The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance.     

Relationship between human immunodeficiency virus type 1 Gag multimerization and membrane binding

The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55(Gag), is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between membrane binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the membrane-binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55(Gag) was truncated at the C terminus of matrix (MAstop), between the N- and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using membrane flotation centrifugation, we observe that MA shows significantly reduced membrane binding relative to full-length Gag but that CA146 displays steady-state membrane-binding properties comparable to those of Pr55(Gag). The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state membrane binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient binding of HIV-1 Gag to membrane.     

Impaired protein folding, dimer formation, and heterotetramer assembly cause intra- and extracellular instability of a Y283C mutant of the A subunit for coagulation factor XIII.

Factor XIII (XIII) is a heterotetramer consisting of two catalytic A subunits (XIIIA) and two noncatalytic B subunits (XIIIB). We examined the molecular mechanisms of a Y283C mutation which had previously been identified in a patient with XIIIA deficiency. The recombinant Y283C protein was labile when expressed in MEG-01 cells, which can endogenously synthesize XIIIA. We also included two other mutants, G562R and I464stop, previously characterized in a non-XIIIA-producing cell line. All these mutants exhibited decreased thermostability and resistance against proteolytic digestion when compared with the wild-type. Gel-filtration analysis revealed that the mutants were in monomer form, while the wild-type formed a dimer. These results were consistent with the prediction by molecular modeling that the mutant molecules would be misfolded. Although assembly of a heterotetramer with XIIIB was demonstrated for Y283C, its binding ability was 10% that of the wild-type. No complex formation was observed for the G562R or I464stop mutants. The wild-type was stabilized in plasma by complex formation with XIIIB, resulting in an increased resistance against proteolytic digestion. In contrast, the mutants were unstable in plasma even in the presence of XIIIB. Thus, impaired folding, dimer formation, and heterotetramer assembly of the mutant XIIIAs lead to both intra- and extracellular instability, which must be responsible for XIIIA deficiency in the patient.     

Heterologous Production of Clostridium cellulovorans engB, Using Protease-Deficient Bacillus subtilis, and Preparation of Active Recombinant Cellulosomes

In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a "designer" cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli. The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.     

Structure and assembly mechanism for heteromeric kainate receptors

Native glutamate receptor ion channels are tetrameric assemblies containing two or more different subunits. NMDA receptors are obligate heteromers formed by coassembly of two or three divergent gene families. While some AMPA and kainate receptors can form functional homomeric ion channels, the KA1 and KA2 subunits are obligate heteromers which?function only in combination with GluR5-7. The mechanisms controlling glutamate receptor assembly involve an initial step in which the amino terminal domains (ATD) assemble as dimers. Here, we establish by sedimentation velocity that the ATDs of GluR6 and KA2 coassemble as a heterodimer of K(d) 11?nM, 32,000-fold lower than the K(d) for homodimer formation by KA2; we solve crystal structures for the GluR6/KA2 ATD heterodimer and heterotetramer assemblies. Using these structures as a guide, we perform a mutant cycle analysis to probe the energetics of assembly and show that high-affinity ATD interactions are required for biosynthesis of functional heteromeric receptors.     

GABA(A) receptor assembly. Identification and structure of gamma(2) sequences forming the intersubunit contacts with alpha(1) and beta(3) subunits

GABA(A) receptors are ligand-gated chloride channels composed of five homologous subunits that specifically recognize one another and assemble around an aqueous pore. To identify domains responsible for the specificity of subunit association, we constructed C-terminal truncated gamma(2) subunits, as well as mutated and chimeric fragments. From their ability to interfere with alpha(1)beta(3)gamma(2) receptor assembly and to associate with full-length subunits, we concluded that amino acid sequences gamma(2)-(91-104) and gamma(2)-(83-90) form the sites mediating assembly with alpha(1) and beta(3) subunits, respectively. Neural network-based secondary structure prediction, Monte Carlo optimization, and hydrophobicity analysis led to the conclusion that these sites also form the intersubunit contacts in the completely assembled receptor and provided important information on the benzodiazepine-binding site and structure of GABA(A) receptors.     

Stoichiometry of complexes between mannose-binding protein and its associated serine proteases. Defining functional units for complement activation

Serum mannose-binding protein (MBP) initiates the lectin branch of the complement cascade by binding to sugars on the surfaces of microorganisms and activating two MBP-associated serine proteases (MASP-1 and MASP-2). Rat serum MBP consists of oligomers containing up to four copies of a subunit that is composed of three identical polypeptide chains. Biophysical analysis of intact and truncated MASPs indicates that each MASP is a homodimer that is stabilized through interactions involving an N-terminal CUB domain. The binding sites for MBP are formed from the three N-terminal MASP domains, in which two CUB modules interact with MBP. Each MASP dimer contains binding sites for two MBP subunits. Both sites must be occupied by subunits from a single MBP oligomer to form a stable complex. Thus, the smallest functional unit for complement activation consists of MBP dimers bound to MASP-1 or MASP-2 homodimers. Trimers and tetramers of MBP form complexes containing up to two MASPs. The results reveal how MASP-1 and MASP-2 can function independently to activate the complement cascade.     

Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Role of the alpha1 and alpha2 integrin cytoplasmic domains in cell morphology, motility and responsiveness to stimulation by the protein kinase C pathway

The alpha1beta1 and alpha2beta1 integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the alpha subunits. We expressed either full-length alpha1 integrin subunit cDNA (X1C1), full-length alpha2 integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of alpha2 subunit and the cytoplasmic domain of alpha1 (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of alpha1 subunit and the cytoplasmic domain of alpha2 (X1C2), alpha1 cDNA truncated after the GFFKR sequence (X1C0) or alpha2 cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the alpha1 and alpha2 subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the alpha1 or alpha2 cytoplasmic tail. Spreading was also influenced by the presence of an alpha subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the alpha2 cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, alpha2-cytoplasmic tail-dependent migration.     

In vivo analysis of conserved C. elegans tomosyn domains

Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo.     

The N-Terminal Domains of Vps3 and Vps8 Are Critical for Localization and Function of the CORVET Tethering Complex on Endosomes

Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.     

Contribution of each membrane binding domain of the CTP:phosphocholine cytidylyltransferase-alpha dimer to its activation, membrane binding, and membrane cross-bridging

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.     

Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (αβ)₂ family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 Å containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 Å. The functional enzyme resembles previously known α₂ and α₄ endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.     

Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86.

DNA ends are generated during double-strand-break repair and recombination. A p70-p86 heterodimer, Ku, accounts for the DNA end binding activity in eukaryotic cell extracts. When one or both subunits of Ku are missing, mammalian cells are deficient in double-strand-break repair and in specialized recombination, such as V(D)J recombination. Little is known of which regions of Ku70 and Ku86 bind to each other to form the heterodimeric complex or of which regions are important for DNA end binding. We have done genetic and biochemical studies to examine the domains within the two subunits important for protein assembly and for DNA end binding. We found that the C-terminal 20-kDa region of Ku70 and the C-terminal 32-kDa region of Ku86 are important for subunit-subunit interaction. For DNA binding, full-length individual subunits are inactive, indicating that heterodimer assembly precedes DNA binding. DNA end binding activity by the heterodimer requires the C-terminal 40-kDa region of Ku70 and the C-terminal 45-kDa region of Ku86. Leucine zipper-like motifs in both subunits that have been suggested as the Ku70-Ku86 interaction domains do not appear to be the sites of such interaction because these are dispensable for both assembly and DNA end binding. On the basis of these studies, we have organized Ku70 into nine sequence regions conserved between Saccharomyces cerevisiae, Drosophila melanogaster, mice, and humans; only the C-terminal three regions are essential for assembly (amino acids [aa] 439 to 609), and the C-terminal four regions appear to be essential for DNA end binding (aa 254 to 609). Within the minimal active fragment of Ku86 necessary for subunit interaction (aa 449 to 732) and DNA binding (aa 334 to 732), a proline-rich region is the only defined motif.     

C-terminal fragments of the alpha 1C (CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha 1C subunits

L-type Ca(2+) channels in native tissues have been found to contain a pore-forming alpha(1) subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminal-truncated alpha(1C) (Ca(V)1.2) subunits, we performed electrophysiological and biochemical experiments. In tsA201 cells expressing either wild type or C-terminal-truncated alpha(1C) subunits in combination with a beta(2a) subunit, truncation of the alpha(1C) subunit by as little as 147 amino acids led to a 10-15-fold increase in currents compared with those obtained from control, full-length alpha(1C) subunits. Dialysis of cells expressing the truncated alpha(1C) subunits with C-terminal fragments applied through the patch pipette reconstituted the inhibition of the channels seen with full-length alpha(1C) subunits. In addition, C-terminal deletion mutants containing a tethered C terminus also exhibited the C-terminal-induced inhibition. Immunoprecipitation assays demonstrated the association of the C-terminal fragments with truncated alpha(1C) subunits. In addition, glutathione S-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the C- terminal fragments of the alpha(1C) subunit can associate with C-terminal-truncated alpha(1C) subunits and inhibit the currents through L-type Ca(2+) channels.     

Common FXIII and Fibrinogen Polymorphisms in Abdominal Aortic Aneurysms

Introduction

Abdominal aortic aneurysms (AAA) are characterized by a progressive dilatation of the abdominal aorta, and are associated with a high risk of rupture once the dilatation exceeds 55 mm in diameter. A large proportion of AAA develops an intraluminal thrombus, which contributes to hypoxia, inflammation and tissue degradation. We have previously shown that patients with AAA produce clots with altered structure which is more resistant to fibrinolysis. The aim of this study was to investigate genetic polymorphisms of FXIII and fibrinogen in AAA to identify how changes to these proteins may play a role in the development of AAA.

Methods

Subjects of Western/European descent, ≥55 years of age (520 AAA patients and 449 controls) were genotyped for five polymorphisms (FXIII-A Val34Leu, FXIII-B His95Arg, FXIII-B Splice Variant (intron K nt29576C-G), Fib-A Thr312Ala and Fib-B Arg448Lys) by RT-PCR. Data were analysed by χ2 test and CubeX.

Results

The FXIII-B Arg95 allele associated with AAA (Relative risk - 1.240, CI 1.093–1.407, P = 0.006). There was no association between FXIII-A Val34Leu, FXIII-B Splice Variant, Fib-A Thr312Ala or Fib-B Arg448Lys and AAA. FXIII-B His95Arg and FXIII-B Splice variant (intron K nt29576C-G) were in negative linkage disequilibrium (D’ = −0.609, p = 0.011).

Discussion

The FXIII-B Arg95 variant is associated with an increased risk of AAA. These data suggest a possible role for FXIII in AAA pathogenesis.