The pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.

Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.

Update of bioRxiv. 2020 Nov 03;:

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T fluorescence, Congo-red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases.

In Escherichia coli, the major bacterial Hsp70 system consists of DnaK, three J-domain proteins (JDPs: DnaJ, CbpA, and DjlA), and nucleotide exchange factor GrpE. JDPs determine substrate specificity for the Hsp70 system; however, knowledge on their specific role in bacterial cellular functions is limited. In this study, we demonstrated the role of JDPs in bacterial survival during heat stress and the DnaK-regulated formation of curli-extracellular amyloid fibers involved in biofilm formation. Genetic analysis demonstrate that only DnaJ is essential for survival at high temperature. On the other hand, either DnaJ or CbpA, but not DjlA, is sufficient to activate DnaK in curli production. Additionally, several DnaK mutants with reduced activity are able to complement the loss of curli production in E. coli ΔdnaK, whereas they do not recover the growth defect of the mutant strain at high temperature. Biochemical analyses reveal that DnaJ and CbpA are involved in the expression of the master regulator CsgD through the solubilization of MlrA, a DNA-binding transcriptional activator for the csgD promoter. Furthermore, DnaJ and CbpA also keep CsgA in a translocation-competent state by preventing its aggregation in the cytoplasm. Our findings support a hierarchical model wherein the role of JDPs in the Hsp70 system differs according to individual cellular functions.

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

Lysine methylation is a key regulator of protein-protein binding. The amine group of lysine can accept up to three methyl groups, and experiments show that protein-protein binding free energies are sensitive to the extent of methylation. These sensitivities have been rationalized in terms of chemical and structural features present in the binding pockets of methyllysine binding domains. However, understanding their specific roles requires an energetic analysis. Here we propose a theoretical framework to combine quantum and molecular mechanics methods, and compute the effect of methylation on protein-protein binding free energies. The advantages of this approach are that it derives contributions from all local non-trivial effects of methylation on induction, polarizability and dispersion directly from self-consistent electron densities, and at the same time determines contributions from well-characterized hydration effects using a computationally efficient classical mean field method. Limitations of the approach are discussed, and we note that predicted free energies of fourteen out of the sixteen cases agree with experiment. Critical assessment of these cases leads to the following overarching principles that drive methylation-state recognition by protein domains. Methylation typically reduces the pairwise interaction between proteins. This biases binding toward lower methylated states. Simultaneously, however, methylation also makes it easier to partially dehydrate proteins and place them in protein-protein complexes. This latter effect biases binding in favor of higher methylated states. The overall effect of methylation on protein-protein binding depends ultimately on the balance between these two effects, which is observed to be tuned via several combinations of local features.

Regulation of bacterial stress responding σS is a sophisticated process and mediated by multiple interacting partners. Controlled proteolysis of σS is regulated by RssB which maintains minimal level of σS during exponential growth but then elevates σS level while facing stresses. Bacteria developed different strategies to regulate activity of RssB, including phosphorylation of itself and production of anti-adaptors. However, the function of phosphorylation is controversial and the mechanism of anti-adaptors preventing RssB-σS interaction remains elusive. Here, we demonstrated the impact of phosphorylation on the activity of RssB and built the RssB-σS complex model. Importantly, we showed that the phosphorylation site - D58 is at the interface of RssB-σS complex. Hence, mutation or phosphorylation of D58 would weaken the interaction of RssB with σS. We found that the anti-adaptor protein IraD has higher affinity than σS to RssB and its binding interface on RssB overlaps with that for σS. And IraD-RssB complex is preferred over RssB-σS in solution, regardless of the phosphorylation state of RssB. Our study suggests that RssB possesses a two-tier mechanism for regulating σS. First, phosphorylation of RssB provides a moderate and reversible tempering of its activity, followed by a specific and robust inhibition via the anti-adaptor interaction.

