9 Sep 2022

Intermediates in SARS-CoV-2 spike-mediated cell entry (Science Advances)

SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.

9 Sep 2022

A public antibody class recognizes an S2 epitope exposed on open conformations of SARS-CoV-2 spike (Nature Communications)

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that similar to 82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (similar to 0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

To fully understand the potential shortcomings of SARS-CoV-2 vaccination, it is necessary to delineate the properties of the antibodies elicited, during immunization, and also infection. Through investigation of the SARS-CoV-2 spike-reactive B cell repertoire, authors identify following infection, a subset of B cells enriched and almost exclusively target a non-neutralizing S2 epitope present in aberrant forms.

9 Sep 2022

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (PNAS)

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of the previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cry-oelectron microscopy (cryo-EM) and X-ray crystallography. We show that mAb J08 has low nanomolar affinity against most VoCs and binds high on the receptor binding domain (RBD) ridge, away from many VoC mutations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy.

10 Jul

Solid-state NMR molecular snapshots of Aspergillus fumigatus cell wall architecture during a conidial morphotype transition)

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatustransit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides β-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

27 Jun

mRNA recognition and packaging by the human transcription-export complex (Nature)

Newly made messenger RNAs are processed and packaged into ribonucleoprotein complexes (mRNPs) and recognized by the essential transcription-export complex (TREX) for nuclear export. However, the mechanisms of mRNP recognition and three-dimensional organization are poorly understood. Here, we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the two-megadalton TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon-junction complexes. Exon-junction complexes can multimerize through ALYREF, suggesting a mechanism for mRNP organization. Endogenous mRNPs form compact ‘globules’ that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact, and protect mRNAs to promote their packaging for nuclear export. The mRNP globule organization provides a framework to understand how mRNP architecture could facilitate mRNA biogenesis and export.

27 Jun

New measures of anisotropy of cryo-EM maps (Nature Methods)

We propose two new measures of resolution anisotropy for cryogenic electron microscopy maps: Fourier shell occupancy (FSO), and the Bingham test (BT). FSO varies from 1 to 0, with 1 representing perfect isotropy, and lower values indicating increasing anisotropy. The threshold FSO = 0.5 occurs at Fourier shell correlation resolution. BT is a hypothesis test that complements the FSO to ensure the existence of anisotropy. FSO and BT allow visualization of resolution anisotropy. We illustrate their use with different experimental cryogenic electron microscopy maps.

27 Jun

Automatic and accurate ligand structure determination guided by cryo-electron microscopy maps (Nature Communications)

Advances in cryo-electron microscopy (cryoEM) and deep-learning guided protein structure prediction have expedited structural studies of protein complexes. However, methods for accurately determining ligand conformations are lacking. In this manuscript, we develop EMERALD, a tool for automatically determining ligand structures guided by medium-resolution cryoEM density. We show this method is robust at predicting ligands along with surrounding side chains in maps as low as 4.5 Å local resolution. Combining this with a measure of placement confidence and running on all protein/ligand structures in the EMDB, we show that 57% of ligands replicate the deposited model, 16% confidently find alternate conformations, 22% have ambiguous density where multiple conformations might be present, and 5% are incorrectly placed. For five cases where our approach finds an alternate conformation with high confidence, high-resolution crystal structures validate our placement. EMERALD and the resulting analysis should prove critical in using cryoEM to solve protein-ligand complexes.

13 Jun

Structure of the Acidobacteria homodimeric reaction center bound with cytochrome c (Nature Communications)

Photosynthesis converts light energy to chemical energy to fuel life on earth. Light energy is harvested by antenna pigments and transferred to reaction centers (RCs) to drive the electron transfer (ET) reactions. Here, we present cryo-electron microscopy (cryo-EM) structures of two forms of the RC from the microaerophilic Chloracidobacterium thermophilum (CabRC): one containing 10 subunits, including two different cytochromes; and the other possessing two additional subunits, PscB and PscZ. The larger form contained 2 Zn-bacteriochlorophylls, 16 bacteriochlorophylls, 10 chlorophylls, 2 lycopenes, 2 hemes, 3 Fe₄S₄ clusters, 12 lipids, 2 Ca²⁺ ions and 6 water molecules, revealing a type I RC with an ET chain involving two hemes and a hybrid antenna containing bacteriochlorophylls and chlorophylls. Our results provide a structural basis for understanding the excitation energy and ET within the CabRC and offer evolutionary insights into the origin and adaptation of photosynthetic RCs.

13 Jun

The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators (Nature Communications)

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3’end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of m⁶A, the most abundant RNA modification. RBM15 positively regulates m⁶A levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.

13 Jun

Structural basis for control of bacterial RNA polymerase pausing by a riboswitch and its ligand (Nature Stuctural & Molecular Biology)

Folding of nascent transcripts can be modulated by the RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ₁riboswitch (termed que-PEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination; however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-PEC in ligand-free and ligand-bound states. In the absence of preQ₁, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, on ligand binding, the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

13 Jun

What Rosalind Franklin truly contributed to the discovery of DNA’s structure (Nature)

Franklin was no victim in how the DNA double helix was solved. An overlooked letter and an unpublished news article, both written in 1953, reveal that she was an equal player. According to journalist Horace Freeland Judson and Franklin’s biographer, Brenda Maddox, Rosalind Franklin has been reduced to the “wronged heroine” of the double helix. She deserves to be remembered not as the victim of the double helix, but as an equal contributor to the solution of the structure.