No abstract

The Glutaminase Interacting Protein (GIP) is composed of a single PDZ domain that interacts with a growing list of partner proteins, including Glutaminase L, that are involved in a number of cell signaling and cancer pathways. Therefore, GIP makes a good target for structure-based drug design. Here we report the solution structures of both free GIP and GIP bound to the C-terminal peptide analog of Glutaminase L. This is the first reported NMR structure of GIP in a complex with one of its binding partners. Our analysis of both free GIP and GIP complexed with the Glutaminase L peptide provides important insights into how a promiscuous binding domain can have affinity for multiple binding partners. Through a detailed chemical shift perturbation analysis and backbone dynamics studies, we demonstrate here that the binding of the Glutaminase L peptide to GIP is an allosteric event. Taken together, the insights reported here lay the groundwork for the future development of a specific inhibitor for GIP.

Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide. The model peptide is known to form a stable, parallel homodimeric coiled coil, which is partially destabilized by Cys substitutions at heptad a and d positions (peptides C30a and C33d). The ProP peptide, a 30-residue synthetic peptide, corresponds to residues 468-497 of osmoregulatory transporter ProP from Escherichia coli. It forms a relatively unstable, homodimeric coiled coil that is predicted to be antiparallel in orientation. Cys was introduced in heptad g positions of the ProP peptide, near the N-terminus (K473C, creating peptide C473g) or closer to the center of the sequence (E480C, creating peptide C480g). In contrast to the destabilizing effect of Cys substitution at the core heptad a or d positions of model peptides C30a and C33d, circular dichroism spectroscopy showed that Cys substitutions at the heptad g positions of the ProP peptide had little or no effect on coiled-coil stability. Thermal denaturation analysis showed that spin labeling increased the stability of the coiled coil for all peptides. Strong exchange broadening was detected for both C30a and C33d, in agreement with a parallel structure. EPR spectra of C480g had a large hyperfine splitting of about 90 G, indicative of strong dipole-dipole interactions and a distance between spin-labeled residues of less than 9 A. Spin-spin interactions were much weaker for C473g. These results supported the hypothesis that the ProP peptide primarily formed an antiparallel coiled coil, since formation of a parallel dimer should result in similar spin-spin interactions for the spin-labeled Cys at both sites.

Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell–cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal.

Our paper reports on the reactivities and orientations of two common phenols, phenol (2) and m-cresol (3), and some of their chlorinated intermediates with aqueous monochloramine, NH2Cl, and dichloramine, NHCl2. We also examined the further reactivity of 2,4,6-trichlorophenol (4) with the chloramines. The phenols are an important area of investigation because they are substituents in the humic acids and are common contaminants in water. m-Cresol (3) was found to be more reactive than phenol (2)with both chlorinating agents. Both NH2Cl and NHCl2were sufficiently reactive to chlorinate all positions ortho and para to the hydroxyl groups. Mono- and dichloramine showed the same orientation with 2 but different orientations in their reactions with the substituent phenols. Indophenol (as its salt) was formed to a minor extent at high pH but not at pH 9. Both NH2Cl and NHCl2 rapidly replaced the parachlorine in 2,4,6-trichlorophenol (4) to give a mixture of 2,6-dichloro-1,4-benzoquinone-4-(N-chloro) imine (5) and 2,6-dichloro-1,4-benzoquinone (18). Similar reactions occurwith 2,4,6-trichloro-m-cresol (17) and 2,4,6-trichloro-3-methoxyphenol (29). The products for 17 were confirmed by mass spectrometry (El and Cl), 1H NMR, 13C NMR, and IR; the products for 29 were confirmed by mass spectrometry (El and Cl) and IR. An ion radical mechanism is suggested to account for the chlorine replacement by the chloramines. [No side chain oxidation of the methyl group in 17 in H20 or ether occurred, with or without ultraviolet radiation.] Both 5 and 18 underwent further chlorination with NH2Cl or NHCl2. Imine 5 did not function as a chlorinated agent.