulate Rates of Substrate Cleavage and Procofactor Activation by Thrombin

Newell-Caito, Jennifer L.; Griffiths, Amy E.; Fay, Philip J.

doi:10.1021/bi300070z

Cited by 6 publications

(13 citation statements)

References 34 publications

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“…Instead, it is likely that the extra occluding region including the Arg-566 residue which notably protrudes into the catalytic cavity functions in substrate selection by ASP, as seen in that by Kex2. In addition, in the thrombin-catalyzed activation of factor VIII, it was reported that P3-P3' residues flanking scissile bonds in the factor VIII modulate substrate was cleaved by thrombin [25]. This report may be another example indicating that the amino acid residues adjacent to the cleavage site of a substrate largely affect the proteolytic action.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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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“…During activation, thrombin cleaves FVIII after Arg372 (A1-A2 junction), Arg740 (A2-B junction), and Arg1689 (N-terminal region of A3 domain), which results in the formation of an FVIIIa heterotrimer A1/A2/A3-C1-C2 [ 9 ]. The initial cleavage site is after Arg740, which subsequently facilitates the cleavage at the other two positions [ 11 ], while cleavage after Arg372 is the rate-limiting step of FVIII activation [ 12 , 13 ]. Besides FVIII, thrombin activates other components of the blood coagulation cascade and is involved in fibrinolysis [ 14 , 15 ], inflammation, atherosclerosis [ 16 , 17 ], and neurodegeneration [ 18 , 19 ], where it has numerous protein substrates [ 20 , 21 ].…”

Section: Introductionmentioning

confidence: 99%

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

Shestopal

Parunov

Olivares

et al. 2022

IJMS

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Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (VL and VH) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into VL and VH as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated VH, a mixture of VL and VH, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated VL and VH assembled into scFv-like heterodimer on FVIII, and the isolated VH alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.

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“…Direct P1-S1 interactions of the substrate with this amino acid explain the strong preference of thrombin for positively charged substrate residues (especially arginine residues) at P1 (C-terminal to the scissile bond). Further requirements have also been described for flanking amino acids [ 31 , 32 ].…”

Section: Introductionmentioning

confidence: 99%

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

et al. 2015

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Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.

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P3–P3′ Residues Flanking Scissile Bonds in Factor VIII Modulate Rates of Substrate Cleavage and Procofactor Activation by Thrombin

Cited by 6 publications

References 34 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

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

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