Structural investigation of C4b-binding protein by molecular modeling: Localization of putative binding sites

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one β-chain and seven α-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its α-chains and with protein S through its β-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP α-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the α-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein–protein interactions. Proteins 31:391–405, 1998. © 1998 Wiley-Liss, Inc.     

Activation of blood coagulation factor VIIa with cleaved tissue factor extracellular domain and crystallization of the active complex

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 × 0.12 × 0.6 mm and showed diffraction to a resolution of 3 Å. © 1995 Wiley-Liss, Inc.     

Interactions of the C2 domain of human factor V with a model membrane

Activated coagulation Factor V is an important cofactor of the coagulation cascade that catalyzes the formation of the prothrombinase complex on the surface of membranes rich in phosphatidyl-L-serine (PS). Here we report molecular dynamics simulations of the two crystallographic structures (the open and closed conformations) of domain C2 of coagulation Factor V (FaVC2). The calculations were performed in water (1.5 ns for each conformation) and in the presence of a neutral phospholipid bilayer model (POPE; 10 ns for each conformation) in order to describe the dynamics of the free (plasma circulating) and membrane bound forms of FaVC2. Water simulations confirmed the hypothesis that the plasma circulating form is in the closed conformation. In contrast, the membrane simulations showed that both conformations are energetically compatible with membrane binding. We have investigated the mechanism, the dynamics, and the energetics of the binding process. Our data are consistent with published estimates of the immersion depth of the aromatic residues (W26 and W27), and with mutagenesis studies involving specific residues located on the spikes at the bottom of the FaVC2 structure. Electrostatic interactions between the phospholipid head groups and hydrophilic residues at the bottom of the structure play a key role in the binding process by creating a large number of hydrogen bonds that anchor the protein to the membrane. The simulations identified a stable phospholipid binding pocket reminiscent of a previously suggested PS interaction site. Our structural data could contribute to the design of potential inhibitors able to disrupt membrane association.     

Increased risk of venous thrombosis by AB alleles of the ABO blood group and Factor V Leiden in a Brazilian population

Most cases of a predisposition to venous thrombosis are caused by resistance to activated protein C, associated in 95% of cases with the Factor V Leiden allele (FVL or R506Q). Several recent studies report a further increased risk of thrombosis by an association between the AB alleles of the ABO blood group and Factor V Leiden. The present study investigated this association with deep vein thrombosis (DVT) in individuals treated at the Hemocentro de Pernambuco in northeastern Brazil. A case-control comparison showed a significant risk of thrombosis in the presence of Factor V Leiden (OR = 10.1), which was approximately doubled when the AB alleles of the ABO blood group were present as well (OR = 22.3). These results confirm that the increased risk of deep vein thrombosis in the combined presence of AB alleles and Factor V Leiden is also applicable to the Brazilian population suggesting that ABO blood group typing should be routinely added to FVL in studies involving thrombosis.     

Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study

Background

Human blood coagulation factor VIII (fVIII) is a large plasma glycoprotein with sequential domain arrangement in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A1, A2 and A3 domains are interconnected by long linker peptides (a1, a2 and a3) that possess the activation sites. Proteolysis of fVIII zymogen by thrombin or factor Xa results in the generation of the activated form (fVIIIa) which serves as a critical co-factor for factor IXa (fIXa) enzyme in the intrinsic coagulation pathway.

Results

In our efforts to elucidate the structural differences between fVIII and fVIIIa, we developed the solution structural models of both forms, starting from an incomplete 3.7 Å X-ray crystal structure of fVIII zymogen, using explicit solvent MD simulations. The full assembly of B-domainless single-chain fVIII was built between the A1-A2 (Ala1-Arg740) and A3-C1-C2 (Ser1669-Tyr2332) domains. The structural dynamics of fVIII and fVIIIa, simulated for over 70 ns of time scale, enabled us to evaluate the integral motions of the multi-domain assembly of the co-factor and the possible coordination pattern of the functionally important calcium and copper ion binding in the protein.

Conclusions

MD simulations predicted that the acidic linker peptide (a1) between the A1 and A2 domains is largely flexible and appears to mask the exposure of putative fIXa enzyme binding loop (Tyr555-Asp569) region in the A2 domain. The simulation of fVIIIa, generated from the zymogen structure, predicted that the linker peptide (a1) undergoes significant conformational reorganization upon activation by relocating completely to the A1-domain. The conformational transition led to the exposure of the Tyr555-Asp569 loop and the surrounding region in the A2 domain. While the proposed linker peptide conformation is predictive in nature and warrants further experimental validation, the observed conformational differences between the zymogen and activated forms may explain and support the large body of experimental data that implicated the critical importance of the cleavage of the peptide bond between the Arg372 and Ser373 residues for the full co-factor activity of fVIII.     

