Biomedical subjects

Philip J Fay

Publications and source records attributed to Philip J Fay.

At least 19 recordsLinked to original sources

PubMed2006

Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa.

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.

Amino Acids, Acidic↗

PubMed2006

pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+.

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.

Copper↗

PubMed2006

Factor VIII structure and function.

Factor VIII, a non-covalent heterodimer comprised of a heavy chain (A1-A2-B domains) and light chain (A3-C1-C2 domains), circulates as an inactive procofactor in complex with von Willebrand factor. Metal ions are critical to the integrity of factor VIII, with Cu and Ca ions stabilizing the heterodimer and generating the active conformation, respectively. Activation of factor VIII catalyzed by thrombin appears dependent upon interactions with both anion-binding exosites I and II, and converts the heterodimer to the active cofactor, factor VIIIa. This protein, comprised of A1, A2, and A3-C1-C2 subunits, is labile due to weak affinity of the A2 subunit. Association of factor VIIIa with factor IXa to form the intrinsic factor Xase complex is membrane-dependent and involves multiple inter-protein contacts that remain poorly characterized. This complex catalyzes the conversion of factor X to factor Xa, a reaction that is essential for the propagation phase of coagulation. The role of factor VIIIa in this complex is to increase the catalytic efficiency for factor Xa generation by several orders of magnitude. Mechanisms for the down-regulation of factor Xase focus upon inactivation of the cofactor and include dissociation of the A2 subunit as well as activated protein C-catalyzed proteolysis.

Blood Coagulation Factors↗

PubMed2005

A Glu113Ala mutation within a factor VIII Ca2+-binding site enhances cofactor interactions in factor Xase.

We recently identified an acidic-rich segment in the A1 domain of factor VIII (residues 110-126) that functions in the coordination of Ca(2+), an ion necessary for cofactor activity [Wakabayashi et al. (2004) J. Biol. Chem. 279, 12677-12684]. Mutagenesis studies showed that replacement of residue Glu113 with Ala (E113A) yielded a factor VIII point mutant possessing increased specific activity as determined by a one-stage clotting assay. Mutagenesis at this site suggested that substitution with relatively small, nonpolar residues was well tolerated, whereas replacement with a number of polar or charged residues appeared detrimental to activity. Ala substitution resulted in the greatest enhancement, yielding an approximately 2-fold increased specific activity. Time course experiments following reaction with thrombin revealed similar rates of activation and inactivation of E113A as observed for the wild type. Results from factor Xa generation assays showed minimal differences in kinetic parameters and factor IXa affinity for E113A and wild-type factor VIIIa when run in the presence of synthetic phospholipid vesicles, whereas factor VIIIa E113A displayed an approximately 4-fold greater affinity for factor IXa compared with factor VIIIa wild type in reactions run on the platelet membrane surface. This latter effect may be attributed, in part, to a 2-fold increased affinity of factor VIIIa E113A for the platelet membrane. Considering that low levels of factors VIIIa and IXa are generated during clotting in plasma, the increased cofactor specific activity observed for E113A factor VIII may result from its enhanced affinity for factor IXa on the physiological membrane.

Alanine↗

PubMed2005

Factor VIII A3 domain residues 1954-1961 represent an A1 domain-interactive site.

Factor VIIIa consists of subunits designated A1, A2, and A3C1C2. Reassociation of the A1 and A3C1C2 subunits monitored by the factor Xa generation assay and fluorescence resonance energy transfer yielded intersubunit affinity values (K(d)) of 58.0 +/- 12.5 and 58.8 +/- 16.8 nM, respectively. These affinity values were equivalent to that previously determined for factor VIII heavy and light chains [Wakabayashi, H., et al. (2001) Biochemistry 40, 10293-10300], suggesting that the A2 domain makes a minimal contribution to the interchain affinity in factor VIII. Ca(2+) showed no effect on A1/A3C1C2 intersubunit affinity (K(d) = 51.6 +/- 16.6 nM), while Cu(2+) enhanced the A1/A3C1C2 intersubunit affinity approximately 5-fold (K(d) = 12.5 +/- 2.3 nM). A synthetic peptide to A3 domain residues 1954-1961 inhibited association of the A1 and A3C1C2 subunits (K(i) = 65.8 +/- 11.9 microM). Three factor VIII point mutants, His1957Ala, Gly1960Val, and His1961Asp, were stably expressed in BHK cells and purified. All mutants exhibited reduced specific activity (39, 42, and 4%, respectively) compared with that of wild-type factor VIII, and their activity was less stable following heat denaturation analysis (t(1/2) values of 13.3 +/- 0.7, 8.7 +/- 0.3, and 8.1 +/- 0.1 min, respectively) compared with that of the wild type (18.8 +/- 0.8 min). This reduced stability appeared to result from an approximately 2-fold increased dissociation rate for the Gly1960Val and His1961Asp dimers as judged by solid-phase binding assays. We propose that residues 1954-1961 of the A3 domain contribute to interactions with the A1 domain, facilitating their association in factor VIII.

