Materials and MethodsAs in the study of the previously determined MscL structure (S1), multiple homologs of MscS [encoded by the yggb gene (S2)] were identified by BLAST searching of the NCBI genome database. Ten homologs from prokaryotes and Archaea were identified and subsequently cloned. The channels were subcloned into expression vectors (pET system, Novagen) and expression screening was carried out. Cells expressing sufficient channels to be identified by Western blotting were subjected to extensive detergent screening utilizing ~50 detergents (Anatrace, Sigma, Aldrich) where both the ability of the channels to be extracted out of the membrane and the ability to remain as a homo-oligomer were determined. Subsequent large-scale expressions, extraction and purification produced sufficient amounts of protein for three channels (E. coli, B. subtilis and C. tepidum) for crystallization trials. Each of these channels was produced recombinantly (vector pet28b, Novagen) in 50-liter fermenter growths in a modified Terrific Broth media containing 1% glucose and 0.4% glycerol. Protein expression was initiated by the addition of 2% lactose and 2 mM IPTG for 2-4 hours, resulting in ~1.5 kg of wet cells. To obtain phase information, selenomethionine-derivatized protein was purified from cells grown in a modified M9 media containing 50 mg/l selenomethionine, and the remaining amino acids at 40 µg/l. Extraction of the E. coli MscS was carried out using sonication and solubilization with 1% Foscholine-14. Ni-affinity chromatography, anion exchange, and size exclusion chromatography in the presence of 0.05% Foscholine-14 were used to purify the protein to homogeneity. The apparent molecular mass of the protein, as indicated by size-exclusion chromatography, was in excess of 200 kD, similar to that reported for recombinant MscS by Sukharev (S3). Crystals were obtained with 10-15 mg/ml MscS by hanging drop vapor diffusion with 100 mM pH 7.2 Hepes buffer, 150 mM Na-formate, 8% glycerol, and 16% PEG-3350 as the precipitant. Crystals grew to ~200 µm in each dimension, and were assigned to space group P4 3 2 1 2 (a = b = 184.7 Å, c = 260.7 Å) with one MscS oligomer in the asymmetric unit (corresponding to ~71% solvent content). Only residues in the extramembrane (water-soluble) regions of MscS participated in lattice contacts.

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.

Antibody drug conjugates (ADCs) are a therapeutic class offering promise for cancer therapy. The attachment of cytotoxic drugs to antibodies can result in an effective therapy with better safety potential than nontargeted cytotoxics. To understand the role of conjugation site, we developed an enzymatic method for site-specific antibody drug conjugation using microbial transglutaminase. This allowed us to attach diverse compounds at multiple positions and investigate how the site influences stability, toxicity, and efficacy. We show that the conjugation site has significant impact on ADC stability and pharmacokinetics in a species-dependent manner. These differences can be directly attributed to the position of the linkage rather than the chemical instability, as was observed with a maleimide linkage. With this method, it is possible to produce homogeneous ADCs and tune their properties to maximize the therapeutic window.

No abstract