No abstract

The KRAB domain is a highly conserved transcription repression module commonly found in eukaryotic zinc finger proteins. KRAB-mediated repression requires binding to the KAP-1 corepressor, which in turn recruits members of the heterochromatin protein 1 (HP1) family. The HP1 proteins are nonhistone chromosomal proteins, although it is unclear how they are targeted to unique chromosomal domains or promoters. In this report, we have reconstituted and characterized the HP1-KAP-1 interaction using purified proteins and have compared KAP-1 to three other known HP1 binding proteins: SP100, lamin B receptor (LBR), and the p150 subunit from chromatin assembly factor (CAF-1 p150). We show that the chromoshadow domain (CSD) of HP1 is a potent repression domain that binds directly to all four previously described proteins. For KAP-1, we have mapped the CSD interaction region to a 15-amino-acid segment, termed the HP1BD, which is also present in CAF-1 p150 but not SP100 or LBR. The region of KAP-1 harboring the HP1BD binds as a monomer to a dimer of the CSD, as revealed by gel filtration, analytical ultracentrifugation, and optical biosensor analyses. The use of a spectrum of amino acid substitutions in the human HP1␣ CSD revealed a strong correlation between CSD-mediated repression and binding to KAP-1, CAF-1 p150, and SP100 but not LBR. Differences among the HP1 binding partners could also be discerned by fusion to a heterologous DNA binding domain and by the potential to act as dominant negative molecules. Together, these results strongly suggest that KAP-1 is a physiologically relevant target for HP1 function.

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the ␤-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.protein conformation ͉ GTP inhibition ͉ transamidase activity A llosteric regulation of enzymes by conformational change is an important means of biological control, involving residues that functionally couple ligand binding at the allosteric site to modification of the catalytic site. Transglutaminase type 2 (TG2), also known as tissue TG or G h (high molecular weight GTP-binding protein), is a multifunctional protein that is allosterically regulated by calcium and GTP (1). TG2 catalyzes calcium-dependent transamidation reactions, resulting in posttranslational amine modification of proteins or cross-linking of interchain glutamine and lysine residues to form N (␥-glutamyl)lysine isopeptide bonds, which confer rigidity and protease resistance on protein complexes (2). TG2 is also a GTPase (3) and mediates intracellular signaling by various G protein-coupled receptors (4-6).GDP-bound human TG2 (7) is comprised of four domains: an N-terminal ␤-sandwich, a core domain in which the transamidase active site catalytic triad (Cys-277, His-335, and Asp-358) and transition-state stabilizing residue (Trp-241) (8) are buried and inaccessible to substrate, and two ␤-barrels. Nucleotide binds mainly to residues from the first and last strands (amino acids 476-482 and 580-583) of ␤-barrel 1 and to two core domain residues (Lys-173 and Phe-174) that protrude on a loop to meet ␤-barrel 1 (7, 9, 10). This is postulated to stabilize two ␤-barrel 1 loops that block access to the catalytic site (7). One of these loops protrudes into the core domain localizing Tyr-516 within hydrogen-bonding distance of Cys-277 (7). This is postulated to prevent Cys-277 interaction with the substrate (7,11,12). Calcium-activated TG2 has unique conformational epitopes (13) and is less compact (14-16) and less resistant to protease digestion (1, 10, 14) than GTP-bound TG2. Allosteric mechanisms governing the conformational switch between transamidase and GTPase functions have yet to be elucidated.Mutation of Arg-579 in rat TG2 (Arg-580 in ...

The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B boxcoiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB⅐KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K d of 142 nM. The fitted R max indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDSpolyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains.

