Structural features associated with the binding of glutamine-containing peptides to Factor XIII

Activated Factor XIII a2 catalyzes the formation of intermolecular gamma-glutamyl- epsilon -lysyl cross-links in the fibrin network. Solution NMR studies were carried out to characterize, the structural features associated with the binding of glutamine-containing peptides to Factor XIII. A coupled uv/vis kinetic assay demonstrated that K9 peptide (1-10), alpha2-antiplasmin (1-15), and alpha2-antiplasmin (1-15 Q4N) all function as glutamine-containing substrates for activated Factor XIII a2. 2D TOCSY spectra of the peptides exhibit upfield chemical shifts for the glutamine protons in the presence of Factor XIII. These results indicate that the reactive peptide glutamines are encountering a distinctive environment within the Factor XIII active site. 1D proton line-broadening and 2D transferred-NOESY studies reveal that the glutamines and residues located C-terminally come in direct contact with the enzyme and adopt an extended conformation. Substrates with sequences similar to alpha2-antiplasmin (1-15) are proposed to bind both at the catalytic site and at a neighboring apolar region.     

Replacing the Factor VIII C1 Domain with a Second C2 Domain Reduces Factor VIII Stability and Affinity for Factor IXa

Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIII_C2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (∼11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIII_C2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (∼2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIII_C2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIII_C2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (∼10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIII_C2C2 variant (∼4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIII_C2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.     

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode. This indicated that the LDLR site for FVIII is located within the LDLR cluster. Similar results were obtained for another ligand of LDLR, α-2-macroglobulin receptor-associated protein (RAP), a common ligand of receptors from the LDLR family. We further generated a set of recombinant fragments of the LDLR cluster and assessed their structural integrity by binding to RAP and by circular dichroism. A number of fragments overlapping CR.2-5 of the cluster were positive for binding RAP and FVIII. The specificity of these interactions was tested by site-directed mutagenesis of conserved tryptophans within the LDLR fragments. For FVIII, the specificity was also tested using a single-chain variable antibody fragment directed against the FVIII light chain as a competitor. Both cases resulted in decreased binding, thus confirming its specificity. The mutagenic study also showed an importance of the conserved tryptophans in LDLR for both ligands, and the competitive binding results showed an involvement of the light chain of FVIII in its interaction with LDLR. In conclusion, the region of CR.2-5 of LDLR was defined as the binding site for FVIII and RAP.     

Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His²⁸¹ (A1 domain) with Ser⁵²⁴ (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His²⁸¹ and Ser⁵²⁴ residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, K_m for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His²⁸¹ and Ser⁵²⁴ are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.     

Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766–Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764–Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.     

The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa

Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2.     

Factor VIIIa A2 Subunit Shows a High Affinity Interaction with Factor IXa: CONTRIBUTION OF A2 SUBUNIT RESIDUES 707-714 TO THE INTERACTION WITH FACTOR IXa*

Factor (F) VIIIa forms a number of contacts with FIXa in assembling the FXase enzyme complex. Surface plasmon resonance was used to examine the interaction between immobilized biotinylated active site-modified FIXa, and FVIII and FVIIIa subunits. The FVIIIa A2 subunit bound FIXa with high affinity (K_d = 3.9 ± 1.6 nm) that was similar to the A3C1C2 subunit (K_d = 3.6 ± 0.6 nm). This approach was used to evaluate a series of baculovirus-expressed, isolated A2 domain (bA2) variants where alanine substitutions were made for individual residues within the sequence 707-714, the C-terminal region of A2 thought to be FIXa interactive. Three of six bA2 variants examined displayed 2- to 4-fold decreased affinity for FIXa as compared with WT bA2. The variant bA2 proteins were also tested in two reconstitution systems to determine activity and affinity parameters in forming FXase and FVIIIa. V_max values for all variants were similar to the WT values, indicating that these residues do not affect cofactor function. All variants showed substantially greater increases in apparent K_d relative to WT in reconstituting the FXase complex (8- to 26-fold) compared with reconstituting FVIIIa (1.3- to 6-fold) suggesting that the mutations altered interaction with FIXa. bA2 domain variants with Ala replacing Lys⁷⁰⁷, Asp⁷¹², and Lys⁷¹³ demonstrated the greatest increases in apparent K_d (17- to 26-fold). These results indicate a high affinity interaction between the FVIIIa A2 subunit and FIXa and show a contribution of several residues within the 707-714 sequence to this binding.     

