Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-Å resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

Supplementary Table 1| Steady-state parameters for cleavage of 5-nitrobenzisoxazole by representative Kemp eliminases a Catalyst k cat (s-1) K M (mM) k cat /K M (M-1 s-1) (k cat /K M)/k AcO-b k cat /k uncat c Ref 34E4 d 0.66 0.12 5.5 x 10 3 9.5 x 10 7 5.7 x 10 5 11

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys 230 Human transglutaminase 2 (TG2) 2 modifies protein-or peptide-bound glutamine residues by either cross-linking their reactive carboxamide side chains to primary amines or by deamidation, converting glutamine residues to glutamate (1, 2). Ca 2ϩ is required for this catalytic activity and induces conformational changes in the enzyme, which arrange the active-site residues for catalysis, including Cys 277 (3-6). As TG2 is abundantly expressed in both the intracellular and extracellular environments of many tissues, its catalytic activity must be tightly regulated to avoid excess modification of cellular and tissue components. GTP and GDP act as allosteric inhibitors by inducing a closed conformation in which the active site is buried (6 -8). Low Ca 2ϩ concentration and high GTP/GDP concentration in the cytosol typically prevent TG2 activation within cells. Despite conditions in the extracellular milieu that favor activation, extracellular TG2 also appears to be predominantly inactive under normal conditions but can be activated by certain types of inflammation and cell injury (9).The catalytic activity of TG2 is implicated in the pathogenesis of several human diseases, including celiac disease (10). Celiac disease is caused by an aberrant immune response to proline-and glutamine-rich peptides from dietary gluten in the small intestine of genetically predisposed individuals (11). In the celiac immune response, the enzymatic activity of TG2 is crucial, as TG2-mediated deamidation of gluten peptides increases their T-cell antigenicity (12, 13).In contrast to our knowledge of the mechanistic basis for TG2 inactivity in the intracellular environment, the mechanisms underlying regulation of TG2 activity in the extracellular compartment remain unclear. TG2 harbors no disulfide bonds in its native state, which is unusual for enzymes in the extracellular environment (14). Previous studies have shown that TG2 is susceptible to oxidation, resulting in inactivation (19 -22). Thus, modulation of enzymatic activity through oxidation presents a plausible mechanism for regulation of extracellular TG2 activity. We have investigated the events underlying oxidative inactivation of TG2 and report the identification of a redox-sensitive cysteine triad consisting of Cys 230 , Cys 370 , and Cys 371 . Within this triad, Cys 230 appears to set the threshold for intramolecular disulfide bond formation and thereby inactivation. Oxidation was influenced by the presence of Ca 2ϩ and substrate, suggesting that the local environment can modulate and fine-tune oxidative inactivation of T...

Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB–BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.