The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S 2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15 N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca 2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.
Copper is an essential nutrient for a variety of biochemical processes; however, the redox properties of copper also make it potentially toxic in the free form. Consequently, the uptake and intracellular distribution of this metal is strictly regulated. This raises the issue of whether specific pathophysiological conditions can promote adaptive changes in intracellular copper distribution. In this study, we demonstrate that oxygen limitation promotes a series of striking alterations in copper homeostasis in RAW264.7 macrophage cells. Hypoxia was found to stimulate copper uptake and to increase the expression of the copper importer, CTR1. This resulted in increased copper delivery to the ATP7A copper transporter and copper-dependent trafficking of ATP7A to cytoplasmic vesicles. Significantly, the ATP7A protein was required to deliver copper into the secretory pathway to ceruloplasmin, a secreted copperdependent enzyme, the expression and activity of which were stimulated by hypoxia. However, the activities of the alternative targets of intracellular copper delivery, superoxide dismutase and cytochrome c oxidase, were markedly reduced in response to hypoxia. Collectively, these findings demonstrate that copper delivery into the biosynthetic secretory pathway is regulated by oxygen availability in macrophages by a selective increase in copper transport involving ATP7A.
Matrix metalloproteinases (MMPs) regulate tissue remodeling, inflammation, and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here, we demonstrate binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labeled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane, and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation.
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 ␣-elastin species cover the primed subsites, a strip across the -sheet from -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.Although protein-protein interactions are universally important, mechanistic understanding of their specificity is often poor (1). An impediment to detailed understanding of proteolytic attack of proteins is the transience and potential heterogeneity of the interactions, which interfere in capturing the structure of a substrate complex by crystallography or other methods. These complications affect characterization of matrix metalloproteinase-12 (MMP-12), 3 the metalloelastase secreted by human macrophages at sites of inflammation. To investigate how MMP-12 achieves specificity for protein fibrils from lungs and arteries, we developed an approach designated BINDSIght, for its combination of bioinformatics and NMR discovery of specificity of interactions.In lungs, arteries, skin, and basement membranes, elastin provides elastic recoil, is heavily cross-linked, and is difficult to digest. Collagens are ubiquitous and comprise ϳ25% of the protein mass of the body. Damage to fibrils of the extracellular matrix by proteases such as MMP-12 contributes to the inflammation and chronic disease states of chronic obstructive pulmonary disease (2-4), atherosclerosis (5-6), abdominal aortic aneurysm (7), multiple sclerosis (8), ulcerative colitis (9), asthma (4), and rheumatoid arthritis (10). The progression of chronic obstructive pulmonary disease/emphysema and (abdominal aortic aneurysm) in smokers depends in large part on MMP-12 expression (11) and its degradation of elastin (7, 12). ...
SUMMARY Matrix metalloproteinase-7 (MMP-7) sheds signaling proteins from cell surfaces to activate bacterial killing, wound healing, and tumorigenesis. The mechanism targeting soluble MMP-7 to membranes has been investigated. NMR structures of the zymogen, free and bound to membrane mimics without and with anionic lipid, reveal peripheral binding to bilayers through paramagnetic relaxation enhancements. Addition of cholesterol sulfate partially embeds the protease in the bilayer, restricts its diffusion, and tips the active site away from the bilayer. Its insertion of hydrophobic residues organizes the lipids, pushing the head groups and sterol sulfate outward towards the enzyme's positive charge on the periphery of the enlarged interface. Fluorescence probing demonstrates a similar mode of binding to plasma membranes and internalized vesicles of colon cancer cells. Binding of bilayered micelles induces allosteric activation and conformational change in the auto-inhibitory peptide and the adjacent scissile site, illustrating a potential intermediate in the activation of the zymogen.
Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a “fly-casting mechanism.” We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light–dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.
Activation by glycosaminoglycans (GAGs) is an emerging trend among extracellular proteases important in disease. ProMMP-7, the zymogen of a matrix metalloproteinase secreted by mucosal epithelial and tumor cells, is activated at their surfaces by sulfated GAGs, but how? ProMMP-7 is activated in trans by representative heparin oligosaccharides in a length-dependent manner, with a large jump in activation at lengths of 16 monosaccharides. Imaging by atomic force microscopy visualized small complexes of proMMP-7 molecules linked by 8-mer lengths of heparinoids and extended assembles formed with 16-mer lengths of heparin. Complexes of proMMP-7 with polydisperse heparin or heparan sulfate were more diverse. Heparinoids evidently accelerate activation by tethering multiple proMMP-7 molecules together for proteolytic attack among neighbors. Removal of either the prodomain or C-terminal peptide sequence of KRSNSRKK from MMP-7 prevents formation of the long arrays induced by heparin 16-mers or heparan sulfate. The role of the C-terminus in activation assays suggests it contributes to remote, allosteric binding of GAGs. Enhancement of proteolytic velocity of MMP-by GAGs indicates them to be effectors of V-type allostery. GAGs from proteoglycans appear to assemble proMMP-7 molecules for activation, an event preceding its tumorigenic or anti-bacterial proteolytic activities at cell surfaces.
How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? NMR suggested soluble elastin to cover surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended out to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II–III loop and β-strand I near the back of the catalytic domain. The high exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, combination of a lesion at each of exosites 1 and 2 and active site decreased catalytic competence towards soluble elastin by 13- to 18-fold to the level of MMP-3, homologue and poor elastase. Double mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 kcat for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced kcat for soluble elastin. In elastin degradation, exosites 1 and 2 contributed independently of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils.
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