Transglutaminase type 2 (TG2; also known as G h ) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and -calpain digestion, R579A is inherently more resistant to -calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated crosslinking activity being suppressed allosterically by guanine nucleotide binding.Transglutaminase type 2 (TG2), 2 also known as tissue transglutaminase or G h (high molecular weight GTP-binding protein), is a multifunctional protein that is involved in diverse physiological processes, including apoptosis, bone ossification, wound repair, cell adhesion, and signal transduction and in the pathophysiology of various diseases, including gluten-induced enteropathy (celiac disease), neurodegenerative disorders, tumor growth, and diabetes (1). It is constitutively expressed by fibroblasts and endothelial and smooth muscle cells as well as a number of organ-specific cell types (2). At the subcellular level, it is found in the cytosol in association with plasma and nuclear membranes and is also found attached to the extracellular matrix (1).TG2 has at least five distinct biological activities. It is a member of the transglutaminase family of cross-linking enzymes that catalyze posttranslational covalent linkages, the best studied of which is transamidation. Transamidation results in either a protein cross-link between a glutamine and lysine residue to form an N⑀-(␥-glutamyl) isopeptide bond, incorporation of an amine into a glutamine residue, or acylation of a lysine residue. TG2 also binds and hydrolyzes GTP (3, 4),thereby mediating signaling by various G-protein-coupled receptors, including ␣ 1B and ␣ 1D -adrenergic (5-7), thromboxane A2 (8, 9), and oxytocin (10) receptors. Additionally, TG2 can act as an adaptor protein that facilitates extracellular interaction between fibronectin and ␤1/3-integrins (11), as a protein disulfide isomerase (12), and as a kinase for insulin growth factor-binding prote...

T ransglutaminase 2 [here designated TG2 (EC2.3.2.13), but also identified as the GTP-binding protein, G h ] differs from its relatives in the family of Ca 2ϩ -dependent protein crosslinking enzymes mainly by its affinity for nucleotides (1-3) and for fibronectin (4-8). GTP-binding and hydrolysis enable TG2 to act as a G protein in signal transduction (G h ; ref. 9); GTP also serves as a potent allosteric inhibitor that suppresses the Ca 2ϩ -activated crosslinking activities of the enzyme. Independently of any of its catalytic functions and GTP-binding activity, TG2 binds fibronectin with very high affinity (7). Thus, when expressed on cell surfaces, TG2 plays an important role in organizing the extracellular matrix as an integrin-binding adhesion coreceptor (10, 11).Crystal structures are available for two related proteins: the A subunit of the coagulation factor XIII zymogen (fXIIIA; ref. 12) and the sea bream liver TG (13). Although neither of these is regulated by GTP (personal information from Kohki Ishikawa regarding the latter enzyme), sequence homologies between TG2 (rat) and these proteins suggest that TG2͞G h also would be organized into four domains: a ␤ sandwich (residues 1-138) followed by a large ␣͞␤ catalytic core (139-471) and two ␤ barrels [barrel 1 (472-584) and barrel 2 (585-686)]. The N-terminal (Ϸ28 kDa) region of TG2 is responsible for binding fibronectin (8) whereas the C-terminal segment of barrel 2 could interact with phospholipase in signal transduction (14). The core domain comprises the papain-like catalytic center (15) and the nucleotidebinding residues (16). Like the monomeric but unlike the heterotrimeric G proteins, TG2 was shown to bind 2Ј-(or 3Ј)-O-(Nmethylanthraniloyl)GTP (mantGTP) with high affinity (17). Binding was evidenced also by the appearance of an energy transfer band from one or more Trp residues of the protein to the mant fluorophore in this nucleotide. Because the core domain contains almost all of the tryptophans conserved throughout the TG family, we evaluated the nucleotide-binding properties and the transamidating activities of candidate tryptophan point mutants of TG2. Specifically, W180, 241, 278, 332, and 337 were targeted because they surround S171, a residue identified to be critical for GTPbinding. On a model of TG2 based on the fXIIIA zymogen crystal structure (16), all of the mutated residues are within 20Å (range 9.3-18.7Å) of both S171 and the active-site cysteine C277. We show that no single Trp residue alone accounts for the observed energy transfer from the protein to the nucleotide but some of the tryptophan mutants displayed greatly diminished mantGTPbinding affinities. Another important finding was that residue W241-that is highly conserved among the TGs but is lacking in the catalytically inactive family member of human red cell, band 4.2-is critical for transamidation by TG2. Materials and MethodsConstructs. Site-directed mutants of rat TG2͞G h cDNA were generated in a temperature-cycling reaction with Pfu DNA polymerase by using two complement...

