Protein oligomerization occurs frequently both in vitro and in vivo, with specific functionalities associated with different oligomeric states. The YqiC protein from Salmonella Typhimurium forms a homotrimer through its C-terminal coiled-coil domain, and the protein is closely linked to the colonization and invasion of the bacteria to the host cells. To elucidate the importance of the oligomeric state of YqiC in vivo and its relation with bacterial infection, we mutated crucial residues in YqiC's coiled-coil region and confirmed the loss of trimer formation using chemical crosslinking and Size Exclusion Chromatography coupled with Multiple Angle Light Scattering (SEC-MALS) techniques. The yqiC-knockout strain complemented with mutant YqiC showed significantly reduced colonization and invasion of Salmonella to host cells, demonstrating the critical role of YqiC oligomerization in bacterial pathogenesis. Furthermore, we conducted a protein-protein interaction study of YqiC using a pulled-down assay coupled with mass spectrometry analysis to investigate the protein's role in bacterial virulence. The results reveal that YqiC interacts with subunits of Complex II of the electron transport chain (SdhA and SdhB) and the β-subunit of F0 F1 -ATP synthase. These interactions suggest that YqiC may modulate the energy production of Salmonella and subsequently affect the assembly of crucial virulence factors, such as flagella. Overall, our findings provide new insights into the molecular mechanisms of YqiC's role in S. Typhimurium pathogenesis and suggest potential therapeutic targets for bacterial infections. This article is protected by copyright. All rights reserved.

Flavodoxins (Flds) mediate the flux of electrons between oxidoreductases in diverse metabolic pathways. To investigate whether Flds can support electron transfer to a sulfite reductase (SIR) that evolved to couple with a ferredoxin, we evaluated the ability of Flds to transfer electrons from a ferredoxin-NADP reductase (FNR) to a ferredoxin-dependent SIR using growth complementation of an Escherichia coli strain with a sulfur metabolism defect. We show that Flds from cyanobacteria complement this growth defect when coexpressed with an FNR and an SIR that evolved to couple with a plant ferredoxin. When we evaluated the effect of peptide insertion on Fld-mediated electron transfer, we observed a sensitivity to insertions within regions predicted to be proximal to the cofactor and partner binding sites, while a high insertion tolerance was detected within loops distal from the cofactor and within regions of helices and sheets that are proximal to those loops. Bioinformatic analysis showed that natural Fld sequence variability predicts a large fraction of the motifs that tolerate insertion of the octapeptide SGRPGSLS. These results represent the first evidence that Flds can support electron transfer to assimilatory SIRs, and they suggest that the pattern of insertion tolerance is influenced by interactions with oxidoreductase partners. This article is protected by copyright. All rights reserved.

Characterizing structural ensembles of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins is essential for studying structure-function relationships. Due to different neutron scattering lengths of hydrogen and deuterium, selective labeling and contrast matching in small-angle neutron scattering (SANS) becomes an effective tool to study dynamic structures of disordered systems. However, experimental timescales typically capture measurements averaged over multiple conformations, leaving complex SANS data for disentanglement. We hereby demonstrate an integrated method to elucidate the structural ensemble of a complex formed by two IDRs. We use data from both full contrast and contrast matching with residue-specific deuterium labeling SANS experiments, microsecond all-atom molecular dynamics (MD) simulations with four molecular mechanics force fields, and an autoencoder-based deep learning (DL) algorithm. From our combined approach, we show that selective deuteration provides additional information that helps characterize structural ensembles. We find that among the four force fields, a99SB-disp and CHARMM36m show the strongest agreement with SANS and NMR experiments. In addition, our DL algorithm not only complements conventional structural analysis methods but also successfully differentiates NMR and MD structures which are indistinguishable on the free energy surface. Lastly, we present an ensemble that describes experimental SANS and NMR data better than MD ensembles generated by one single force field and reveal three clusters of distinct conformations. Our results demonstrate a new integrated approach for characterizing structural ensembles of IDPs. This article is protected by copyright. All rights reserved.