Intrinsically disordered proteins (IDPs) lack stable tertiary structure under physiological conditions. The unique composition and complex dynamical behaviour of IDPs make them a challenge for structural biology and molecular evolution studies. Using NMR ensembles, we found that IDPs evolve under a strong site-specific evolutionary rate heterogeneity, mainly originated by different constraints derived from their inter-residue contacts. Evolutionary rate profiles correlate with the experimentally observed conformational diversity of the protein, allowing the description of different conformational patterns possibly related to their structure-function relationships. The correlation between evolutionary rates and contact information improves when structural information is taken not from any individual conformer or the whole ensemble, but from combining a limited number of conformers. Our results suggest that residue contacts in disordered regions constrain evolutionary rates to conserve the dynamic behaviour of the ensemble and that evolutionary rates can be used as a proxy for the conformational diversity of IDPs.

Anti-CRISPRs are protein inhibitors of CRISPR-Cas systems. They are produced by phages and other mobile genetic elements to evade CRISPR-Cas-mediated destruction. Anti-CRISPRs are remarkably diverse in sequence, structure, and functional mechanism; thus, structural and mechanistic investigations of anti-CRISPRs continue to yield exciting new insights. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AcrIE2, an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. Guided by the structure, we used site-directed mutagenesis to identify key residues that are required for AcrIE2 function. Using affinity purification experiments, we found that AcrIE2 binds the type I-E CRISPR-Cas complex (Cascade). In vivo transcriptional assays, in which Cascade was targeted to promoter regions, demonstrated that Cascade still binds to DNA in the presence of AcrIE2. This is the first instance of a type I anti-CRISPR that binds to a CRISPR-Cas complex but does not prevent DNA-binding. Another unusual property of AcrIE2 is that the effect of Cascade:AcrIE2 complex binding to promoter regions varied depending on the position of the binding site. Most surprisingly, Cascade:AcrIE2 binding led to transcriptional activation in some cases rather than repression, which did not occur when Cascade alone bound to the same sites. We conclude that AcrIE2 operates through a distinct mechanism compared to other type I anti-CRISPRs. While AcrIE2 does not prevent Cascade from binding DNA, it likely blocks subsequent recruitment of the Cas3 nuclease to Cascade thereby preventing DNA cleavage.

Legionella pneumophila infects alveolar macrophages and can cause life-threatening pneumonia in humans. Upon internalization into the host cell, L. pneumophila injects numerous effector proteins into the host cytoplasm as a part of its pathogenesis. LegK7 is an effector kinase of L. pneumophila that functionally mimics the eukaryotic Mst kinase and phosphorylates the host MOB1 protein to exploit the Hippo pathway. To elucidate the LegK7 activation mechanism, we determined the apo structure of LegK7 in an inactive form and performed a comparative analysis of LegK7 structures. LegK7 is a non-RD kinase that contains an activation segment that is ordered, irrespective of stimulation, through a unique β-hairpin-containing segment, and it does not require phosphorylation of the activation segment for activation. Instead, bacterial LegK7 becomes an active kinase via its heterologous molecular interaction with the host MOB1 protein. MOB1 binding triggers reorientation of the two lobes of the kinase domain, as well as a structural change in the interlobe hinge region in LegK7, consequently reshaping the LegK7 structure into an ATP binding-compatible closed conformation. Furthermore, we reveal that LegK7 is an atypical kinase that contains an N-terminal capping domain and a hydrophilic interlobe linker motif, which play key roles in the MOB1-induced activation of LegK7.

Forkhead box O4 (FOXO4) is a human transcription factor (TF) that participates in cell homeostasis. While the structure and DNA binding properties of the conserved forkhead domain (FHD) have been thoroughly investigated, how the transactivation domain (TAD) regulates the DNA binding properties of the protein remains elusive. Here, we investigated the role of TAD in modulating the DNA binding properties of FOXO4 using solution NMR. We found that TAD and FHD form an intramolecular complex mainly governed by hydrophobic interaction. Remarkably, TAD and DNA share the same surface of FHD for binding. While FHD did not differentiate binding to target and non-target DNA, the FHD-TAD complex showed different behaviors depending on the DNA sequence. In the presence of TAD, free and DNA-bound FHD exhibited a slow exchange with target DNA and a fast exchange with non-target DNA. The interaction of the two domains affected the kinetic function of FHD depending on the type of DNA. Based on these findings, we suggest a transcription initiation model by which TAD modulates FOXO4 recognition of its target promoter DNA sequences. This study describes the function of TAD in FOXO4 and provides a new kinetic perspective on target sequence selection by TFs.

