Transferrin receptor 1 (TfR) delivers iron across cellular membranes by shuttling the ion carrier protein transferrin. This ability to deliver large protein ligands inside cells is taken advantage of by pathogens to infiltrate human cells. Notably, the receptor's outermost ectodomain, the apical domain, is used as a point of attachment for several viruses including hemorrhagic arenaviruses. To better understand interactions with the receptor it would be advantageous to probe sequence determinants in the apical domain with viral spike proteins. Here, we carried out affinity maturation of our computationally designed apical domain from human TfR to identify underlying driving forces that lead to better binding. The improved variants were confirmed by in vitro surface plasmon resonance measurements with dissociation constants obtained in the lower nanomolar range. It was found that the strong binding affinities for the optimized variants matched the strength of interactions with the native receptor. The structure of the best variant was determined experimentally indicating that the conformational change in the hairpin binding motif at the protein-protein interface plays a crucial role. The experimental methodology can be straightforwardly applied to other arenavirus or pathogens that use the apical domain. It can further be useful to probe host-virus compatibility or therapeutic strategies based on the transferrin receptor decoys.

Chromatin in the nucleus undergoes mechanical stresses from different sources during the various stages of cell life. Here a trinucleosome array is used as the minimal model to study the mechanical response to applied stress at the molecular level. By using large-scale, all-atom steered-molecular dynamics simulations, we show that the largest part of mechanical stress in compression is accommodated by the DNA linkers joining pairs of nucleosomes, which store the elastic energy accumulated by the applied force. Different mechanical instabilities (Euler bending, Brazier kinking, twist-bending) can deform the DNA canonical structure, as a function of the increasing force load. An important role of the histone tails in assisting the DNA deformation is highlighted. The overall response of the smallest chromatin fragment to compressive stress leaves the nucleosome assembly with a substantial plastic deformation and localised defects, which can have a potential impact on DNA transcription, downstream signaling pathways, the regulation of gene expression, and DNA repair.

Approximately 70% of bacteriophage-encoded proteins are of unknown function. Elucidating these protein functions represents opportunities to discover new phage-host interactions and mechanisms by which the phages modulate host activities. Here, we describe a pipeline for prioritizing phage-encoded proteins for structural analysis and characterize the gp82 protein encoded by mycobacteriophage Phaedrus. Structural and solution studies of gp82 show it is a trimeric protein containing two domains. Co-precipitation studies with the host Mycobacterium smegmatis identified the ATPase MoxR as an interacting partner protein. Phaedrus gp82-MoxR interaction requires the presence of a loop sequence within gp82 that is highly exposed and disordered in the crystallographic structure. We show that Phaedrus gp82 overexpression in M. smegmatis retards the growth of M. smegmatis on solid medium, resulting in a small colony phenotype. Overexpression of gp82 containing a mutant disordered loop or the overexpression of MoxR both rescue this phenotype. Lastly, we show that recombinant gp82 reduces levels of MoxR-mediated ATPase activity in vitro that is required for its chaperone function, and that the disordered loop plays an important role in this phenotype. We conclude that Phaedrus gp82 binds to and reduces mycobacterial MoxR activity, leading to reduced function of host proteins that require MoxR chaperone activity for their normal activity.

Short tandem repeats (STRs) are consecutive repetitions of one to six nucleotide motifs. They are hypervariable due to the high prevalence of repeat unit insertions or deletions primarily caused by polymerase slippage during replication. Genetic variation at STRs has been shown to influence a range of traits in humans, including gene expression, cancer risk, and autism. Until recently STRs have been poorly studied since they pose significant challenges to bioinformatics analyses. Moreover, genome-wide analysis of STR variation in population-scale cohorts requires large amounts of data and computational resources. However, the recent advent of genome-wide analysis tools has resulted in multiple large genome-wide datasets of STR variation spanning nearly two million genomic loci in thousands of individuals from diverse populations. Here we present WebSTR, a database of genetic variation and other characteristics of genome-wide STRs across human populations. WebSTR is based on reference panels of more than 1.7 million human STRs created with state of the art repeat annotation methods and can easily be extended to include additional cohorts or species. It currently contains data based on STR genotypes for individuals from the 1000 Genomes Project, H3Africa, the Genotype-Tissue Expression (GTEx) Project and colorectal cancer patients from the TCGA dataset. WebSTR is implemented as a relational database with programmatic access available through an API and a web portal for browsing data. The web portal is publicly available at https://webstr.ucsd.edu.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-21 screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.

