MUS81 is an important structure-specific endonuclease responsible for the processing of stalled replication forks and recombination intermediates. In human, MUS81 functions by forming complexes with its regulatory subunits EME1 and EME2, playing distinct roles in G2/M and S phases. Although the structures of MUS81-EME1 have been intensively studied, there is no structure information available about MUS81-EME2. Here, we report the crystal structure of MUS81-EME2, which reveals an overall protein fold similar to that of MUS81-EME1 complex. Further biochemical and structural characterization shows that the MUS81-EME1 and MUS81-EME2 complexes are identical in substrate recognition and endonuclease activities in vitro, implying that the distinct cellular roles of the two complexes could arise from temporal controls in cells. Finally, an extensive structure-guided mutagenesis analysis provides implications for the molecular basis of how the MUS81-EME endonucleases recognize various DNA substrates in a structure-selective manner.

Plant defensins demonstrate high structural stability at extreme temperatures and pH values and, in general, are non-toxic to mammalian cells. These properties make them attractive candidates for use in biotechnology and biomedicine. Knowing the structure-function relationship is desirable to guide the design of plant defensin-based applications. Thus far, the broad range of biological activities was described only for one defensin from gymnosperms, the defensin PsDef1 from Scots pine. Here, we report that closely related defensin from the same taxonomy group, PsDef2, differing from PsDef1 by six amino acids, also possesses antimicrobial, antibacterial, and insect α-amylase inhibitory activities. We also report the solution structure and dynamics properties of PsDef2 assessed using a combination of experimental nuclear magnetic resonance (NMR) techniques. Lastly, we perform a comparative analysis of PsDef2 and PsDef1 gaining a molecular-level insight into their structure-dynamics-function relationship.

Comment in Structure. 2022 May 5;30(5):653-654.

Comment on Structure. 2022 May 5;30(5):671-684.e5.

Because of the evolutionary variants of SARS-CoV-2, development of broad-spectrum neutralizing antibodies resilient to virus escape is urgently needed. We identified a group of high-affinity nanobodies from camels immunized with receptor-binding domain (RBD) of SARS-CoV-2 spike protein and resolved the structures of two non-competing nanobodies (NB1A7 and NB1B11) in complex with RBD using X-ray crystallography. The structures show that NB1A7 targets the highly conserved cryptic epitope shared by SARS-CoV-2 variants and some other coronaviruses and blocks ACE2 receptor attachment of the spike protein, and NB1B11 epitope overlaps with the contacting surface of ACE2 and is different from the binding site of NB1A7. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, which significantly improved the avidity and neutralization potency and may further inhibit viral escape. The results contribute to the structure-guided design of antibodies against future variants of SARS-CoV-2 virus to combat coronavirus epidemics and pandemics.

Connexins form large-pore channels that function either as dodecameric gap junctions or hexameric hemichannels to allow the regulated movement of small molecules and ions across cell membranes. Opening or closing of the channels is controlled by a variety of stimuli, and dysregulation leads to multiple diseases. An increase in the partial pressure of carbon dioxide (PCO2) has been shown to cause connexin26 (Cx26) gap junctions to close. Here, we use cryoelectron microscopy (cryo-EM) to determine the structure of human Cx26 gap junctions under increasing levels of PCO2. We show a correlation between the level of PCO2 and the size of the aperture of the pore, governed by the N-terminal helices that line the pore. This indicates that CO2 alone is sufficient to cause conformational changes in the protein. Analysis of the conformational states shows that movements at the N terminus are linked to both subunit rotation and flexing of the transmembrane helices.

Carcinoembryonic cellular adhesion molecules (CEACAMs) serve diverse roles in cell signaling, proliferation, and survival and are made up of one or several immunoglobulin (Ig)-like ectodomains glycosylated in vivo. The physiological oligomeric state and how it contributes to protein function are central to understanding CEACAMs. Two putative dimer conformations involving different CEACAM1 N-terminal Ig-like domain (CCM1) protein faces (ABED and GFCC'C″) were identified from crystal structures. GFCC'C″ was identified as the dominant CCM1 solution dimer, but ambiguity regarding the effect of glycosylation on dimer formation calls its physiological relevance into question. We present the first crystal structure of minimally glycosylated CCM1 in the GFCC'C″ dimer conformation and characterization in solution by continuous-wave and double electron-electron resonance electron paramagnetic resonance spectroscopy. Our results suggest the GFCC'C″ dimer is dominant in solution with different levels of glycosylation, and structural conservation and co-evolved residues support that the GFCC'C″ dimer is conserved across CEACAMs.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

Influenza viruses pose severe public health threats globally. Influenza viruses are extensively pleomorphic, in shape, size, and organization of viral proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships and aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we leveraged convolutional neural networks and cryoET to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza morphology, glycoprotein density, and conducted subtomogram averaging of influenza glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells.

Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. These results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens.

Serine beta-lactamase-like protein (LACTB) is a mammalian mitochondrial serine protease that can specifically hydrolyze peptide bonds adjacent to aspartic acid residues and is structurally related to prokaryotic penicillin-binding proteins. Here, we determined the cryoelectron microscopy structures of human LACTB (hLACTB) filaments from wild-type protein, a middle region deletion mutant, and in complex with the inhibitor Z-AAD-CMK at 3.0-, 3.1-, and 2.8-Å resolution, respectively. Structural analysis and activity assays revealed that three interfaces are required for the assembly of hLACTB filaments and that the formation of higher order helical structures facilitates its cleavage activity. Further structural and enzymatic analyses of middle region deletion constructs indicated that, while this region is necessary for substrate hydrolysis, it is not required for filament formation. Moreover, the inhibitor-bound structure showed that hLACTB may cleave peptide bonds adjacent to aspartic acid residues. These findings provide the structural basis underlying hLACTB catalytic activity.

Comment in Structure. 2022 May 5;30(5):655-657.

Comment on Structure. 2022 May 5;30(5):721-732.e4.