Gillian E. Begg

Molecular Determinants for Targeting Heterochromatin Protein 1-Mediated Gene Silencing: Direct Chromoshadow Domain–KAP-1 Corepressor Interaction Is Essential

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 HP1α 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.

Mechanism of allosteric regulation of transglutaminase 2 by GTP

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 β-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.

Mutation of a Critical Arginine in the GTP-binding Site of Transglutaminase 2 Disinhibits Intracellular Cross-linking Activity *

Transglutaminase type 2 (TG2; also known as Gh) 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) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPγS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry 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 μ-calpain digestion, R579A is inherently more resistant to μ-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 cross-linking activity being suppressed allosterically by guanine nucleotide binding.

Conserved tryptophan in the core domain of transglutaminase is essential for catalytic activity

Transglutaminase 2 (TG2) is a distinctive member of the family of Ca2+-dependent enzymes recognized mostly by their abilities to catalyze the posttranslational crosslinking of proteins. TG2 uniquely binds and hydrolyzes GTP; binding GTP inhibits its crosslinking activity but allows it to function in signal transduction (hence the Gh designation). The core domain of TG2 (residues 139–471, rat) comprises the papain-like catalytic triad and the GTP-binding domain (residues 159–173) and contains almost all of the conserved tryptophans of the protein. Examining point mutations at Trp positions 180, 241, 278, 332, and 337 showed that, upon binding 2′-(or 3′)-O-(N-methylanthraniloyl)GTP (mantGTP), the Phe-332 mutant was the weakest (35% less than wild type) in resonance energy transfer from the protein (λexc, max = 290 nm) to the mant fluorophore (λem = 444 nm) and had a reduced affinity for mantGTP. Trp-332, situated near the catalytic center and the nucleotide-binding area of TG2, may be part of the allosteric relay machinery that transmits negative effector signals from nucleotide binding to the active center of TG2. A most important observation was that, whereas no enzyme activity could be detected when Trp-241 was replaced with Ala or Gln, partial preservation of catalytic activity was seen with substitutions by Tyr > Phe > His. The results indicate that Trp-241 is essential for catalysis, possibly by stabilizing the transition states by H-bonding, quadrupole–ion, or van der Waals interactions. This contrasts with the evolutionarily related papain family of cysteine proteases, which uses Gln-19 (papain) for stabilizing the transition state.

Wide Turn Diversity in Protein Transmembrane Helices Implications for G-Protein-Coupled Receptor and Other Polytopic Membrane Protein Structure and Function

Previously, we showed that perturbations of protein transmembrane helices are manifested as one of three types of noncanonical structures (wide turns, tight turns, and kinks), which, compared with α-helices, are evident by distinctive Cαi→Cαx distances. In this study, we report the analysis of more than 3000 transmembrane helices in 244 crystal structures from which we identified 70 wide turns (29 proline- and 41 nonproline-induced). Based on differences in the Cαi→Cαi-4 and Cαi→Cαi-5 profiles, we show that wide turns can be subclassified into three distinct subclasses (W1, W2, and W3) that differ with regard to the number and position of backbone i → i-5 H-bonds formed N-terminal to the perturbing or signature proline or nonproline residue. Although wide turns generally produce changes in helical direction of 20° to 30° and a lateral shift in the helical axis, some of the W3 subclass are associated with changes of <5°. We also show that the distinct architectural features of wide turns allow the carbonyl bond of the i-4th residue, which is located on the widened loop of a wide turn, to be directed away from the helical axis. This provides regions of flexibility within helical regions allowing, for example, unique opportunities for interhelical H-bonding, including interactions with glycine zipper motifs, and for ion and cofactor binding. Furthermore, differences in wide-turn subtype usage by related protein family members, such as the G-protein-coupled receptors rhodopsin and the β2-adrenergic receptor, can significantly affect the orientation and position of residues critical for ligand binding and receptor activation.

Cross-Linking Transglutaminases with G Protein–Coupled Receptor Signaling

Transglutaminases are a family of calcium- and thiol-dependent acyl transferases that catalyze the formation of an amide bond between the γ-carboxamide groups of peptide-bound glutamine residues and the primary amino groups in various compounds, including the ε-amino group of lysines in certain proteins. As a result, these enzymes effect posttranslational modification of proteins by amine incorporation, or stabilization of protein assemblies by their cross-linking; such actions profoundly influence critical biological processes such as blood clotting and protection from infection and dehydration by establishing the barrier function of skin. In addition, transglutaminases have other more diverse actions, including involvement in signaling by the superfamily of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) in one of three ways: (i) through actions as guanosine triphosphate–binding proteins that activate intracellular effectors, such as phospholipase C; (ii) by cross-linking GPCR monomers to enhance signaling as a result of covalent dimer formation; or (iii) by interacting with an apparent growth inhibitory orphan GPCR, GPR56, to limit metastatic spread of melanoma cells. The implications of these receptor-coupled actions of transglutaminases are discussed.

Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry

Recombinant proteins are subject to many forms of heterogeneity, including aggregation, proteolytic degradation, chemical modification, mutation, and incorrect translation. This unit describes methods for the detection and identification of these problems using analytical HPLC gel filtration and MALDI‐MS. Preliminary characterization of recombinants is necessary before the structure or function of the protein can be investigated.