Eukaryotic N-glycosylation occurs via the membrane-anchored C-terminal domain of the Stt3p subunit of oligosaccharyltransferase.

N-glycosylation is an essential and highly conserved protein modification. In eukaryotes, it is catalyzed by a multisubunit membrane-associated enzyme, oligosaccharyltransferase (OT). We report the high resolution structure of the C-terminal domain of eukaryotic Stt3p. Unlike its soluble ß-sheet-rich prokaryotic counterparts, our model reveals that the C-terminal domain of yeast Stt3p is highly helical and has an overall oblate spheroid-shaped structure containing a membrane-embedded region. Anchoring of this protein segment to the endoplasmic reticulum membrane is likely to bring the membrane-embedded donor substrate closer, thus facilitating glycosylation efficiency. Structural comparison of the region near the WWDYG signature motif revealed that the acceptor substrate-binding site of yeast OT strikingly resembles its prokaryotic counterparts, suggesting a conserved mechanism of N-glycosylation from prokaryotes to eukaryotes. Furthermore, comparison of the NMR and cryo-EM structures of yeast OT revealed that the molecular architecture of this acceptor substrate-recognizing domain has interesting spatial specificity for interactions with other essential OT subunits.

Promiscuous binding at the crossroads of numerous cancer pathways: insight from the binding of glutaminase interacting protein with glutaminase L.

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 analogue of glutaminase L. This is the first reported nuclear magnetic resonance structure of GIP in a complex with one of its binding partners. Our analysis of both free GIP and GIP in a complex 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.

NMR and bioinformatics discovery of exosites that tune metalloelastase specificity for solubilized elastin and collagen triple helices.

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that α-elastin species cover the primed subsites, a strip across the ß-sheet from ß-strand IV to the II-III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the "primed" side in the III-IV, V-B, and S1' specificity loops. Two map to the "unprimed" side in the IV-V and B-C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.

Apparent tradeoff of higher activity in MMP-12 for enhanced stability and flexibility in MMP-3.

The greater activity of MMP-12 than MMP-3 toward substrates from protein fibrils has been quantified. Why is MMP-12 the more active protease? We looked for behaviors associated with the higher activity of MMP-12 than MMP-3, using nuclear magnetic resonance to monitor backbone dynamics and residue-specific stabilities of their catalytic domain. The proteolytic activities are likely to play important roles in inflammatory diseases of arteries, lungs, joints, and intestines. Nuclear magnetic resonance line broadening indicates that regions surrounding the active sites of both proteases sample conformational substates within milliseconds. The more extensive line broadening in MMP-3 suggests greater sampling of conformational substates, affecting the full length of helix B and beta-strand IV forming the active site, and more remote sites. This could suggest more excursions to functionally incompetent substates. MMP-3 also has enhanced subnanosecond fluctuations in helix A, in the beta-hairpin of strands IV and V, and before and including helix C. Hydrogen exchange protection in the EX2 regime suggests that MMP-3 possesses 2.8 kcal/mol higher folding stability than MMP-12(E219A). The beta-sheet of MMP-3 appears to be stabilized still more. The higher stability of MMP-3 relative to MMP-12 coincides with the former's considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.

A structural biology approach to understand human lymphatic filarial infection.