The spectrin heterodimer is formed by the antiparallel lateral association of an ␣ and a ␤ subunit, each of which comprises largely a series of homologous triplehelical motifs. Initiation of dimer assembly involves strong binding between complementary motifs near the actin-binding end of the dimer. In this study, the mechanism of lateral spectrin association at this dimer nucleation site was investigated using the analytical ultracentrifuge to analyze heterodimers formed from recombinant peptides containing two or four homologous motifs from each subunit (␣20 -21/␤1-2; ␣18 -21/␤1-4). Both the two-motif and four-motif dimer associations were weakened substantially with increasing salt concentration, indicating that electrostatic interactions are important for the dimer initiation process. Modeling of the electrostatic potential on the surface of the ␣20 and ␤2 motifs showed that the side of the motifs comprising the A and B helices is the most favorable for association, with an area of positive electrostatic potential on the AB face of the ␤2 motif opposite negative potential on the AB face of the ␣20 motif and vise versa. Protease protection analysis of the ␣20 -21/␤1-2 dimer showed that multiple trypsin and proteinase K sites in the A helices of the ␤2 and ␣21 motifs become buried upon dimer formation. Together, these data support a model where complementary long range electrostatic interactions on the AB faces of the triple-helical motifs in the dimer nucleation site initiate the correct pairing of motifs, i.e. ␣21-␤1 and ␣20-␤2. After initial docking of these complementary triple-helical motifs, this association is probably stabilized by subsequent formation of stronger hydrophobic interactions in a complex involving the A helices of both subunits and possibly most of the AB faces. The ␤ subunit A helix in particular appears to be buried in the dimer interface.Members of the spectrin family of membrane skeleton proteins are widely expressed in vertebrates as well as in lower organisms. In erythrocytes, spectrin is the major component of the membrane skeleton, a network of spectrin oligomers crosslinked with short actin filaments that is bound to the membrane and provides cell membrane stability. The most common form of spectrin on intact cell membranes is the tetramer, which is formed by the "head-to-head" association of two elongated heterodimers, each comprising an ␣ subunit and a ␤ subunit with molecular masses of 280 kDa (1) and 246 kDa (2), respectively. Both the ␣ and ␤ subunits consist largely of a series of homologous motifs, each approximately 106 residues in length (3). The tertiary structure of these motifs is a triplehelical bundle, as determined by x-ray crystallography (4) and NMR spectroscopy (5). Head-to-head tetramers form by binding of complementary partial motifs at the ends of the ␣ and ␤ subunits to form a complete triple-helical bundle (6 -8).The antiparallel lateral association of the ␣ and ␤ subunits to form a heterodimer is initiated near the actin-binding end of the molecule at a dim...

The GA733-2 antigen (GA733) is a homotypic calciumindependent cell adhesion molecule (CAM) present in most normal human epithelial cells and gastrointestinal carcinomas. Because oligomerization of some CAMs regulates cell adhesion and signal transduction, the correlation between GA733 oligomeric state and cell-cell adhesion was investigated. Sedimentation equilibrium studies showed that full-length (-FL) GA733 exists as dimers and tetramers in solution, whereas the GA733 extracellular domain (-EC) is a monomer. The K d of GA733-FL is less than 10 nM for the monomer-dimer association, whereas the dimer-tetramer association is about 1000-fold weaker (K d ϳ10 M). Chemical crosslinking of purified GA733-FL in solution resulted in a major product corresponding to GA733 dimers, and minor amounts of trimers and tetramers. However, GA733-EC cross-linked under the same conditions was consistently a monomer. Chemical cross-linking of dissociated colon carcinoma cells produced predominantly GA733 dimers, whereas cross-linking of cells in monolayers yielded some tetramers as well. GA733-FL retained its cell-cell adhesion function as shown by inhibition of cell aggregation, whereas monomeric GA733-EC was inactive. These data show that GA733 exists predominantly as high affinity noncovalent cisdimers in solution and on dissociated colon carcinoma cells. The lower affinity association of dimers to form tetramers is most likely the head-to-head interaction between GA733 cis-dimers on opposing cells that represents its cell-cell adhesion activity.