Role of calcium in the conformational dynamics of factor XIII activation examined by hydrogen-deuterium exchange coupled with MALDI-TOF MS

Factor XIII catalyzes formation of γ-glutamyl-ε-lysyl crosslinks within fibrin clots. FXIII A₂ can be activated proteolytically with thrombin and low mM Ca²⁺ or nonproteolytically with high monovalent/divalent cations along with low mM Ca²⁺. Physiologically, FXIII A₂ is poised to respond to transient influxes of Ca²⁺ in a Na⁺ containing environment. A successful strategy to monitor FXIII conformational events is hydrogen–deuterium exchange (HDX) coupled with mass spectrometry. FXIII A₂ was examined in the presence of different cations (Ca²⁺, Mg²⁺, Ba²⁺, Cu²⁺, Na⁺, TMAC⁺, and EDA²⁺) ranging from 1 to 2 mM, physiological Ca²⁺ concentration, to 50–500 mM for nonproteolytic activation. Increases in FXIII solvent exposure could already be observed at 1 mM Ca²⁺ for the dimer interface, the catalytic site, and glutamine substrate regions. By contrast, solvent protection was observed at the secondary cleavage site. These events occurred even though 1 mM Ca²⁺ is insufficient for FXIII activation. The metals 1 mM Mg²⁺, 1 mM Ba²⁺, and 1 mM Cu²⁺ each led to conformational changes, many in the same FXIII regions as Ca²⁺. FXIII could also be activated nonproteolytically with 500 mM tetramethylammonium chloride (TMAC⁺) and 500 mM ethylenediamine (EDA²⁺), both with 2 mM Ca²⁺. These different HDX studies help reveal the first FXIII segments that respond to physiological Ca²⁺ levels.     

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.     

Temporal Variations of Coagulation Factor VII Activity in Mice Are Influenced by Lighting Regime

It was recently reported that the circadian clock machinery controls plasma levels of factor (F) VII, the serine protease triggering blood coagulation. Here, by exploiting the mouse model, this study showed that variations of photoperiod (i.e., winter or summer conditions or simulated chronic jetlag conditions) have a strong impact on plasma FVII activity levels. Under conditions mimicking summer or winter photoperiods, FVII activity showed a clear 24 h rhythmicity. Interestingly, mean daily FVII activity levels were significantly reduced in mice exposed to summer photoperiods. Behavioral activity rhythms under both photoperiods were synchronized to LD cycles, and the amount of activity per 24 h was comparable. The authors also investigated the influence of chronic jetlag (CJL) on the FVII activity rhythms, which can be easily mimicked in mice through continuous abrupt shifts in the lighting schedule. The exposure of mice to simulated CJL of either consecutive westward or consecutive westward and eastward flights for 15 days did not abolish the behavioral activity rhythms but was associated with a period significantly different from 24 h. Intriguingly, both types of CJL exerted a strong influence on FVII activity rhythms, which were virtually suppressed. Moreover, the mean daily FVII activity was significantly lower in the CJL than in the winter photoperiod condition. Taken together, these findings in mice provide novel insights into the modulation of FVII activity levels, which might have implications for human pathophysiology.     

Reversible activation of cellular factor XIII by calcium

Factor XIII (FXIII) is a pro-transglutaminase found in the plasma as well as intracellularly in platelets and macrophages. Plasma FXIII is activated by thrombin cleavage (FXIIIa*) and acts in the final stages of blood coagulation cascade. In contrast, the function and activation of cellular FXIII are less characterized. Cellular FXIII relies on a conformational activation of the protein. The nonproteolytic activation of FXIII to FXIIIa° induced by Ca(2+) alone is well known, but up until now it has been discussed under which conditions the process can be induced and whether it can be reversed. Here, we study the nature of the Ca(2+)-induced FXIII activation. Previously used methods to evaluate FXIII activity detect both FXIIIa* and FXIIIa° because they rely on occurrence of enzyme activity or on active site Cys-314 solvent accessibility. Therefore, an analytical HPLC method was developed that separates zymogen recombinant FXIII (rFXIII) from rFXIIIa°. The data demonstrate that nonproteolytic activation and deactivation are highly dependent on Ca(2+) concentration, buffer, and salt components. Moreover, it is established that Ca(2+) activation of rFXIII is fully reversible, and only 2-5 mm CaCl(2) is sufficient to retain full rFXIIIa° activity. However, below 2 mm CaCl(2) the rFXIIIa° molecule deactivates. The deactivated molecule can subsequently undergo a new activation round. Furthermore, it is demonstrated that thermal stress of freeze-dried rFXIII can induce a new predisposed form that activates faster than nonstressed rFXIII.     

Interactions Outside the Proteinase-binding Loop Contribute Significantly to the Inhibition of Activated Coagulation Factor XII by Its Canonical Inhibitor from Corn

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (K_i = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a K_i of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20–45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (K_i = 11.7 ± 1.2 μm) and activated factor XI (K_i = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a K_i of 1.3 ± 0.2 nm and activated factor XI with a K_i of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.     