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans.

Multiple copies of WW domains and PPXY motif sequences are often reciprocally presented by regulatory proteins that interact at crucial regulatory steps in the cell life cycle. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. By way of investigating such assemblies, we characterized the multivalent association of the entire cognate binding domains, two WW sequences and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor. Isothermal titration calorimetry, sedimentation velocity, size-exclusion chromatography coupled to multi-angle light scattering and native-state mass spectrometry of Yorkie WW domains interactions with the full-length Warts PPXY domain, and numerous PPXY motif variants of Warts show that the two proteins assemble via binding of tandem WW domains to adjacent PPXY pairs to produce an ensemble of interconverting complexes of variable stoichiometries, binding energetics and WW domain occupancy. Apparently, the Yorkie tandem WW domains first target the two adjacent PPXY motifs at the C-terminus of the Warts polypeptide and additional WW domains bind unoccupied motifs. Similar ensembles of interconverting conformers may be common in multivalent WW domain-PPXY interactions to promote the adaptability and versatility of WW domain-PPXY mediated cellular processes.

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na+ as the driving force. The crystal structures of two bacterial homologues ASBTNM and ASBTYf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na+-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na+ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBTNM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBTNM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na+ ions shift the conformational equilibrium of ASBTNM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase β to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol β in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.

The Rossmann-like fold is the most prevalent and diversified doubly-wound superfold of ancient evolutionary origin. Rossmann-like domains are present in a variety of metabolic enzymes and are capable of binding diverse ligands. Discerning evolutionary relationships among these domains is challenging because of their diverse functions and ancient origin. We defined a minimal Rossmann-like structural motif (RLM), identified RLM-containing domains among known 3D structures (20%) and classified them according to their homologous relationships. New classifications were incorporated into our Evolutionary Classification of protein Domains (ECOD) database. We defined 156 homology groups (H-groups), which were further clustered into 123 possible homology groups (X-groups). Our analysis revealed that RLM-containing proteins constitute approximately 15% of the human proteome. We found that disease-causing mutations are more frequent within RLM domains than within non-RLM domains of these proteins, highlighting the importance of RLM-containing proteins for human health.

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP6). IP6 interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP6 on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP6, arrestin-2 forms "infinite" chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP6; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP6, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP6 effect. Because IP6 is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.

Interleukin-1β (IL-1β) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1β regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1β to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1β initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1β neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1β. We validated IgG26AW-neutralizing antibodies specific for IL-1β in vivo to prevent human IL-1β-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1β also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1β complex structure. Meanwhile, SPR experiments showed that IL-1β bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1β/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1β, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1β is critical to pathogenesis.

Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.

The severity of SARS-CoV-2 infection is highly variable and yet the molecular basis for this effect remains elusive. One potential contribution are differences in the glycosylation of target human cells, particularly as SARS-CoV-2 has the capacity to bind sialic acid which is a common, and highly variable, terminal modification of glycans. The viral spike glycoprotein (S) of SARS-CoV-2 and the human cellular receptor, angiotensin-converting enzyme 2 (ACE2) are both densely glycosylated. We therefore sought to investigate whether the glycosylation state of ACE2 impacts the interaction with SARS-CoV-2 viral spike. We generated a panel of engineered ACE2 glycoforms which were analyzed by mass spectrometry to reveal the site-specific glycan modifications. We then probed the impact of ACE2 glycosylation on S binding and revealed a subtle sensitivity with hypersialylated or oligomannose-type glycans slightly impeding the interaction. In contrast, deglycosylation of ACE2 did not influence SARS-CoV-2 binding. Overall, ACE2 glycosylation does not significantly influence viral spike binding. We suggest that any role of glycosylation in the pathobiology of SARS-CoV-2 will lie beyond its immediate impact of receptor glycosylation on virus binding.