Engineering of human coagulation factor X variants activated by prostate-specific antigen

In this study, we present a novel approach for the induction of tumor vessel thrombosis using genetically modified coagulation factor X. Human factor X was engineered in its activation peptide in a way that it can be specifically activated by prostate-specific antigen (PSA), a tumor-specific proteinase secreted into the bloodstream by prostate cancer cells. For this purpose we inserted different sequences of known PSA cleavage sites from the natural substrate of PSA, semenogelin I, into the activation peptide of factor X. One FX variant (FX-V4) was further optimized by site-directed mutagenesis of the P2 position and the P5 position (FX-V4-P2YP5R). After preincubation with PSA, FX-V4-P2YP5R was able to efficiently induce coagulation in vitro. These FX variants should be useful for site-specific induction of blood coagulation in the tumor vasculature.     

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF_1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF_1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.     

Identification of the N-terminal functional domains of Cdk5 by molecular truncation and computer modeling

Cyclin dependent kinase (Cdk) 5, an atypical member of the Cdk family, plays a fundamental role in the development of the nervous system, and may also be involved in the pathogenesis of certain neurodegenerative diseases. Further, Cdk5 is activated by the specific regulatory proteins p39, p35, or p25 rather than cyclins, and in contrast to other members of the Cdk family is not involved in the progression of the cell cycle. A three-dimensional computer model of Cdk5-p25-ATP has been generated previously [Chou et al., Biochem Biophys Res Commun 1999;259:420-428], providing a structural basis for the study of the mechanisms of Cdk5 activation. To assess the predicted ATP and p25 binding domains at the N-terminal of Cdk5, two mutants of Cdk5 were prepared in which amino acids 9-15 (Delta9-15) or 9-47 (Delta9-47) were deleted. The results of these studies clearly demonstrate that an N-terminal loop and the PSSALRE helix are indispensable for Cdk5-p25 interactions, and amino acids 9-15 are necessary for ATP binding but are not involved in Cdk5-p25 interactions. Predicted models of Delta9-15 Cdk5 and Delta9-47 Cdk5 were generated, and were used to interpret the experimental data. The experimental and molecular modeling results confirm and extend specific aspects of the original predicted computer model, and may provide useful information for the design of highly selective inhibitors of Cdk5, which could be used in the treatment of certain neurodegenerative conditions.     

The effect of calcium (II) on the binding of anticoagulation factor I with activated coagulation factor X

Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca²⁺-dependent fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca²⁺ ions was quantitatively analyzed by HPLC, and the result showed that the maximal binding of ACF I to FXa occurred at concentration of Ca²⁺ ions of about 1 mM. The binding of Ca²⁺ ions to ACF I was investigated by equilibrium dialysis and two Ca²⁺-binding sites with different affinities were identified. At pH 7.6, the apparent association constants K₁ and K₂ for these two sites were (1.8 ± 0.5) × 10⁵ and (2.7 ± 0.6) × 10⁴ M^–1 (mean ± SE, n = 4), respectively. It was evident from the observation of Ca²⁺-induced changes in the intrinsic fluorescence of ACF I that ACF I underwent a conformational change upon binding of Ca²⁺ ions. The occupation of both Ca²⁺-binding sites in ACF I required a concentration of Ca²⁺ ions of about 1 mM, which is equal to the effective concentration of Ca²⁺ ions required both for maximal binding of ACF I to FXa and for the maximal enhancement of emission fluorescence of ACF I. It could be deduced from these results that the occupation of both Ca²⁺-binding sites in ACF I with Ca²⁺ ions and subsequent conformational rearrangement might be essential for the binding of ACF I to FXa.     