Amino Acid Sequence↗

PubMed2005

Exosite-interactive regions in the A1 and A2 domains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain.

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation.

Catalysis↗

PubMed2005

Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site.

Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type. N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar Michaelis-Menten constant (K(m), apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (V(max)) for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild-type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P(1)Arg, and this property likely reflects the severity of the hemophilia A phenotype.

Amino Acid Substitution↗

PubMed2005

Mutating factor VIII: lessons from structure to function.

Factor VIII, a metal ion-dependent heterodimer, circulates in complex with von Willebrand factor. At sites of vessel wall damage, this procofactor is activated to factor VIIIa by limited proteolysis and assembles onto an anionic phospholipid surface in complex with factor IXa to form the intrinsic factor Xase; an enzyme complex that efficiently converts factor X to factor Xa during the propagation phase of coagulation. Factor Xase activity is down-regulated by mechanisms that include self-dampening by dissociation of a critical factor VIIIa subunit and proteolytic inactivation by the activated protein C pathway. Recent studies identify putative metal ion coordination sites as well as ligands involved in the catabolism of the activated and procofactor forms of the protein. Our knowledge of these multiple intra- and inter-molecular interactions has been facilitated by the application of naturally occurring and site-directed mutations to study factor VIII structure and function. In this review, we document important and novel contributions following this line of investigation.

Cysteine Endopeptidases↗

PubMed2004

Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit.

Factor VIIIa can be reconstituted from A2 subunit and A1/A3-C1-C2 dimer in a reaction that is facilitated by slightly acidic pH. We recently demonstrated that a truncated A1 (A1(37-336)) possessed markedly reduced affinity for A2 compared with intact A1, but retained 30% of native factor VIIIa activity in the presence of A3-C1-C2. We now identify A1-interactive regions for A2 using A1 fragments derived from a limited tryptic digest. Unfractionated trypsin-cleaved A1 inhibited reconstituted factor VIIIa activity. Two fragments, designated A1(37-121) and A1(221-336), markedly inhibited factor VIIIa reconstitution with either native A1 (K(i)=340 and 194 nM, respectively) or with A1(37-336) (K(i)=69 and 116 nM, respectively) at pH 6.0. A third fragment designated A1(122-206) did not possess inhibitory activity. At pH 7.2, the A1(221-336) partially inhibited reconstitution, whereas the A1(37-121) possessed little if any inhibitory activity. Both fragments inhibited factor VIIIa reconstitution as judged by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1 forms at pH 6.0. Furthermore, covalent cross-linking between A2 and A1(37-121) but not A1(221-336) was observed following reaction with a zero-length cross-linker. These findings demonstrate the presence of an extended, pH-dependent A2-interactive surface within regions 37-121 and 221-336 of A1. This interactive surface appears conformationally labile in the truncated A1 as judged by its apparent stabilization following association with A3-C1-C2.

Binding Sites↗

PubMed2004

Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain.

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys36 cleavage site, generating the A137-372 intermediate with approximately 20% the catalytic efficiency of wild type. This defect likely resulted from an approximately 4-fold increase in Km for the A1 substrate because kcat values for the wild type and mutant were equivalent. Cleavage of the A1-A2 domain junction by factor Xa R240A was not blocked by the 337-372 peptide. Studies using mutant factor VIII where clustered acidic residues in the 337-372 segment were replaced by Ala showed that a factor VIIIa D361A/D362A/D363A mutant possessed a approximately 1.6-fold increase in Km for factor X compared with wild type. However, similar Km values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase.

Alanine↗

PubMed2004

Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase.

Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective competitor of FVIIIa binding to FIXa as judged by inhibition of FXa generation performed in the absence of vesicles (K(i) approximately 1.6K(d) for FVIIIa-FIXa). However, the capacity for A3-C1-C2 to inhibit FVIIIa-dependent FXa generation in the presence of phospholipid was poor with a K(i) values (approximately 400 nM) that were approximately 100-fold greater than the K(d) for FVIIIa-FIXa interaction (4.2 +/- 0.6 nM). These results indicated that a significant component of the interprotein affinity is contributed by FVIIIa subunits other than A3-C1-C2 in the membrane-dependent complex. The isolated A2 subunit of FVIIIa interacts weakly with FIXa, and recent modeling studies have implicated a number of residues that potentially contact the FIXa protease domain (Bajaj et al. (2001) J. Biol. Chem. 276, 16302-16309). Site-directed mutagenesis of candidate residues in the A2 domain was performed, and recombinant proteins were stably expressed and purified. Functional affinity determinations demonstrated that one mutant, FVIII/Asp712Ala exhibited an 8-fold increased K(d) (35 +/- 1.5 nM) relative to wild-type suggesting a contribution by this residue of approximately 10% of the FVIIIa-FIXa binding energy. Thus both A2 and A3-C1-C2 subunits contribute to the affinity of FVIIIa for FIXa in the membrane-dependent FXase.

Binding, Competitive↗

PubMed2004

Identification of a factor Xa-interactive site within residues 337-372 of the factor VIII heavy chain.

We recently demonstrated that the residues 337-372, comprising the acidic C-terminal region in A1 subunit, interact with factor Xa during the proteolytic inactivation of factor VIIIa (Nogami, K., Wakabayashi, H., and Fay, P. J. (2003) J. Biol. Chem. 278, 16502-16509). We now show this sequence is important for factor Xa-catalyzed activation of factor VIII. Peptide 337-372 markedly inhibited cofactor activation, consistent with a delay in the rate of cleavage at the A1-A2 junction. Studies using the isolated factor VIII heavy chain indicated that the peptide completely blocked cleavage at the A1-A2 junction (IC50 = 11 microm) and partially blocked cleavage at the A2-B junction (IC50 = 100 microm). Covalent cross-linking was observed between the 337-372 peptide and factor Xa following reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the peptide quenched the fluorescence of dansyl-Glu-Gly-Arg active site-modified factor Xa, suggesting that residues 337-372 directly interact with factor Xa. Studies using a monoclonal antibody recognizing residues 351-365 as well as the peptide to this sequence further restricted the interactive region. Mutant factor VIII molecules in which clustered acidic residues in the 337-372 segment were converted to alanine were evaluated for activation by factor Xa. Of the mutants tested, only factor Xa-catalyzed activation of the D361A/D362A/D363A mutant was inhibited with peak activity of approximately 50% and an activation rate constant of approximately 30% of the wild type values. These results indicate that the 337-372 acidic region separating A1 and A2 domains and, in particular, a cluster of acidic residues at position 361-363 contribute to a unique factor Xa-interactive site within the factor VIII heavy chain that promotes factor Xa docking during cofactor activation.

Amino Acid Sequence↗

PubMed2004

Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity.

Generation of factor VIII cofactor activity requires divalent metal ions such as Ca2+ or Mn2+. Evaluation of cofactor reconstitution from isolated factor VIIIa subunits revealed the presence of a functional Ca2+ binding site within the A1 subunit. Isothermal titration calorimetry demonstrated at least two Ca2+ binding sites of similar affinity (K(d) = 0.74 microm) within the A1 subunit. Mutagenesis of an acidic residue-rich region in the A1 domain (residues 110-126) homologous to a putative Ca2+ binding site in factor V (Zeibdawi, A. R., and Pryzdial, E. L. (2001) J. Biol. Chem. 276, 19929-19936) and expression of B-domainless factor VIII molecules yielded reagents to probe Ca2+ and Mn2+ binding in a functional assay. Basal activity observed for wild type factor VIII in a metal ion-free buffer was enhanced approximately 2-fold with saturating Ca2+ or Mn2+ and yielded functional K(d) values of 1.2 and 1.40 microm, respectively. Ca2+ binding affinity was greatly reduced (or lost) in several mutants including E110A, E110D, D116A, E122A, D125A, and D126A. Alternatively, E113A, D115A, and E124A showed wild type-like activity with little or no reduction in Ca2+ affinity. However, Mn2+ affinity was minimally altered except for mutant D125A (and D116A). These results are consistent with region 110-126 serving a critical role for Ca2+ coordination with selected residues capable of contributing to a partially overlapping site for Mn2+, and that occupancy of either site is required for maximal cofactor activity.

