Patricia J. Anderson

Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting. Here we present the 2.2 Å crystal structures of human α-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as ‘molecular sexuality’.

Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease

Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr1605–Met1606 scissile bond in the VWF A2 domain depends on a Glu1660–Arg1668 segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme–substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage ≈20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.

Zinc and Calcium Ions Cooperatively Modulate ADAMTS13 Activity

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) multimers. The metal ion dependence of ADAMTS13 activity was examined with multimeric VWF and a fluorescent peptide substrate based on Asp1596–Arg1668 of the VWF A2 domain, FRETS-VWF73. ADAMTS13 activity in citrate-anticoagulated plasma was enhanced ∼2-fold by zinc ions, ∼3-fold by calcium ions, and ∼6-fold by both ions, suggesting cooperative activation. Cleavage of VWF by recombinant ADAMTS13 was activated up to ∼200-fold by zinc ions (KD app ∼0.5 μm), calcium ions (KD app ∼4.8 μm), and barium ions (KD app ∼1.7 mm). Barium ions stimulated ADAMTS13 activity in citrated plasma but not in citrate-free plasma. Therefore, the stimulation by barium ions of ADAMTS13 in citrated plasma appears to reflect the release of chelated calcium and zinc ions from complexes with citrate. At optimal zinc and calcium concentrations, ADAMTS13 cleaved VWF with a Km app of 3.7 ± 1.4 μg/ml (∼15 nm for VWF subunits), which is comparable with the plasma VWF concentration of 5–10 μg/ml. ADAMTS13 could cleave ∼14% of VWF pretreated with guanidine HCl, suggesting that this substrate is heterogeneous in susceptibility to proteolysis. ADAMTS13 cleaved FRETS-VWF73 with a Km app of 3.2 ± 1.1 μm, consistent with an ∼200-fold decrease in affinity compared with VWF. ADAMTS13 cleaved VWF and FRETS-VWF73 with roughly comparable catalytic efficiency of 55 μm–1 min–1 and 18 μm–1 min–1, respectively. The striking preference of ADAMTS13 for VWF suggests that substrate recognition depends on structural features or exosites on multimeric VWF that are missing from FRETS-VWF73.

Thrombotic thrombocytopenic purpura directly linked with ADAMTS13 inhibition in the baboon ( Papio ursinus )

Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor-cleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n = 6) induced severe thrombocytopenia (< 12 × 10(9)/L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet- and von Willebrand factor-rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n = 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n = 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Furthermore, we present a reliable animal model of this disease as an opportunity for the development and validation of novel treatment strategies.

Multi-step binding of ADAMTS-13 to von Willebrand factor

Summary.  Background: ADAMTS‐13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non‐catalytic domains in ADAMTS‐13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS‐13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS‐13. Methods: Immunoprecipiation of VWF–ADAMTS‐13 complexes using anti‐VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Results: Co‐immunoprecipitation demonstrated that ADAMTS‐13 binds to native multimeric VWF (Kd of 79 ± 11 nmol L−1) with no measurable proteolysis. Upon shear‐induced unfolding of VWF, binding increased 3‐fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50–200). Moreover, results with ADAMTS‐13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS‐13 spacer, probably thrombospondin‐1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS‐13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). Conclusions: ADAMTS‐13 binds to circulating VWF and may therefore be incorporated into a platelet‐rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.

A novel endogenous inhibitor of the secreted streptococcal NAD-glycohydrolase

The Streptococcus pyogenes NAD-glycohydrolase (SPN) is a toxic enzyme that is introduced into infected host cells by the cytolysin-mediated translocation pathway. However, how S. pyogenes protects itself from the self-toxicity of SPN had been unknown. In this report, we describe immunity factor for SPN (IFS), a novel endogenous inhibitor that is essential for SPN expression. A small protein of 161 amino acids, IFS is localized in the bacterial cytoplasmic compartment. IFS forms a stable complex with SPN at a 1:1 molar ratio and inhibits SPNs NAD-glycohydrolase activity by acting as a competitive inhibitor of its β-NAD+ substrate. Mutational studies revealed that the gene for IFS is essential for viability in those S. pyogenes strains that express an NAD-glycohydrolase activity. However, numerous strains contain a truncated allele of ifs that is linked to an NAD-glycohydrolase−deficient variant allele of spn. Of practical concern, IFS allowed the normally toxic SPN to be produced in the heterologous host Escherichia coli to facilitate its purification. To our knowledge, IFS is the first molecularly characterized endogenous inhibitor of a bacterial β-NAD+−consuming toxin and may contribute protective functions in the streptococci to afford SPN-mediated pathogenesis.

Characterization of Streptococcus pyogenes β-NAD+ Glycohydrolase RE-EVALUATION OF ENZYMATIC PROPERTIES ASSOCIATED WITH PATHOGENESIS

