Fig 1.
Cartoon representation of the model proteins used for the molecular dynamics (MD) study.
The disulfide bonded cysteine residues are highlighted as coloured beads and listed on the left of each snapshot.
Fig 2.
TCEP and Trx reduce plasma proteins important for coagulation and haemostasis.
(A) Protein samples were reduced with thioredoxin or TCEP and free thiols labelled with Alexa 488-maleimide. Samples were separated on a 4–20% SDS-PAGE gel and visualised for the incorporation of the fluorescent label (top). Coomassie stains to show equal loading is also shown (bottom). (B) FVIII undergoes reduction by both thioredoxin and TCEP (Alexa 488-maleimide staining on the left, total protein [Coomassie staining] on the right).
Fig 3.
Selecting proximity criteria from parallel bias metadynamics.
Boxplots showing (A) the average distance, (B) the minimum distance, or (C) the number of TCEP molecules closer than 0.8 nm to the disulfide bonds of CD44. In the boxplots, the top bar is the maximum observation, the lower bar is the minimum observation, the top of the box is the third quartile, the bottom of the box is the first quartile, while the middle bar represents the median. The medians are connected by a straight line to guide the eye.
Fig 4.
Distance- or distance+energy-based criteria to identify redox-labile disulfide bonds.
Histograms showing the probability that each disulfide is reduced by TCEP according to the distance- (plain black bars) or distance+energy-based (dashed bars) criteria. The different panels refer to CD44 (A), tissue factor (B), CD132 (C), IL-4 (D), the D1 and D2 domains of GP130 (E), and FVIII light chains (F).
Table 1.
Structural parameters of disulfide bonds using online prediction analysis.
Four published FVIII structures were evaluated in parallel using the UNSW DSB prediction tool [58]. Allosteric disulfide bonds tend to be solvent accessible and have short alpha carbon distances. Stable structural disulphide bonds tend to have a -LHspiral conformation; these have the most common DSB conformation and have the lowest dihedral strain energy.
Fig 5.
Experimental validation of in silico MD simulations by quantitative mass spectroscopy and ELISA.
(A) Native or reduced (TRX 1) BDD rFVIII was alkylated with iodoacetamide (IAA) and subjected to tryptic digest and LC-MS/MS. Peak areas of IAA labelled cysteine peptides were compared to a control sample of BDD rFVIII which had been fully denatured, reduced and alkylated. The x-axis labels are as follows: cysteine 198 (from the Cys153-Cys179 bond); cysteines 267 and 348 (from the Cys248-Cys329 bond); cysteine 573 (from the Cys528-Cys554 bond); cysteine 1877 (from the Cys1832-Cys1858 bond); cysteine 1922 (from the Cys1899-Cys1903 bond); cysteine 2040 (from the Cys2021-Cys2169 bond) and cysteine 2345 (from the Cys2174-Cys2326 bond).
Fig 6.
Modelled conformational changes in FVIII upon reduction of disulfide bond Cys1899-Cys1903.
(A) Evolution of FVIII radius of gyration Rg, number of intra-molecular hydrogen bonds HBpp, solvent accessible surface area SASA, and backbone RMSD with respect to the crystal structure, as function of simulation time, for both full-oxidized FVIII (black curve) and reduced FVIII (red curve). (B-C) Cartoon representation and superimposition of full-oxidized and reduced (Cys1899-Cys1903) FVIII. The alignment has been performed either on the whole structure (B), or on the separate domains (C). The cartoons are coloured according to the Q parameter, which is 1 for identical structures; the blue areas indicate structural similarity between full-oxidized and reduced FVIII, where Q = 1. Areas of poor structural similarity (Q = 0.1–0.3) are shown in red. The figure was obtained using the VMD software [59] and the STAMP algorithm [60], that works by minimizing the Cα distance between aligned residues of each molecule. The Cys1899-Cys1903 disulfide bond is represented as yellow beads.
Fig 7.
ELISA analysis of FVIII-mAb binding in solid state.
Reduction of rFL-FVIII decreases binding of the A2- and A3-specific anti-FVIII antibodies NB11B2 and NB41. The binding of the C1 domain-specific antibody NB33 also decreases, while no change in binding is observed for the C2-specific antibody NB2C11.
Fig 8.
TCEP-mediated reduction of a selection of FVIII products.
A selection of FVIII products were incubated with PBS or reduced with 1 mM TCEP and free -SH groups visualised with the Alexa Fluor 488-maleimide fluorescent label. FVIII products tested include recombinant porcine FVIII (rpFVIII) produced in Baby Hamster Kidney (BHK) cells, two recombinant full-length FVIII (rFL FVIII) products also produced in BHK cells, a rFL-FVIII product produced in Chinese Hamster Ovary (CHO) cells, and a recombinant B domain deleted FVIII product produced in BHK cells.
Fig 9.
Chromogenic activity assay of native and reduced (1mM TCEP) FVIII products.
Different FVIII products (porcine and human, FL and recombinant, CHO and BHK expressed) were incubated with either PBS (black circles), 10 mM DTT (squares), 1 mM TCEP (triangles), or 10 nM Trx (grey diamonds) before being assessed for activity (A–F). Activity was determined by parallel line analysis (G).