Restoring the Procofactor State of Factor Va-like Variants by Complementation with B-domain Peptides

Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism of this FV autoinhibition. We show that a fragment encoding the basic region (BR) of the B-domain binds with high affinity to cofactor-like FV(a) variants that harbor an intact acidic region. Furthermore, the BR inhibits procoagulant function of the variants, thereby restoring the procofactor state. The BR competes with FXa for binding to FV(a), and limited proteolysis of the B-domain, specifically at Arg¹⁵⁴⁵, ablates BR binding to promote high affinity association between FVa and FXa. These results provide new insight into the mechanism by which the B-domain stabilizes FV as an inactive procofactor and reveal how limited proteolysis of FV progressively destabilizes key regulatory regions of the B-domain to produce an active form of the molecule.     

Reactivity of the N-terminal region of fibronectin protein to transglutaminase 2 and factor XIIIA

Transglutaminase 2 (TG2) is secreted by a non-classical pathway into the extracellular space, where it has several activities pertinent to fibronectin (FN), including binding to the gelatin-binding domain of FN and acting as an integrin co-receptor. Glutamines in the N-terminal tail of FN are known to be susceptible to transamidation by both TG2 and activated blood coagulation factor XIII (FXIIIa). We used immunoblotting, limited proteolysis, and mass spectrometry to localize glutamines within FN that are subject to TG2-catalyzed incorporation of dansylcadaverine in comparison to residues modified by FXIIIa. Such analysis of plasma FN indicated that Gln-3, Gln-7, and Gln-9 in the N-terminal tail and Gln-246 of the linker between fifth and sixth type I modules ((5)F1 and (6)F1) are transamidated by both enzymes. Only minor incorporation of dansylcadaverine was detected elsewhere. Labeling of C-terminally truncated FN constructs revealed efficient TG2- or FXIIIa-catalyzed dansylcadaverine incorporation into the N-terminal residues of constructs as small as the 29-kDa fragment that includes (1-5)F1 and lacks modules from the adjacent gelatin-binding domain. However, when only (1-3)F1 were present, dansylcadaverine incorporation into the N-terminal residues of FN was lost and instead was in the enzymes, near the active site of TG2 and terminal domains of FXIIIa. Thus, these results demonstrate that FXIIIa and TG2 act similarly on glutamines at either end of (1-5)F1 and transamidation specificity of both enzymes is achieved through interactions with the intact 29K fragment.     

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase^{1, 2}. Manual FV assays have been described ^{3, 4}, but they are time consuming and subjective. Automated FV assays have been reported ^5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput ^{8, 9}. Microplate assays have been reported for clot lysis ¹⁰, platelet aggregation ¹¹, and coagulation Factors ¹², but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) ¹³. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections ¹⁴. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology ¹⁵. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.     

凝血因子及血浆蛋白类制品的研究现状及发展前景

血浆制品主要是以血浆为原料,经特殊的分离提纯而得到的血浆单一组分,也有部分血浆制品可以通过生物工程技术合成。不同的组分具有相应的功能,血浆制品是一种特殊的药品。目前市场上分离且临床上使用的血浆制品主要有凝血因子、白蛋白、免疫球蛋白以及凝血酶、蛋白C、α1-抗胰蛋白酶、α2-巨球蛋白、组织纤溶酶原激活剂、补体脂酶抑制剂、血清胆碱酯酶、铜蓝蛋白、转铁蛋白、纤维结合蛋白\高密度脂蛋白等微量血浆蛋白等。凝血因子根据不同的功能分为FI、FII、FIII、FIV、FV、FVII、FVIII、FIX、FX、FXI、FXII以及因子FXIII。随着技术的发展已经人们对血浆制品的认知度越来越高,血浆制品正已前所未有的速度发展。     

Trp2313-His2315 of Factor VIII C2 Domain Is Involved in Membrane Binding: STRUCTURE OF A COMPLEX BETWEEN THE C2 DOMAIN AND AN INHIBITOR OF MEMBRANE BINDING*

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 Å) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed β-strand of the C2 domain, Trp²³¹³-His²³¹⁵. This result indicates that the Trp²³¹³-His²³¹⁵ segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.     

Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin

The NH2-terminal 12-residue peptide of alpha 2-plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Gly-Leu-Lys-NH2 . AcOH, was found to be a good substrate for plasma transglutaminase (activated blood coagulation factor XIII) and rapidly incorporated into fibrin by the enzyme. A high concentration of the peptide inhibited the enzyme-mediated cross-linking of alpha 2-plasmin inhibitor to fibrin probably by competing with the inhibitor for the same site of fibrin alpha-chain.     

Tissue transglutaminase-mediated glutamine deamidation of beta-amyloid peptide increases peptide solubility, whereas enzymatic cross-linking and peptide fragmentation may serve as molecular triggers for rapid peptide aggregation

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGase-mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of β-amyloid peptide (Aβ), resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of Aβ monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGase-mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation.     

The Structure of Human Apolipoprotein A-IV as Revealed by Stable Isotope-assisted Cross-linking,Molecular Dynamics,and Small Angle X-ray Scattering

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a “clasp” mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.