FRET studies with Factor X mutants provide insight into the topography of the membrane-bound Factor X/Xa

Qureshi, Shabir H.; Yang, Likui; Yegneswaran, Subramanian; Rezaie, Alireza R.

doi:10.1042/bj20070735

Cited by 16 publications

(34 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the context of this model, the first EGF domain of ZPI could play a spacer function to properly accommodate ZPI into the active site pocket of the membrane-bound enzyme without making any specific interaction with fXa in the ternary complex. A similar role for the EGF1 domain of fXa has been postulated in the prothrombinase complex (14). Thus, the loss of the spacer function of EGF1 most likely accounts for ϳ4-fold lower second-order rate constant observed for the PZ-mediated ZPI inhibition of fXadesEGF1 on PC/PS vesicles.…”

Section: Proteasementioning

confidence: 90%

“…Because of a very poor yield in the mammalian expression system, the Gla-domainless PZ (GD-PZ lacking residues 1-47) was expressed in the InsectSelect system (Invitrogen) using the pIZ/V5-His expression vector transfected to sf21 insect cells according to the manufacturer's instruction. The construction strategy was such that the sequence of His tag in the vector remained downstream of the stop codon for GD-PZ; thus it was not incorporated into the sequence of the mutant cofactor as we described previously for the expression of other proteins in this system (14). The expression, purification, and characterization of recombinant ZPI in HEK-293 cells has been described previously (15).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Rezaie

Bae

Manithody

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the ␥-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGFlike domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K D ) of ϳ7 nM. However, the apparent K D for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes ϳ6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes. Protein Z (PZ)2 is a vitamin K-dependent coagulation glycoprotein, which has a genetic organization that is identical to that of factor Xa (fXa) and other similar vitamin K-dependent coagulation factors (1, 2). Thus, PZ has an N-terminal ␥-carboxyglutamic acid (Gla) domain that is followed by two epidermal growth factor (EGF)-like domains (light chain homologue) and a C-terminal pseudo catalytic domain (heavy chain homologue) (1, 2). Unlike fXa and other vitamin K-dependent coagulation proteases, two of the catalytic triad residues of the C-terminal domain have not been conserved in the homologous regions of PZ (1, 2). Thus, PZ has no catalytic function, but instead it binds to PZ-dependent protease inhibitor (ZPI) with a high affinity, thereby promoting the ZPI inhibition of fXa in the presence of PC/PS vesicles and Ca 2ϩ by approximately three orders of magnitude (3-5). ZPI is a 72-kDa serpin with a plasma concentration of 2.6 -2.9 g/ml that appears to interact with the active site pocket of fXa by a covalent mechanism similar to that observed with antithrombin and most other inhibitory serpins (4 -6). However, unlike antithrombin, which can inhibit all coagulation proteases, ZPI is a specific inhibitor of fXa and factor XIa (4, 7, 8), though recent results have indicated that it may also inhibit factor IXa (9). ZPI may play a critical role in the regulation of the coagulation cascade because its deficiency is associated with venous thromboembolic diseases (10, 11). ZPI by itself is a poor inhibitor of fXa, unless i...

show abstract

Section: Proteasementioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Rezaie

Bae

Manithody

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…All spectral measurements were performed using an Aminco-Bowman series 2 Spectrophotometer (Spectronic Unicam, Rochester, NY) as described [19]. The anisotropy of the fluorescein-labeled proteins (25-50 nM) was measured both in the absence and presence of saturating concentrations of PCPS vesicles (35 μM) and protein S (300 nM) using the excitation and emission wavelengths of 490 nm and 520 nm as described [19].…”

Section: Spectral Measurementsmentioning

confidence: 99%

“…Phospholipid vesicles at a molar ratio of PC to PS of 4:1 containing or lacking different amounts of OR were prepared by the extrusion method as described [19]. Briefly, dried phospholipid mixtures were suspended at 1 mg/mL in 0.1 M NaCl, 0.05 M Hepes, pH 7.4, mixed for 10 min and passed twenty times through a 100 nm pore diameter polycarbonate membrane.…”

Section: Phospholipid Vesicle Preparationsmentioning

confidence: 99%

Autolysis loop restricts the specificity of activated protein C: Analysis by FRET and functional assays

Qureshi

Manithody

Bae

et al. 2008

Biophysical Chemistry

Self Cite

View full text Add to dashboard Cite

We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S. The activity of APC-Tryp 143-154 toward fVa was identical to that of wild-type APC both in the presence and absence of protein S. However, the reactivity of APC-Tryp 143-154 with plasma inhibitors was preferentially improved independent of protein S. The FRET analysis revealed a dramatic change in the active-site topography of APC both in the absence and presence of protein S. Anisotropy measurements revealed that the fluorescein dye has a remarkable degree of rotational freedom in the active-site of APC-Tryp 143-154. These results suggest that the autolysis loop of APC may not be a target for modulation by protein S. This loop, however, plays a critical role in restricting both the specificity and spatial environment of the active-site groove of APC.

show abstract

“…Phospholipid vesicles containing 80% phosphatidylcholine and 20% phosphatidylserine (PC/PS) were prepared as described (17). TFPI was from Monsanto Chemical Co. (St. Louis, MO).…”

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

Residues of the 39-Loop Restrict the Plasma Inhibitor Specificity of Factor IXa

Yang

Rezaie

2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

FRET studies with Factor X mutants provide insight into the topography of the membrane-bound Factor X/Xa

Cited by 16 publications

References 36 publications

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Protein Z-dependent Protease Inhibitor Binds to the C-terminal Domain of Protein Z

Autolysis loop restricts the specificity of activated protein C: Analysis by FRET and functional assays

Residues of the 39-Loop Restrict the Plasma Inhibitor Specificity of Factor IXa

Contact Info

Product

Resources

About