Serum autoantibody profiles are unique to individuals and reflect the level and history of autoimmunity and tumor immunity. The identification of autoantibody biomarkers is critical for the development of immune monitoring systems for immune-related disorders. Here, we present a practical method for large-scale autoantibody discovery using total cellular proteins from cultured mammalian cells. We found that nucleic acid-free and fully denatured water-soluble total cellular proteins from mammalian cells were superior, allowing precise separation by reversed-phase HPLC after preparing a large set of homogeneous total cellular proteins. After separating the proteins based on hydrophobicity, the fractionated samples were subjected to molecular mass analysis using conventional SDS-PAGE. The resulting two-dimensional gel electrophoresis was successfully employed for immune blotting and LC-MS/MS analysis. All procedures, including TRIzol-based total cellular protein extraction, solubilization of denatured proteins, reversed-phase HPLC separation, and SDS-PAGE, were highly reproducible and easily scalable. We propose this novel two-dimensional gel electrophoresis system as an alternative proteomics-based methodology suitable for large-scale autoantibody discovery. This article is protected by copyright. All rights reserved.

Targeted intracellular delivery via receptor-mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane lysis tightly restricted to endosomal membranes upon internalization to avoid general membrane insertion and lysis. Here we describe the design of small monomeric proteins with buried histidine containing pH-responsive hydrogen bond networks and membrane permeating amphipathic helices. Of 30 designs that were experimentally tested, all expressed in E. coli, 13 were monomeric with the expected secondary structure, and 4 designs disrupted artificial liposomes in a pH-dependent manner. Mutational analysis showed that the buried histidine hydrogen bond networks mediate pH-responsiveness and control lysis of model membranes within a very narrow range of pH (6.0 - 5.5) with almost no lysis occurring at neutral pH. These tightly controlled lytic monomers could help mediate endosomal escape in designed targeted delivery platforms. This article is protected by copyright. All rights reserved.

The catalytic activity of human glutathione S-transferase A1-1 (hGSTA1-1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C-terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand-free state of the hGSTA1-1 homodimer has not been resolved. Here, we used a combination of EPR distance measurements and weighted ensemble simulations to characterize the conformational ensemble of the ligand-free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand-free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. Weighted ensemble simulations generated unbiased pathways for the seconds-timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand-free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1-1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1-1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with weighted ensemble rare-events sampling strategy to gain mechanistic information on protein function at the atomic level. This article is protected by copyright. All rights reserved.

Ubiquitin (Ub)-conjugating enzymes (E2s) transfer activated Ub from Ub-activating enzymes (E1s) to substrates and are associated with various cancers and neurological disorders. In this study, the unique property of E2-binding and auto-ubiquitination of artificial RING fingers (ARFs) were demonstrated in ubiquitination assays. Circular dichroism spectra indicated the characteristic structures of ARFs. Point mutations of 31 PKLTC35 in ARF by tryptophan (Trp) resulted in dramatic changes in E2 specificity and the type of Ub chain elongation of mono- and polyubiquitination. The Trp residue was a cue that changed the ubiquitination activity of ARF via E2-binding. Furthermore, the ARF mutants interacted with all 11 E2s, and then promoted auto-ubiquitination. Thus, the use of the ARF mutants allowed specific detection of E2 activities during ubiquitination. The present study opens up a new avenue for researching E2 activities related to the fatal diseases. This article is protected by copyright. All rights reserved.

Eukaryotic proteins often feature modular domain structures comprising of globular domains that are connected by linker regions and intrinsically disordered regions that may contain important functional motifs. The intramolecular interactions of globular domains and nonglobular regions can play critical roles in different aspects of protein function. However, studying these interactions and their regulatory roles can be challenging due to the flexibility of nonglobular regions, the long insertions separating interacting modules, and the transient nature of some interactions. Obtaining the experimental structures of multiple domains and functional regions is more difficult than determining structures of individual globular domains. High-quality structural models generated by AlphaFold offer a unique opportunity to study intramolecular interactions in eukaryotic proteins. In this study, we systematically explored intramolecular interactions between human protein kinase domains and potential regulatory regions, including globular domains, N- and C-terminal tails, long insertions, and distal nonglobular regions. Our analysis identified intramolecular interactions between human kinase domains and 35 different types of globular domains, exhibiting a variety of interaction modes that could contribute to orthosteric or allosteric regulation of kinase activity. We also identified prevalent interactions between human kinase domains and their flanking regions (N- and C-terminal tails). These interactions exhibit group-specific characteristics and can vary within each specific kinase group. Although long-range interactions between kinase domains and nonglobular regions are relatively rare, structural details of these interactions offer new insights into the regulation mechanisms of several kinases, such as HASPIN, MAPK7, MAPK15 and SIK1B. This article is protected by copyright. All rights reserved.