Structural insight into how an anti-idiotypic antibody against D3H44 (anti-tissue factor antibody) restores normal coagulation

6A6 is a murine monoclonal antibody raised against the humanized anti-tissue factor antibody D3H44. 6A6 is able to completely neutralize the anticoagulant activity of D3H44 in tissue factor-dependent functional assays, such as endotoxin-induced whole blood clotting, prothrombin time, as well as factor X and factor IX activation. ELISA-type assays further showed that 6A6 binds to an epitope with critical determinants on the V(L) domain of D3H44. The possibility that the anti-idiotypic 6A6 might carry an "internal image" of the original antigen (tissue factor) was examined using the X-ray structure of the 6A6-Fab/D3H44-Fab complex determined at 2.5A resolution. We find that 6A6 structurally mimics tissue factor only so far as it combines with the antigen recognition surface of D3H44. While 6A6 contacts both V(L) and V(H) domains of D3H44, as does tissue factor, there is more contact with the D3H44 V(L) domain and less with the D3H44 V(H) domain relative to the tissue factor contacts on D3H44. Additionally, there is an almost total lack of correspondence between 6A6 and tissue factor at the level of amino acid side-chain functional groups. Despite the fact that both tissue factor and 6A6 are composed largely of beta-sheets, they present fundamentally different elements of secondary structure to D3H44; tissue factor presents beta-sheets edge-on, while 6A6 uses mostly loops. Finally, the finding that 6A6 competes with tissue factor for D3H44 binding raises the possibility of using 6A6 as an antidote for D3H44 anticoagulant therapy. To this end, we constructed a chimeric murine/human 6A6-Fab, which effectively neutralized D3H44 and fully restored tissue factor function in enzymatic assays.     

A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation

Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF-bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365-374{216-225}) of the so-called activation domain and the 170-loop (residues 313-322{170A-175}) succeeding the TF-binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active-site inhibitor. Based on MD simulations, a key switch of the TF-induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S(1) and S(3) substrate pockets, the activation pocket, and N-terminal insertion.     

Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.     

The tissue factor/factor VIIa/factor Xa complex: a model built by docking and site-directed mutagenesis

Factor X is activated to factor Xa (fXa) in the extrinsic coagulation pathway by the tissue factor (TF)/factor VIIa (fVIIa) complex. Upon activation, the fXa molecule remains associated with the TF/fVIIa complex, and this ternary complex is known to activate protease-activated receptors (PARs) 1 and 2. Activation of fVII in the TF complex by fXa is also seen at physiologic concentrations. The ternary complexes TF/fVII/fXa, TF/fVIIa/fX, and TF/fVIIa/fXa are therefore all physiologically relevant and of interest as targets for inhibition of both coagulation and cell-signaling pathways that are important in cardiovascular disease and inflammation. We therefore present a model of the TF/fVIIa/fXa complex, built with the use of the available structures of the TF/fVIIa complex and fXa by protein-protein docking calculations with the program Surfdock. The fXa model has an extended conformation, similar to that of fVIIa in the TF/fVIIa complex, with extensive interactions with TF and the protease domain of fVIIa. All four domains of fXa are involved in the interaction. The gamma-carboxyglutamate (Gla) and epithelial growth factor (EGF1 and EGF2) domains of fVIIa are not significantly involved in the interaction. Docking of the Gla domain of fXa to TF/fVIIa has been reported previously. The docking results identify potential interface residues, allowing rational selection of target residues for site-directed mutagenesis. This combination of docking and mutagenesis confirms that residues Glu51 and Asn57 in the EGF1 domain, Asp92 and Asp95 in the EGF2 domain, and Asp 185a, Lys 186, and Lys134 in the protease domain of factor Xa are involved in the interaction with TF/fVIIa. Other fX protease domain residues predicted to be involved in the interaction come from the 160s loop and the N-terminus of the fX protease domain, which is oriented in such a way that activation of both fVII by fXa, and the reciprocal fX activation by fVIIa, is possible.     

Identification of the oligosaccharide structures of human coagulation factor X activation peptide at each glycosylation site

Human blood coagulation factor X has two N-linked oligosaccharides at Asn³⁹ and Asn⁴⁹ residues and two O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues in the region of the factorX activationpeptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn³⁹ and Asn⁴⁹ residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri-and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Ac-26 residue. In addition, the structures of O-linked oligosaccharides at Thr¹⁷ and Thr²⁹ residues were suggested to be disialylated Gal/3GalNAc sequences by their component analyses.Abbreviations Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose - HPLC high-performance liquid chromatography - NDV Newcastle disease virus - Neu5Ac 5-N-acetylneuraminic acid - ODS octadecylsilyl - PA pyridylamino - RVV-X Russell's viper venom factor X activator - TBS Tris-buffered saline - XAP factor X activation peptide.     

