BiP is a major endoplasmic reticulum (ER) chaperone and is suggested to act as primary sensor in the activation of the unfolded protein response (UPR). How BiP operates as a molecular chaperone and ...as an ER stress sensor is unknown. Here, by reconstituting components of human UPR, ER stress and BiP chaperone systems, we discover that the interaction of BiP with the luminal domains of UPR proteins IRE1 and PERK switch BiP from its chaperone cycle into an ER stress sensor cycle by preventing the binding of its co-chaperones, with loss of ATPase stimulation. Furthermore, misfolded protein-dependent dissociation of BiP from IRE1 is primed by ATP but not ADP. Our data elucidate a previously unidentified mechanistic cycle of BiP function that explains its ability to act as an Hsp70 chaperone and ER stress sensor.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo-electron microscopy ...(cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)-up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.
The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, ...isolates with the D614G substitution in the S protein appeared early during the pandemic and are now the dominant form worldwide. Here, we explore S conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryoelectron microscopy (cryo-EM) structures reveal altered receptor binding domain (RBD) disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the RBDs in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, which harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 S conformational landscape and allostery and have implications for vaccine design.
Display omitted
•SARS-CoV-2 S 2P mutations do not impact its structure, stability, or antigenicity•D614G mutation increases RBD “up” state and enhances S1/S2 junction proteolysis•Structure and antigenicity reveal allostery between the S1/S2 junction and RBD•SD2 anchors the mobile RBD and NTD, separating large S1 subunit motions from S2
SARS-CoV-2 spike undergoes large conformational changes during cell fusion. Gobeil et al. identify a subdomain anchor that limits large motions in the receptor binding subunit of the pre-fusion spike from propagating to its fusion subunit. They demonstrate that the D614G mutation increases the rate of furin cleavage, which may impact infectivity.
The endoplasmic reticulum (ER) is an important site for protein folding and maturation in eukaryotes. The cellular requirement to synthesize proteins within the ER is matched by its folding capacity. ...However, the physiological demands or aberrations in folding may result in an imbalance which can lead to the accumulation of misfolded protein, also known as "ER stress." The unfolded protein response (UPR) is a cell-signaling system that readjusts ER folding capacity to restore protein homeostasis. The key UPR signal activator, IRE1, responds to stress by propagating the UPR signal from the ER to the cytosol. Here, we discuss the structural and molecular basis of IRE1 stress signaling, with particular focus on novel mechanistic advances. We draw a comparison between the recently proposed allosteric model for UPR induction and the role of Hsp70 during polypeptide import to the mitochondrial matrix.
The coronavirus (CoV) spike (S) protein, involved in viral-host cell fusion, is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The ...highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available β-CoV S-protein structures. Despite an overall similarity in domain organization, we found that S-proteins from different β-CoVs display distinct configurations. Based on this analysis, we developed two soluble ectodomain constructs for the SARS-CoV-2 S-protein, in which the highly immunogenic and mobile receptor binding domain (RBD) is either locked in the all-RBDs 'down' position or adopts 'up' state conformations more readily than the wild-type S-protein. These results demonstrate that the conformation of the S-protein can be controlled via rational design and can provide a framework for the development of engineered CoV S-proteins for vaccine applications.
The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response ...in order to maintain homeostasis. How cells detect the accumulation of misfolded proteins remains unclear. In this study, we identify a noncanonical interaction between the ATPase domain of the ER chaperone BiP and the luminal domains of the UPR sensors Ire1 and Perk that dissociates when authentic ER unfolded protein CH1 binds to the canonical substrate binding domain of BiP. Unlike the interaction between chaperone and substrates, we found that the interaction between BiP and UPR sensors was unaffected by nucleotides. Thus, we discover that BiP is dual functional UPR sensor, sensing unfolded proteins by canonical binding to substrates and transducing this event to noncanonical, signaling interaction to Ire1 and Perk. Our observations implicate BiP as the key component for detecting ER stress and suggest an allosteric mechanism for UPR induction.