The presence of aspartic protease inhibitor in filarial parasite Brugia malayi (Bm-Aspin) makes it interesting to study because of the fact that the filarial parasite never encounters the host digestive system. Here, the aspartic protease inhibition kinetics of Bm-Aspin and its NMR structural characteristics have been investigated. The overall aim of this study is to explain the inhibition and binding properties of Bm-Aspin from its structural point of view. UV-spectroscopy and multi-dimensional NMR are the experiments that have been performed to understand the kinetic and structural properties of Bm-Aspin respectively. The human aspartic proteases that are considered for this study are pepsin, renin, cathepsin-E and cathepsin-D. The results of this analysis performed with the specific substrate [Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu (4-pyridylmethyl) ester] against aspartic proteases suggest that Bm-Aspin inhibits the activities of all four human aspartic proteases. The kinetics studies indicate that Bm-Aspin follows a competitive mode of inhibition for pepsin and cathepsin-E, non-competitive for renin and mixed mode for cathepsin-D. The triple resonance NMR experiments on Bm-Aspin suggested the feasibility of carrying out NMR studies to obtain its solution structure. The NMR titration studies on the interactions of Bm-Aspin with the proteases indicate that it undergoes fast-exchange phenomena among themselves. In addition to this, the chemical shift perturbations for some of the residues of Bm-Aspin observed from (15)N-HSQC spectra upon the addition of saturated amounts of aspartic proteases suggest the binding between Bm-Aspin and human aspartic proteases. They also provide information on the variations in the intensities and mode of binding between the proteases duly corroborating with the results from the protease inhibition assay method.

MMP-12 catalytic domain recognizes triple helical peptide models of collagen V with exosites and high activity.

Matrix metalloproteinase (MMP)-12 (or metalloelastase) efficiently hydrolyzed the gelatinase-selective alpha1(V)436-447 fluorescent triple helical peptide (THP) when the substrate was submicromolar. The sequence of this THP was derived from collagen V, a component of collagen I fibrils. The hemopexin domains of MMP-12 and -9 each increased k(cat)/K(m) toward this substrate by decreasing K(m), just as the hemopexin domain of MMP-1 enhances its triple helical peptidase activity. Non-fluorescent alpha1(V) THP subtly perturbed amide NMR chemical shifts of MMP-12 not only in the active site cleft but also at remote sites of the beta-sheet and adjoining loops. The alpha1(V) THP protected MMP-12 from the NMR line broadening effects of Gd .EDTA in the active site cleft and more dramatically in the V-B loop next to the primed subsites. Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary among MMP sequences established that this site supports the high specific activity toward alpha1(V) fluorescent THP without affecting general MMP activity. Surprisingly the alpha1(V) THP also protected novel surfaces in the S-shaped metal-binding loop and beta-strands III and V that together form a pocket on the remote side of the zinc binding site. The patterns of protection suggest bending of the triple helical peptide partly around the catalytic domain to reach novel exosites. Partial unwinding or underwinding of the triple helix could accompany this to facilitate its hydrolysis.

Solution structure of inhibitor-free human metalloelastase (MMP-12) indicates an internal conformational adjustment.

Macrophage metalloelastase or matrix metalloproteinase-12 (MMP-12) appears to exacerbate atherosclerosis, emphysema, aortic aneurysm, rheumatoid arthritis, and inflammatory bowel disease. An inactivating E219A mutation, validated by crystallography and NMR spectra, prevents autolysis of MMP-12 and allows us to determine its NMR structure without an inhibitor. The structural ensemble of the catalytic domain without an inhibitor is based on 2813 nuclear Overhauser effects (NOEs) and has an average RMSD to the mean structure of 0.25 A for the backbone and 0.61 A for all heavy atoms for residues Trp109-Gly263. Compared to crystal structures of MMP-12, helix B (hB) at the active site is unexpectedly more deeply recessed under the beta-sheet. This opens a pocket between hB and beta-strand IV in the active-site cleft. Both hB and an internal cavity are shifted toward beta-strand I, beta-strand III, and helix A on the back side of the protease. About 25 internal NOE contacts distinguish the inhibitor-free solution structure and indicate hB's greater depth and proximity to the sheet and helix A. Line broadening and multiplicity of amide proton NMR peaks from hB are consistent with hB undergoing a slow conformational exchange among subtly different environments. Inhibitor-binding-induced perturbations of the NMR spectra of MMP-1 and MMP-3 map to similar locations across MMP-12 and encompass the internal conformational adjustments. Evolutionary trace analysis suggests a functionally important network of residues that encompasses most of the locations adjusting in conformation, including 18 residues with NOE contacts unique to inhibitor-free MMP-12. The conformational change, sequence analysis, and inhibitor perturbations of NMR spectra agree on the network they identify between structural scaffold and the active site of MMPs.