Open Access

Show Abstract ▼

Abstract: Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.

Show Abstract ▼

Abstract: This investigation examines the validity of employing single-solute theory to interpret SAXS measurements on buffered protein solutions—the current practice despite the necessity to regard the buffer components as additional non-scattering solutes rather than as part of the solvent. The present study of bovine serum albumin in phosphate-buffered saline supplemented with 20–100 g/L sucrose as small cosolute has certainly verified the prediction that the experimentally obtained second virial coefficient should contain protein–cosolute contributions. Nevertheless, the second virial coefficient determined for protein solutions supplemented with high cosolute concentrations on the basis of single-solute theory remains a valid means for identifying conditions conducive to protein crystallization, because the return of a slightly negative second virial coefficient based on single-solute theory 𝐴app2 still establishes the existence of slightly associative interactions between protein molecules, irrespective of the molecular source–protein self-interactions and/or protein–cosolute contributions.

Open Access

Show Abstract ▼

Abstract: Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity.

Show Abstract ▼

Abstract: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3,4,5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication—which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7,8,9,10—remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0–4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3′ cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs.

Show Abstract ▼

Abstract: The Proliferating Cell Nuclear Antigen-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, binds to PCNA as non-ubiquitinated p15PAF does, but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by 8 disordered residues, form transient dimers due to the high local effective concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1for maintenance of DNA methylation during replication.