Daniel O. Beck

Structural Requirements for Expression of Factor Va Activity

Thrombin activated factor Va (factor VIIa, residues 1–709 and 1546–2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of ∼0.5 nm. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1–696) and a Mr 80,000 light chain (amino acid residues 1509/1514–2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses ∼60–80% of its clotting activity. An enzyme from Russells viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1–1018 and 1546–2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by α-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app ∼ 0.5 nm). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697–709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697–709 of the heavy chain of the cofactor.

The contribution of amino acid region Asp695-Tyr698 of factor V to procofactor activation and factor Va function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC50 values of ∼12 and ∼10 μm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with Ki values of ∼6.3 and ∼5.3 μm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp695-Tyr-Asp-Tyr-Gln699 (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC50 of 1.6 μm (with a KD for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp695 → Lys, Tyr696 → Phe, Asp697 → Lys, and Tyr698 → Phe (factor V2K2F) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor VaRVV2K2F) and factor Xa (factor VaXa2K2F). Factor VaRVV2K2F and factor VaXa2K2F had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.

Structure of TatA Paralog, TatE, suggests a structurally homogeneous form of Tat protein translocase that transports folded proteins of differing diameter

Background: The Tat system transports folded proteins in bacteria. Results: Unlike TatA, the paralogous TatE is found as small, homogeneous complexes. Conclusion: TatE cannot form a variable translocation channel as suggested for TatA. Significance: This work suggests a new model in which a structurally homogeneous form of translocase uses a flexible channel. The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70–90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6–8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed.

The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII

α-Thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. α-Thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by α-thrombin. We have used plasma-derived α-thrombin, β-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg320 (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{DY}(\mathrm{SO}_{3}^{-})\mathrm{DY}(\mathrm{SO}_{3}^{-})\mathrm{Q}\) \end{document}, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with β-thrombin was increased by ∼8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than α-thrombin under similar experimental conditions. α-Thrombin readily activated factor V following cleavages at Arg709, Arg1018, and Arg1545 and factor VIII following proteolysis at Arg372, Arg740, and Arg1689. Cleavage of both procofactors byα-thrombin was significantly inhibited by D5Q1,2. In contrast, β-thrombin was unable to cleave factor V at Arg1545 and factor VIII at both Arg372 and Arg1689. The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. β-Thrombin was found to cleave factor V at Arg709 and factor VIII at Arg740, albeit less efficiently than α-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by β-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of α-thrombin can account for the interaction of both procofactors with α-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of α-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

Ultrastructural characterisation of Bacillus subtilis TatA complexes suggests they are too small to form homooligomeric translocation pores

Tat-dependent protein transport permits the traffic of fully folded proteins across membranes in bacteria and chloroplasts. The mechanism by which this occurs is not understood. Current theories propose that a key step requires the coalescence of a substrate-binding TatC-containing complex with a TatA complex, which forms pores of varying sizes that could accommodate different substrates. We have studied the structure of the TatAd complex from Bacillus subtilis using electron microscopy to generate the first 3D model of a TatA complex from a Gram-positive bacterium. We observe that TatAd does not exhibit the remarkable heterogeneity of Escherichia coli TatA complexes but instead forms ring-shaped complexes of 7.5–9 nm diameter with potential pores of 2.5–3 nm diameter that are occluded at one end. Such structures are consistent with those seen for E. coli TatE complexes. Furthermore, the small diameter of the TatAd pore, and the homogeneous nature of the complexes, suggest that TatAd cannot form the translocation channel by itself. Biochemical data indicate that another B. subtilis TatA complex, TatAc, has similar properties, suggesting a common theme for TatA-type complexes from Bacillus.

Contribution of Amino Acid Region 334−335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase

We have demonstrated that amino acids E323, Y324, E330, and V331 from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332−336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D334 and Y335 as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D334 → K and Y335 → F (factor VKF) and D334 → A and Y335 → A (factor VAA). Kinetic studies showed that while factor VaKF and factor VaAA had a KD for factor Xa similar to the KD observed for wild-type factor Va (factor VaWT), the clotting activities of the mutant molecules were impaired and the kcat of prothrombinase assembled with factor VaKF and factor VaAA was reduced. The second-order rate constant of prothrombinase assembled with factor VaKF or factor VaAA for prothrombin activation was ∼10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor VaWT. We also created quadruple mutants combining mutations in the amino acid region 334–335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E323Y324 and E330V331). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor VaWT. The data demonstrate that amino acid region 334–335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.

A mutation leading to super-assembly of twin-arginine translocase (Tat) protein complexes

The Tat system transports folded proteins across the bacterial plasma membrane. The mechanism is believed to involve coalescence of a TatC-containing unit with a separate TatA complex, but the full translocation complex has never been visualised and the assembly process is poorly defined. We report the analysis of the Bacillus subtilis TatAyCy system, which occurs as separate TatAyCy and TatAy complexes at steady state, using single-particle electron microscopy (EM) and advanced atomic force microscopy (AFM) approaches. We show that a P2A mutation in the TatAy subunit leads to apparent super-assembly of Tat complexes. Purification of TatCy-containing complexes leads to a large increase in the TatA:TatC ratio, suggesting that TatAy(P2A) complexes may have attached to the TatAyCy complex. EM and AFM analyses show that the wild-type TatAyCy complex purifies as roughly spherical complexes of 9-16nm diameter, whereas the P2A mutation leads to accumulation of large (up to 500nm long) fibrils that are chains of numerous complexes. Time lapsed AFM imaging, recorded on fibrils under liquid, shows that they adopt a variety of tightly curved conformations, with radii of curvature of 10-12nm comparable to the size of single TatAy(P2A) complexes. The combined data indicate that the mutation leads to super-assembly of TatAy(P2A) complexes and we propose that an individual TatAy(P2A) complex assembles initially with a TatAy(P2A)Cy complex, after which further TatAy(P2A) complexes attach to each other. The data further suggest that the N-terminal extracytoplasmic domain of TatAy plays an essential role in Tat complex interactions.