Fortuitous somatic mutations during antibody evolution endow broad neutralization against SARS-CoV-2 Omicron variants.

Striking antibody evasion by emerging circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identify a clonally related antibody family from a convalescent individual. One of the members, XG005, exhibits potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members show significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface reveals how crucial somatic mutations endow XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality exhibits a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provide a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

Fortuitous Somatic Mutations during Antibody Evolution Endow Broad Neutralization against SARS-CoV-2 Omicron Variants.

Striking antibody evasion by emerging circulating SARS-CoV-2 variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identified a clonally-related antibody family from a convalescent individual. One of the members, XG005, exhibited potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members showed significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface revealed how crucial somatic mutations endowed XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality, exhibited a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provided a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) play a central role in tryptophan metabolism and are involved in many cellular and disease processes. Here we report the crystal structure of human TDO (hTDO) in a ternary complex with the substrates L-Trp and O2 and in a binary complex with the product N-formylkynurenine (NFK), defining for the first time the binding modes of both substrates and the product of this enzyme. The structure indicates that the dioxygenation reaction is initiated by a direct attack of O2 on the C2 atom of the L-Trp indole ring. The structure also reveals an exo binding site for L-Trp, located ~42 Å from the active site and formed by residues conserved among tryptophan-auxotrophic TDOs. Biochemical and cellular studies indicate that Trp binding at this exo site does not affect enzyme catalysis but instead it retards the degradation of hTDO through the ubiquitin-dependent proteasomal pathway. This exo site may therefore provide a novel L-Trp-mediated regulation mechanism for cellular degradation of hTDO, which may have important implications in human diseases.

Spectroscopic and mutagenesis studies of human PGRMC1.

Progesterone receptor membrane component 1 (PGRMC1) is a 25 kDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Although the general definition of PGRMC1 is as a progesterone receptor, progesterone-binding activity has not been directly demonstrated in any of the purified PGRMC1 proteins fully loaded with heme. Here, we show that the human homologue of PGRMC1 (hPGRMC1) binds heme in a five-coordinate (5C) high-spin (HS) configuration, with an axial tyrosinate ligand, likely Y95. The negatively charged tyrosinate ligand leads to a relatively low redox potential of approximately -331 mV. The Y95C or Y95F mutation dramatically reduces the ability of the protein to bind heme, supporting the assignment of the axial heme ligand to Y95. On the other hand, the Y95H mutation retains ∼90% of the heme-binding activity. The heme in Y95H is also 5CHS, but it has a hydroxide axial ligand, conceivably stabilized by the engineered-in H95 via an H-bond; CO binding to the distal ligand-binding site leads to an exchange of the axial ligand to a histidine, possibly H95. We show that progesterone binds to hPGRMC1 and introduces spectral changes that manifest conformational changes to the heme. Our data offer the first direct evidence supporting progesterone-binding activity of PGRMC1.

Glutathionylspermidine in the modification of protein SH groups: the enzymology and its application to study protein glutathionylation.

Cysteine is very susceptible to reactive oxygen species. In response; posttranslational thiol modifications such as reversible disulfide bond formation have arisen as protective mechanisms against undesired in vivo cysteine oxidation. In Gram-negative bacteria a major defense mechanism against cysteine overoxidation is the formation of mixed protein disulfides with low molecular weight thiols such as glutathione and glutathionylspermidine. In this review we discuss some of the mechanistic aspects of glutathionylspermidine in prokaryotes and extend its potential use to eukaryotes in proteomics and biochemical applications through an example with tissue transglutaminase and its S-glutathionylation.

Characteristic tandem mass spectral features under various collision chemistries for site-specific identification of protein S-glutathionylation.

Protein S-glutathionylation is a reversible post-translational modification widely implicated in redox regulated biological functions. Conventional biochemical methods, however, often do not allow such a mixed disulfide modification to be reliably identified on specific cysteine residues or be distinguished from other related oxidized forms. To develop more efficient mass spectrometry (MS)-based analytical strategies for this purpose, we first investigated the MS/MS fragmentation pattern of S-glutathionylated peptides under various dissociation modes, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD), using synthetic peptides derived from protein tyrosine phosphatase as models. Our results indicate that a MALDI-based high energy CID MS/MS on a TOF/TOF affords the most distinctive spectral features that would facilitate rapid and unambiguous identification of site-specific S-glutathionylation. For more complex proteomic samples best tackled by LC-MS/MS approach, we demonstrate that HCD performed on an LTQ-Orbitrap hybrid instrument fairs better than trap-based CID and ETD in allowing more protein site-specific S-glutathionylation to be confidently identified by direct database searching of the generated MS/MS dataset using Mascot. Overall, HCD afforded more peptide sequence-informative fragment ions retaining the glutathionyl modification with less neutral losses of side chains to compromise scoring. In conjunction with our recently developed chemo-enzymatic tagging strategy, our nanoLC-HCD-MS/MS approach is sufficiently sensitive to identify endogenous S-glutathionylated peptides prepared from non-stressed cells. It is anticipated that future applications to global scale analysis of protein S-glutathionylation will benefit further from current advances in both speed and mass accuracy afforded by HCD MS/MS mode on the Orbitrap series.

An acyloxymethyl ketone-based probe to monitor the activity of glutathionylspermidine amidase in Escherichia coli.

Cellular redox conditions affect Gsp amidase activity in Escherichia coli. Guided by the structure and catalytic mechanism of the amidase, we designed and synthesized an acyloxymethyl ketone-based activity probe containing a biotin handle. This probe was used to monitor Gsp amidase activity in E. coli lysates that had been subjected to oxidative or methylglyoxal-induced stress.

Protein S-thiolation by Glutathionylspermidine (Gsp): the role of Escherichia coli Gsp synthetASE/amidase in redox regulation.

Certain bacteria synthesize glutathionylspermidine (Gsp), from GSH and spermidine. Escherichia coli Gsp synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Prior to the work reported herein, the physiological role(s) of Gsp or how the two opposing GspSA activities are regulated had not been elucidated. We report that Gsp-modified proteins from E. coli contain mixed disulfides of Gsp and protein thiols, representing a new type of post-translational modification formerly undocumented. The level of these proteins is increased by oxidative stress. We attribute the accumulation of such proteins to the selective inactivation of GspSA amidase activity. X-ray crystallography and a chemical modification study indicated that the catalytic cysteine thiol of the GspSA amidase domain is transiently inactivated by H(2)O(2) oxidation to sulfenic acid, which is stabilized by a very short hydrogen bond with a water molecule. We propose a set of reactions that explains how the levels of Gsp and Gsp S-thiolated proteins are modulated in response to oxidative stress. The hypersensitivities of GspSA and GspSA/glutaredoxin null mutants to H(2)O(2) support the idea that GspSA and glutaredoxin act synergistically to regulate the redox environment of E. coli.

Dual binding sites for translocation catalysis by Escherichia coli glutathionylspermidine synthetase.

Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.