Amino Acid Sequence↗

PubMed2004

Activation of factor VIII and mechanisms of cofactor action.

The factor VIII procofactor circulates as a metal ion-dependent heterodimer of a heavy chain and light chain. Activation of factor VIII results from limited proteolysis catalyzed by thrombin or factor Xa, which binds the factor VIII substrate over extended interactive surfaces. The proteases efficiently cleave factor VIII at three sites, two within the heavy and one within the light chain resulting in alteration of its covalent structure and conformation and yielding the active cofactor, factor VIIIa. The role of factor VIIIa is to markedly increase the catalytic efficiency of factor IXa in the activation of factor X. This effect is manifested in a dramatic increase in the catalytic rate constant, k(cat), by mechanisms that remain poorly understood.

Cysteine Endopeptidases↗

PubMed2003

Mechanisms of factor Xa-catalyzed cleavage of the factor VIIIa A1 subunit resulting in cofactor inactivation.

Activation of factor VIII by factor Xa is followed by proteolytic inactivation resulting from cleavage within the A1 subunit (residues 1-372) of factor VIIIa. Factor Xa attacks two sites in A1, Arg(336), which precedes the highly acidic C-terminal region, and a recently identified site at Lys(36). By using isolated A1 subunit as substrate for proteolysis, production of the terminal fragment, A1(37-336), was shown to proceed via two pathways identified by the intermediates A1(1-336) and A1(37-372) and generated by initial cleavage at Arg(336) and Lys(36), respectively. Appearance of the terminal product by the former pathway was 7-8-fold slower than the product obtained by the latter pathway. The isolated A1 subunit was cleaved slowly, independent of the presence of phospholipid. The A1/A3-C1-C2 dimer demonstrated an approximately 3-fold increased cleavage rate constant, and inclusion of phospholipid further enhanced this value by approximately 2-fold. Although association of A1 or A1(37-372) with A3-C1-C2 enhanced the rate of cleavage at Arg(336), inclusion of A3-C1-C2 did not affect the cleavage at Lys(36) in A1(1-336). A synthetic peptide 337-372 blocked the cleavage at Lys(36) (IC(50) = 230 microm) while showing little if any effect on cleavage at Arg(336). Proteolysis at Lys(36), and to a lesser extent Arg(336), was inhibited in a dose-dependent manner by heparin. These results suggest that inactivating cleavages catalyzed by factor Xa at Lys(36) and Arg(336) are regulated in part by the A3-C1-C2 subunit. Furthermore, cleavage at Lys(36) appears to be selectively modulated by the C-terminal acidic region of A1, a region that may interact with factor Xa via its heparin-binding exosite.

Blood Coagulation↗

PubMed2003

Exosite-dependent regulation of factor VIIIa by activated protein C.

Activated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80-loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

Arginine↗

PubMed2003

Mn2+ binding to factor VIII subunits and its effect on cofactor activity.

Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.

Binding Sites↗

PubMed2003

Template dimerization promotes an acceptor invasion-induced transfer mechanism during human immunodeficiency virus type 1 minus-strand synthesis.

The biochemical mechanism of template switching by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the role of template dimerization were examined. Homologous donor-acceptor template pairs derived from the HIV-1 untranslated leader region and containing the wild-type and mutant dimerization initiation sequences (DIS) were used to examine the efficiency and distribution of transfers. Inhibiting donor-acceptor interaction was sufficient to reduce transfers in DIS-containing template pairs, indicating that template dimerization, and not the mere presence of the DIS, promotes efficient transfers. Additionally, we show evidence that the overall transfer process spans an extended region of the template and proceeds through a two-step mechanism. Transfer is initiated through an RNase H-facilitated acceptor invasion step, while synthesis continues on the donor template. The invasion then propagates towards the primer terminus by branch migration. Transfer is completed with the translocation of the primer terminus at a site distant from the invasion point. In our system, most invasions initiated before synthesis reached the DIS. However, transfer of the primer terminus predominantly occurred after synthesis through the DIS. The two steps were separated by 60 to 80 nucleotides. Sequence markers revealed the position of primer terminus switch, whereas DNA oligomers designed to block acceptor-cDNA interactions defined sites of invasion. Within the region of homology, certain positions on the template were inherently more favorable for invasion than others. In templates with DIS, the proximity of the acceptor facilitates invasion, thereby enhancing transfer efficiency. Nucleocapsid protein enhanced the overall efficiency of transfers but did not alter the mechanism.

5' Untranslated Regions↗