Abstract
Multidrug resistance protein 1 (MDR1), a phase III drug transporter that exports substrates out of cells, has been discovered in both cancerous and normal tissues. The over expression of ...MDR1 in cancer cells contributes to multiple drug resistance, whereas the MDR1 in normal tissues protects them from chemical-induced toxicity. Currently, the role of MDR1 in the ovary has not been entirely understood. Our objective is to determine the function of MDR1 in protecting against chemotherapy-induced ovarian toxicity. Using both the in vivo transgenic mouse model and in vitro follicle culture model, we investigated the expression of MDR1 in the ovary, the effect of MDR1 deficiency on doxorubicin (DOX)-induced ovarian toxicity, and the ovarian steroid hormonal regulation of MDR1. Results showed that the MDR1 was expressed in the ovarian epithelial cells, stroma cells, theca cell layers, endothelial cells, and luteal cells. The lack of MDR1 did not affect female ovarian function and fertility; however, its deficiency significantly exacerbated the DOX-induced ovarian toxicity in both in vivo and in vitro models. The MDR1 showed significantly higher expression levels in the ovaries at estrus and metestrus stages than those at proestrus and diestrus stages. However, this dynamic expression pattern was not regulated by the ovarian steroid hormones of estrogen (E2) and progesterone (P4) but correlated to the number and status of corpus luteum. In conclusion, our study demonstrates that the lack of MDR1 promotes DOX-induced ovarian toxicity, suggesting the critical role of MDR1 in protecting female ovarian functions during chemotherapy.
The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its receptor binding domain in two conformations, the receptor-accessible 'up' or receptor-inaccessible ...'down' states. Here we report that the commonly used stabilized S ectodomain construct '2P' is sensitive to cold temperatures, and this cold sensitivity is abrogated in a 'down' state-stabilized ectodomain. Our findings will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.
The genes coding for the enzymes of oxidative degradation of nicotinic acid have recently been identified in several species of aerobic bacteria, namely, Pseudomonas putida KT2440, Bordetella ...bronchiseptica RB50, and Bacillus niacini. One of the enzymes involved in an early step of this pathway is a flavin-dependent monooxygenase (6-hydroxynicotinic acid 3-monooxygenase; NicC) that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP), with concomitant oxidation of NADH to NAD+. The nicC genes from B. bronchiseptica RB50 and P. putida have been cloned, and the purified enzymes have been characterized functionally and structurally. Global fits of the steady-state kinetic data show that both enzymes are efficient catalysts, with an apparent k cat/K M 6‑HNA of 5.0 × 104 M–1 s–1 for B. bronchiseptica NicC. The pH dependence of V max/E t fits a double-bell model showing an optimum around pH 8 with apparent pK as of 7.24 ± 0.08 and 8.64 ± 0.08, whereas the apparent catalytic efficiency (k cat/K M 6‑HNA) is maximal around pH 7 and decreases at high pH with an apparent pK a of 7.60 ± 0.06. The enzyme’s relative affinity for 6-hydroxynicotinaldehyde, a neutral analogue that shows competitive inhibition with respect to 6-HNA, is weak (K i = 3000 ± 400 μM) in comparison to the apparent binding of 6-HNA (K M = 85 ± 13 μM). The crystal structure for P. putida NicC has been solved to 2.1 Å using SAD phasing, and the 6-HNA substrate has been modeled into the active site. Together these data provide insight into potential reaction mechanisms for this novel decarboxylative hydroxylation reaction.
Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To ...understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.
Display omitted
•Glycan276-dependent antibodies with up to 81% neutralization breadth are isolated•Structure for three glycan276-dependent antibodies with envelope trimer is determined•Glycan276-dependent antibodies bind glycan276 with diverse modes of recognition•Glycan276-dependent antibodies maintain ligand-free orientation of glycan276
CD4 binding site-directed antibodies can broadly neutralize circulating HIV strains but often need to overcome glycan shielding, especially from glycan276. Cottrell et al. isolate two lineages of antibodies that depend on glycan276 for neutralization and elucidate the mechanistic difference between glycan276-dependent and glycan276-independent antibodies, with implications for HIV vaccine design.
PDF
