Specific length and structure rather than high thermodynamic stability enable regulatory mRNA stem-loops to pause translation.

Translating ribosomes unwind mRNA secondary structures by three basepairs each elongation cycle. Despite the ribosome helicase, certain mRNA stem-loops stimulate programmed ribosomal frameshift by inhibiting translation elongation. Here, using mutagenesis, biochemical and single-molecule experiments, we examine whether high stability of three basepairs, which are unwound by the translating ribosome, is critical for inducing ribosome pauses. We find that encountering frameshift-inducing mRNA stem-loops from the E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) hinders A-site tRNA binding and slows down ribosome translocation by 15-20 folds. By contrast, unwinding of first three basepairs adjacent to the mRNA entry channel slows down the translating ribosome by only 2-3 folds. Rather than high thermodynamic stability, specific length and structure enable regulatory mRNA stem-loops to stall translation by forming inhibitory interactions with the ribosome. Our data provide the basis for rationalizing transcriptome-wide studies of translation and searching for novel regulatory mRNA stem-loops.

Molecular orientation of factor VIIIa on the phospholipid membrane surface determined by fluorescence resonance energy transfer.

F (Factor) VIIIa binds to phospholipid membranes during formation of the FXase complex. Free thiols from cysteine residues of isolated FVIIIa A1 and A2 subunits and the A3 domain of the A3C1C2 subunit were labelled with PyMPO maleimide {1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)-oxazol-2-yl]pyridinium methanesulfonate} or fluorescein (fluorescence donors). Double mutations of the A3 domain (C2000S/T1872C and C2000S/D1828C) were also produced to utilize Cys(1828) and Cys(1872) residues for labelling. Labelled subunits were reacted with complementary non-labelled subunits to reconstitute FVIIIa. Octadecylrhodamine incorporated into phospholipid vesicles was used as an acceptor for distance measurements between FVIII residues and membrane surface by fluorescence resonance energy transfer. The results of the present study indicate that a FVIII axis on a plane that intersects the approximate centre of each domain is orientated with a tilt angle of ~30-50° on the membrane surface. This orientation predicted the existence of contacts mediated by residues 1713-1725 in the A3 domain in addition to a large area of contacts within the C domains. FVIII variants where Arg(1719) or Arg(1721) were mutated to aspartate showed a >40-fold reduction in membrane affinity. These results identify possible orientations for FVIIIa bound to the membrane surface and support a new interaction between the A3 domain and the membrane probably mediated in part by Arg(1719) and Arg(1721).

Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase.

Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having µM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 µM) and directly bound to FX (K(d) = 7.7 µM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.

Membrane-binding properties of the Factor VIII C2 domain.

Factor VIII functions as a cofactor for Factor IXa in a membrane-bound enzyme complex. Membrane binding accelerates the activity of the Factor VIIIa-Factor IXa complex approx. 100000-fold, and the major phospholipid-binding motif of Factor VIII is thought to be on the C2 domain. In the present study, we prepared an fVIII-C2 (Factor VIII C2 domain) construct from Escherichia coli, and confirmed its structural integrity through binding of three distinct monoclonal antibodies. Solution-phase assays, performed with flow cytometry and FRET (fluorescence resonance energy transfer), revealed that fVIII-C2 membrane affinity was approx. 40-fold lower than intact Factor VIII. In contrast with the similarly structured C2 domain of lactadherin, fVIII-C2 membrane binding was inhibited by physiological NaCl. fVIII-C2 binding was also not specific for phosphatidylserine over other negatively charged phospholipids, whereas a Factor VIII construct lacking the C2 domain retained phosphatidyl-L-serine specificity. fVIII-C2 slightly enhanced the cleavage of Factor X by Factor IXa, but did not compete with Factor VIII for membrane-binding sites or inhibit the Factor Xase complex. Our results indicate that the C2 domain in isolation does not recapitulate the characteristic membrane binding of Factor VIII, emphasizing that its role is co-operative with other domains of the intact Factor VIII molecule.

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.

Ca(2+) binding to both the heavy and light chains of factor VIII is required for cofactor activity.

Previously, we demonstrated that Ca(2+) was necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC) but that Ca(2+) did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca(2+) followed by factor Xa generation assay showed a two-site binding pattern, with indicated high-affinity (K(d) = 8.9 +/- 1.8 microM) and low-affinity (K(d) = 4.0 +/- 0.6 mM) sites. Analysis by equilibrium dialysis using (45)Ca and <400 microM free Ca(2+) verified a high-affinity binding (K(d) = 18.9 +/- 3.7 microM). Preincubation of either HC or LC with 6 mM Ca(2+) followed by reassociation with the untreated complementary chain in the presence of 0.12 mM Ca(2+) failed to generate significant cofactor activity (<0.5 nM min(-1) (nM LC)(-1)). However, pretreatment of both HC and LC with 6 mM Ca(2+) followed by reassociation (at 0.12 mM Ca(2+)) generated high activity (7.5 +/- 0.4 nM min(-1) (nM LC)(-1)). Progress curves for activity regain following factor VIII-Ca(2+) association kinetics fitted well to a series reaction scheme rather than one of simple association (p < 0.0001), suggesting a multistep process which may include a Ca(2+)-dependent conformational change. These results suggest that factor VIII contains two Ca(2+) binding sites with different affinities and that active factor VIII can be reconstituted from HC and LC only when both chains are preactivated by Ca(2+).