The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore, which gives insight into the inhibition mechanism of HMA for SARS-CoV-2 E. This article is protected by copyright. All rights reserved.

Cyclization can stabilize the structure of proteins, as previously demonstrated in single-domain proteins. Although Lys48-linked polyubiquitin, a multi-domain protein, is also known to be cyclized in human cells, the structural effects of cyclization remain unclear. Here, we examined the impact of cyclization on the structural stability and dynamics of cyclic Lys48-linked diubiquitin (Ub2 ). As expected, cyclization increased the thermal stability of Ub2 and its resistance to proteolytic digestion, indicating that cyclization stabilized the structure of Ub2 . Furthermore, cyclization repressed the interdomain motion in Ub2 , but cyclic Ub2 still exhibited microsecond conformational exchange in NMR relaxation dispersion experiments. A series of long coarse-grained (CG) MD simulations visualized how cyclization slowed down the intrinsic nanosecond open-closed domain motion of Ub2 to microseconds. Thus, CG-MD analysis helped to explain the unexpected NMR relaxation results, thereby facilitating characterization of the structural stabilization of cyclic Ub2 . This article is protected by copyright. All rights reserved.

In structural biology, peptide bonds, fundamental linkages between hundreds of amino acids, of which a protein molecule is composed, have been commonly treated as a plane structure just as Linus Pauling et al. proposed. In this paper, a site-specific peptide bond relaxation mechanism by deuterons whose localization has been suggested by neutron crystallography is proposed. Such deuteron was observed as an arm of neutron scattering length density protruding from the carbonyl oxygen atoms in the main chain in the omit map drawn by neutron crystallography of human lysozyme. Our comprehensive study using X-ray and neutron diffraction and 15 N chemical shifts of individual amide nitrogen atoms within the same peptide bond strongly suggests the relaxation of the electronic resonance structure because of site-specific modulation by protons/deuterons localized on the electron orbital of the carbonyl oxygen. All experimental data used in this examination were obtained at room temperature, which is preferable for enzymatic activity. Such a close interaction between the electron resonance structure of a peptide bond and the exchangeable protons/deuterons well agreed with that observed in an intermediate state in an amide hydrolytic reaction simulated by the ab-initio calculation including water molecules. This article is protected by copyright. All rights reserved.

Alcohols and urea are widely used as effective protein denaturants. Among monohydric alcohols, 2,2,2-trifluoroethanol (TFE) has large co-solvent effects as a helix stabilizer in proteins. In contrast, urea efficiently denatures ordered native structures, including helices, into coils. These opposing co-solvent effects of TFE and urea are well known, even though both preferentially bind to proteins; however, the underlying molecular mechanism remains controversial. Co-solvent-dependent relative stability between native and denatured states is rigorously related to the difference in preferential binding parameters (PBPs) between these states. In this study, GCN4-p1 with two-stranded coiled coil helices was employed as a model protein, and molecular dynamics simulations for the helix dimer and isolated coil were conducted in aqueous solutions with 2 M TFE and urea. As 2 M co-solvent aqueous solutions did not exhibit clustering of co-solvent molecules, we were able to directly investigate the molecular origin of the excess PBP without considering the enhancement effect of PBPs arising from the concentration fluctuations. The calculated excess PBPs of TFE for the helices and those of urea for the coils were consistent with experimentally observed stabilization of helix by TFE and that of coil by urea. The former was caused by electrostatic interactions between TFE and side chains of the helices, while the latter was attributed to both electrostatic and dispersion interactions between urea and the main chains. Unexpectedly, reverse-micelle-like orientations of TFE molecules strengthened the electrostatic interactions between TFE and the side chains, resulting in strengthening of TFE solvation. This article is protected by copyright. All rights reserved.

RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4 and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS. This article is protected by copyright. All rights reserved.

Weak molecular interactions play an important role in protein structure and function. Computational tools that identify weak molecular interactions are, therefore, valuable for the study of proteins. Here, we present AQcalc, a web server (https://aqcalcbiocomputing.com/) that can be used to identify anion-quadrupole (AQ) interactions, which are weak interactions involving aromatic residue (Trp, Tyr, and Phe) ring edges and anions (Asp, Glu, and phosphate ion) both within proteins and at their interfaces (protein-protein, protein-nucleic acids, and protein-lipid bilayer). AQcalc identifies AQ interactions as well as clusters involving AQ, cation-π, and salt bridges, among others. Utilizing AQcalc we analyzed weak interactions in protein models, even in the absence of experimental structures, to understand the contributions of weak interactions to deleterious structural changes, including those associated with oncogenic and germline disease variants. We identified several deleterious variants with disrupted AQ interactions (comparable in frequency to cation-π disruptions). Amyloid fibrils utilize AQ to bury anions at frequencies that far exceed those observed for globular proteins. AQ interactions were detected three and five times more frequently than the hydrogen-bonded AQ (HBAQ) in fibril structures and protein-lipid bilayer interfaces, respectively. By contrast, AQ and HBAQ interactions were detected with similar frequencies in globular proteins. Collectively, these findings suggest AQcalc will be effective in facilitating fine structural analysis. As other web utilities designed to identify protein residue interaction networks do not report AQ interactions, wide use of AQcalc will enrich our understanding of residue interaction networks and facilitate hypothesis testing by identifying and experimentally characterizing these comparably weak but important interactions. This article is protected by copyright. All rights reserved.

The pupal cuticle protein from Aedes aegypti (AaPC) inhibits dengue virus (DENV) infection; however, the underlying mechanism of this inhibition remains unknown. Here, we report that AaPC is an intrinsically disordered protein and interacts with domain I/II of the DENV envelope protein via residues Asp59, Asp61, Glu71, Asp73, Ser75 and Asp80. AaPC can directly bind to and cause the aggregation of DENV, which in turn blocks virus infection during the virus-cell fusion stage. AaPC may also influence viral recognition and attachment by interacting with human immune receptors DC-SIGN and CD4. These findings enhance our understanding of the role of AaPC in mitigating viral infection and suggest that AaPC is a potential target for developing inhibitors or antibodies to control dengue virus infection. This article is protected by copyright. All rights reserved.

Fungal infections have become a significant global health issue, affecting millions worldwide. Antifungal peptides (AFPs) have emerged as a promising alternative to conventional antifungal drugs due to their low toxicity and low propensity for inducing resistance. In this study, we developed a deep learning-based framework called DeepAFP to efficiently identify AFPs. DeepAFP fully leverages and mines composition information, evolutionary information, and physicochemical properties of peptides by employing combined kernels from multiple branches of convolutional neural network (CNN) with bi-directional long short-term memory (BiLSTM) layers. In addition, DeepAFP integrates a transfer learning strategy to obtain efficient representations of peptides for improving model performance. DeepAFP demonstrates strong predictive ability on carefully curated datasets, yielding an accuracy of 93.29% and an F1-score of 93.45% on the DeepAFP-Main dataset. The experimental results show that DeepAFP outperforms existing AFP prediction tools, achieving state-of-the-art performance. Finally, we provide a downloadable AFP prediction tool to meet the demands of large-scale prediction and facilitate the usage of our framework by the public or other researchers. Our framework can accurately identify AFPs in a short time without requiring significant human and material resources, and hence can accelerate the development of AFPs as well as contribute to the treatment of fungal infections. Furthermore, our method can provide new perspectives for other biological sequence analysis tasks. This article is protected by copyright. All rights reserved.

Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but a mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and X-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 Å and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates. This article is protected by copyright. All rights reserved.