Cross-strand pair correlations are calculated for residue pairs in anti-parallel beta-sheet for two cases: pairs whose backbone atoms are hydrogen bonded together (H-bonded site) and pairs which are not (non-H-bonded site). The statistics show that this distinction is important. When glycine is located on the edge of a sheet, it shows a 3:1 preference for the H-bonded site. The strongest observed correlations are for pairs of disulfide-bonded cystines, many of which adopt a close-packed conformation with each cystine in a spiral conformation of opposite chirality to its partner. It is likely that these pairs are a signature for the family of small, cystine-rich proteins. Most other strong positive and negative correlations involve charged and polar residues. It appears that electrostatic compatibility is the strongest factor affecting pair correlation. Significant correlations are observed for beta- and gamma-branched residues in the non-H-bonded site. An examination of the structures shows a directionality in side chain packing. There is a correlation between (1) the directionality in the packing interactions of non-H-bonded beta- and gamma-branched residue pairs, (2) the handedness of the observed enantiomers of chiral beta-branched side chains, and (3) the handedness of the twist of beta-sheet. These findings have implications for the formation of beta-sheets during protein folding and the mechanism by which the sheet becomes twisted.

The molecular mechanisms underlying thiol-based redox control are poorly defined. Disulfide bonds between Cys residues are commonly thought to confer extra rigidity and stability to their resident protein, forming a type of proteinaceous spot weld. Redox biologists have been redefining the role of disulfides over the last 30-40 years. Disulfides are now known to form in the cytosol under conditions of oxidative stress. Isomerization of extracellular disulfides is also emerging as an important regulator of protein function. The current paradigm is that the disulfide proteome consists of two subproteomes: a structural group and a redox-sensitive group. The redox-sensitive group is less stable and often associated with regions of stress in protein structures. Some characterized redox-active disulfides are the helical CXXC motif, often associated with thioredoxin-fold proteins; and forbidden disulfides, a group of metastable disulfides that disobey elucidated rules of protein stereochemistry. Here we discuss the role of redox-active disulfides as switches in proteins.

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

Cysteine is susceptible to a variety of modifications by reactive oxygen and nitrogen oxide species, including glutathionylation; and when two cysteines are involved, disulfide formation. Glutathione-cysteine adducts may be removed from proteins by glutaredoxin, whereas disulfides may be reduced by thioredoxin. Glutaredoxin is homologous to the disulfide-reducing thioredoxin and shares similar binding modes of the protein substrate. The evolution of these systems is not well characterized. When a single Cys is present in a protein, conjugation of the redox buffer glutathione may induce conformational changes, resulting in a simple redox switch that effects a signaling cascade. If a second cysteine is introduced into the sequence, the potential for disulfide formation exists. In favorable protein contexts, a bistable redox switch may be formed. Because of glutaredoxin's similarities to thioredoxin, the mutated protein may be immediately exapted into the thioredoxin-dependent redox cycle upon addition of the second cysteine. Here we searched for examples of protein substrates where the number of redox-active cysteine residues has changed throughout evolution. We focused on cross-strand disulfides (CSDs), the most common type of forbidden disulfide. We searched for proteins where the CSD is present, absent and also found as a single cysteine in protein orthologs. Three different proteins were selected for detailed study—CD4, ERO1, and AKT. We created phylogenetic trees, examining when the CSD residues were mutated during protein evolution. We posit that the primordial cysteine is likely to be the cysteine of the CSD which undergoes nucleophilic attack by thioredoxin. Thus, a redox-active disulfide may be introduced into a protein structure by stepwise mutation of two residues in the native sequence to Cys. By extension, evolutionary acquisition of structural disulfides in proteins can potentially occur via transition through a redox-active disulfide state.

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 ...