Ssp DnaB intein大肠杆菌中介导FVIII重链和轻链的连接

研究利用intein的蛋白质反式剪接功能在大肠杆菌中对凝血VIII因子(FVIII)重链和轻链的连接作用,将B结构域大部分缺失型FVIII(BDD-FVIII)于满足剪接所需的保守性氨基酸Ser1657前断裂为重链和轻链,分别与split mini Ssp DnaB intein的106个氨基酸的N端(Int-N)和48个氨基酸的C端(Int-C)融合,构建到原核表达载体pBV220。诱导表达后SDS-PAGE分析可见预期大小的BDD-FVIII蛋白条带,Western blotting用FVIII特异性抗体证明其为剪接所产生的BDD-FVIII蛋白,表明intein可有效连接BDD-FVIII的重链和轻链。为进一步甲型血友病基因治疗研究应用intein以双腺相关病毒载体(AAV)携带FVIII基因,克服单个AAV载体的容量限制提供了依据。     

Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina

Blood coagulation factor V (FV) is a multifunctional protein that circulates in human plasma as a precursor molecule which can be activated by thrombin or activated factor X (FXa) in order to express its cofactor activity in prothrombin activation. FV activation is achieved by limited proteolysis after Arg709, Arg1018, and Arg1545 in the FV molecule. The venoms of Daboia russelli and Daboia lebetina contain a serine protease that specifically activates FV by a single cleavage at Arg1545. We have predicted the three-dimensional structure of these enzymes using comparative protein modeling techniques. The plasminogen activator from Agkistrodon acutus, which shows a high degree of homology with the venom FV activators and for which a high-quality crystallographic structure is available, was used as the molecular template. The RVV-V and LVV-V models provide for the first time a detailed and accurate structure of a snake venom FV activator and explain the observed sensitivity or resistance toward a number of serine protease inhibitors. Finally, electrostatic potential calculations show that two positively charged surface patches are present on opposite sides of the active site. We propose that both FV activators achieve their exquisite substrate specificity for the Arg1545 site via interactions between these exosites and FV.     

The function of the human factor V carbohydrate moiety in blood coagulation

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acid-specific Limax flavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.     

Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% -sheets, 20% -turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IXa and factor IXa. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% -structures, 22% -turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% -structures, 24% -turns, and 29% coils. None of these values is substantially altered as a result of increase of thepH from 7.4 to 10.5, or upon addition of Ca²⁺ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their -carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the abovepH adjustment and Ca²⁺ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the abovepH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by -sheets and -turns. Their overall secondary structures are not influenced by Ca²⁺ binding and are stable to alkalinepH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the -carboxyglutamic acid regions.     

Inhibition of human cytochrome P450 1A2 by flavones: A molecular modeling study

Cytochrome P450 1A2 metabolizes a number of important drugs, procarcinogens, and endogenous compounds. Several flavones, a class of phytochemicals consumed in the human diet, have been shown to differentially inhibit human P450 1A2-mediated methoxyresorufin demethylase. A molecular model of this P450 was constructed in order to elucidate the molecular basis of the P450-flavone interaction. Flavone and its 3,5,7-trihydroxy and 3,5,7-trimethoxy derivatives were docked into the active site to assess their mode of binding. The site is hydrophobic and includes several residues that hydrogen bond with substituents on the flavone nucleus. The binding interactions of these flavones in the modeled active side are consistent with their relative inhibitory potentials, namely 3,5,7-trihydroxylflavone > flavone >3,5,7-trimethoxylflavone, toward P450 1A2-mediated methoxyresorufin demethylation.     

Inhibition of human cytochrome P450 1A2 by flavones: A molecular modeling study

Cytochrome P450 1A2 metabolizes a number of important drugs, procarcinogens, and endogenous compounds. Several flavones, a class of phytochemicals consumed in the human diet, have been shown to differentially inhibit human P450 1A2-mediated methoxyresorufin demethylase. A molecular model of this P450 was constructed in order to elucidate the molecular basis of the P450-flavone interaction. Flavone and its 3,5,7-trihydroxy and 3,5,7-trimethoxy derivatives were docked into the active site to assess their mode of binding. The site is hydrophobic and includes several residues that hydrogen bond with substituents on the flavone nucleus. The binding interactions of these flavones in the modeled active side are consistent with their relative inhibitory potentials, namely 3,5,7-trihydroxylflavone > flavone >3,5,7-trimethoxylflavone, toward P450 1A2-mediated methoxyresorufin demethylation.