The Gram-positive pathogen Streptococcus pyogenes injects a β-NAD+ glycohydrolase (SPN) into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. In this compartment, SPN accelerates the death of the host cell by an unknown mechanism that may involve its β-NAD+-dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of β-NAD+, but also carrying out ADP-ribosyl cyclase and ADP-ribosyltransferase activities, making SPN the only β-NAD+ glycohydrolase that can catalyze all of these reactions. With the long term goal of understanding how these activities may contribute to pathogenesis, we have further characterized the enzymatic activity of SPN using highly purified recombinant protein. Kinetic studies of the multiple activities of SPN revealed that SPN possessed only β-NAD+ hydrolytic activity and lacked detectable ADP-ribosyl cyclase and ADP-ribosyltransferase activities. Similarly, SPN was unable to catalyze cyclic ADPR hydrolysis, and could not catalyze methanolysis or transglycosidation. Kinetic analysis of product inhibition by recombinant SPN demonstrated an ordered uni-bi mechanism, with ADP-ribose being released as a second product. SPN was unaffected by product inhibition using nicotinamide, suggesting that this moiety contributes little to the binding energy of the substrate. Upon transformation, SPN was toxic to Saccharomyces cerevisiae, whereas a glycohydrolase-inactive SPN allowed for viability. Taken together, these data suggest that SPN functions exclusively as a strict β-NAD+ glycohydrolase during pathogenesis.The gram-positive pathogen Streptococcus pyogenes injects a beta-NAD(+) glycohydrolase (SPN) into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. In this compartment, SPN accelerates the death of the host cell by an unknown mechanism that may involve its beta-NAD(+)-dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of beta-NAD(+), but also carrying out ADP-ribosyl cyclase and ADP-ribosyltransferase activities, making SPN the only beta-NAD(+) glycohydrolase that can catalyze all of these reactions. With the long term goal of understanding how these activities may contribute to pathogenesis, we have further characterized the enzymatic activity of SPN using highly purified recombinant protein. Kinetic studies of the multiple activities of SPN revealed that SPN possessed only beta-NAD(+) hydrolytic activity and lacked detectable ADP-ribosyl cyclase and ADP-ribosyltransferase activities. Similarly, SPN was unable to catalyze cyclic ADPR hydrolysis, and could not catalyze methanolysis or transglycosidation. Kinetic analysis of product inhibition by recombinant SPN demonstrated an ordered uni-bi mechanism, with ADP-ribose being released as a second product. SPN was unaffected by product inhibition using nicotinamide, suggesting that this moiety contributes little to the binding energy of the substrate. Upon transformation, SPN was toxic to Saccharomyces cerevisiae, whereas a glycohydrolase-inactive SPN allowed for viability. Taken together, these data suggest that SPN functions exclusively as a strict beta-NAD(+) glycohydrolase during pathogenesis.

Effects of Activation Peptide Bond Cleavage and Fragment 2 Interactions on the Pathway of Exosite I Expression during Activation of Human Prethrombin 1 to Thrombin

Activation of prothrombin (Pro) by factor Xa to form thrombin occurs by proteolysis of Arg271-Thr272 and Arg320-Ile321, resulting in expression of regulatory exosites I and II. Cleavage of Pro by thrombin liberates fragment 1 and generates the zymogen analog, prethrombin 1 (Pre 1). The properties of exosite I on Pre 1 and its factor Xa activation intermediates were characterized in spectroscopic and equilibrium binding studies using the fluorescein-labeled probe, hirudin54–65 (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \([5\mathrm{F}]\mathrm{Hir}^{54-65}-(\mathrm{SO}_{3}^{-})\) \end{document}). Prethrombin 2 (Pre 2), formed by factor Xa cleavage of Pre 1 at Arg271-Thr272, had the same affinity for hirudin54–65 peptides as Pre 1 in the absence or presence of near-saturating fragment 2 (F2). Pre 2 and thrombin also had indistinguishable affinities for F2. By contrast, cleavage of Pre 1 at Arg320-Ile321, to form active meizothrombin des-fragment 1 MzT(-F1), showed a 11- to 20-fold increase in affinity for hirudin54–65, indistinguishable from the 13- to 20-fold increase seen for conversion of Pre 2 to thrombin. Thus, factor Xa cleavage of Pre 1 at Arg271-Thr272 does not effect exosite I expression, whereas cleavage at Arg320-Ile321 results in concomitant activation of the catalytic site and exosite I. Furthermore, expression of exosite I on the Pre 1 activation intermediates is not modulated by F2, and exosite II is not activated conformationally. The differential expression of exosite I affinity on the Pre 1 activation intermediates and the previously demonstrated role of (pro)exosite I in factor Va-dependent substrate recognition suggest that changes in exosite I expression may regulate the rate and direction of the Pre 1 activation pathway.

Rearranging Exosites in Noncatalytic Domains Can Redirect the Substrate Specificity of ADAMTS Proteases

Background: ADAMTS metalloproteases are multidomain proteins with remarkable substrate specificity. Results: Swapping noncatalytic domains between ADAMTS13 and ADAMTS5 causes reciprocal changes in the cleavage of their natural substrates. Conclusion: ADAMTS exosites in noncatalytic domains are portable modifiers of proteolytic activity. Significance: Shuffling and recombination of ADAMTS ancillary structural domains may be exploited to evolve or engineer new protease functions. ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu373–Ala374 and Glu1480–Gly1481 bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr1605–Met1606 bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu1615–Ile1616 bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.

Modality-specific interference with verbal and nonverbal stimulus information

Subjects recalled both letters and the locations of letters in 2 by 3 and 1 by 6 matrices after either no interfering activity or visual, auditory, or kinesthetic interfering activity. Results for each type of matrix indicated the presence of selective auditory (verbal) interference with the recall of letter identity and selective visual and kinesthetic interference with the recall of letter location. Supplementary correlational analyses indicated that the presence of such a dual encoding strategy was most consistent across subjects for the 2 by 3 matrix. Although the results indicated that use of different modes of representation was related to the verbal-nonverbal nature of the information, it was shown that the structure of the stimulus array also affected the nature of the representation.