Characterizing the effects of mutations on stability is critical for understanding the function and evolution of proteins and improving their biophysical properties. High throughput folding and abundance assays have been successfully used to characterize missense mutations associated with reduced stability. However, screening for increased thermodynamic stability is more challenging since such mutations are rarer and their impact on assay readout is more subtle. Here, a multiplex assay for high throughput screening of protein folding was developed by combining deep mutational scanning, fluorescence-activated cell sorting, and deep sequencing. By analyzing a library of 2000 variants of Adenylate kinase we demonstrate that the readout of the method correlates with stability and that mutants with up to 13 °C increase in thermal melting temperature could be identified with low false positive rate. The discovery of many stabilizing mutations also enabled the analysis of general substitution patterns associated with increased stability in Adenylate kinase. This high throughput method to identify stabilizing mutations can be combined with functional screens to identify mutations that improve both stability and activity.

RNA tertiary structures from experiments or computational predictions often contain missing atoms, which prevent analyses requiring full atomic structures. Current programs for RNA reconstruction can be slow, inaccurate, and/or require specific atoms to be present in the input. We present Arena (Atomic Reconstruction of RNA), which reconstructs a full atomic RNA structure from residues that can have as few as one atom. Arena first fills in missing atoms and then iteratively refines their placement to reduce nonideal geometries. We benchmarked Arena on a dataset of 361 RNA structures, where Arena achieves high accuracy and speed compared to other structure reconstruction programs. For example, Arena was used to reconstruct full atomic structures from a single phosphorus atom per nucleotide to, on average, within 3.63 Å RMSD of the experimental structure, while virtually removing all clashes and running in <3 s, which is 353× and 46× faster than state-of-the-art programs PDBFixer and C2A, respectively. The Arena source code is available at https://github.com/pylelab/Arena and the webserver at https://zhanggroup.org/Arena/.

Immunoglobulin light chain amyloidosis (AL) is caused by the aberrant production of amyloidogenic light chains (LC) that accumulate as amyloid deposits in vital organs. Distinct LC sequences in each patient yield distinct amyloid structures. However different tissue microenvironments may also cause identical protein precursors to adopt distinct amyloid structures. To address the impact of the tissue environment on the structural polymorphism of amyloids, we extracted fibrils from the kidney of an AL patient (AL55) whose cardiac amyloid structure was previously determined by our group. Here we show that the 4.0 Å resolution cryo-EM structure of the renal fibril is virtually identical to that reported for the cardiac fibril. These results provide the first structural evidence that LC amyloids independently deposited in different organs of the same AL patient share a common fold.

The intramembrane protease γ-secretase activates important signaling molecules, such as Notch receptors. It is still unclear, however, how different elements within the primary structure of substrate transmembrane domains (TMDs) contribute to their cleavability. Using a newly developed yeast-based cleavage assay, we identified three crucial regions within the TMDs of the paralogs Notch1 and Notch3 by mutational and gain-of-function approaches. The AAAA or AGAV motifs within the N-terminal half of the TMDs were found to confer strong conformational flexibility to these TMD helices, as determined by mutagenesis coupled to deuterium/hydrogen exchange. Crucial amino acids within the C-terminal half may support substrate docking into the catalytic cleft of presenilin, the enzymatic subunit of γ-secretase. Further, residues close to the C-termini of the TMDs may stabilize a tripartite β-sheet in the substrate/enzyme complex. NMR structures reveal different extents of helix bending as well as an ability to adopt widely differing conformational substates, depending on the sequence of the N-terminal half. The difference in cleavability between Notch1 and Notch3 TMDs is jointly determined by the conformational repertoires of the TMD helices and the sequences of the C-terminal half, as suggested by mutagenesis and building molecular models. In sum, cleavability of a γ-secretase substrate is enabled by different functions of cooperating TMD regions, which deepens our mechanistic understanding of substrate/non-substrate discrimination in intramembrane proteolysis.

Enterococci are normal human commensals and major causes of hospital-acquired infections. Enterococcal infections can be difficult to treat because enterococci harbor intrinsic and acquired antibiotic resistance, such as resistance to cephalosporins. In Enterococcus faecalis, the transmembrane kinase IreK, a member of the bacterial PASTA kinase family, is essential for cephalosporin resistance. The activity of IreK is boosted by the cytoplasmic protein GpsB, which promotes IreK autophosphorylation and signaling to drive cephalosporin resistance. A previous phosphoproteomics study identified eight putative IreK-dependent phosphorylation sites on GpsB, but the functional importance of GpsB phosphorylation was unknown. Here we used genetic and biochemical approaches to define three sites of phosphorylation on GpsB that functionally impact IreK activity and cephalosporin resistance. Phosphorylation at two sites (S80 and T84) serves to impair the ability of GpsB to activate IreK in vivo, suggesting phosphorylation of these sites acts as a means of negative feedback for IreK. The third site of phosphorylation (T133) occurs in a segment of GpsB termed the C-terminal extension that is unique to enterococcal GpsB homologs. The C-terminal extension is highly mobile in solution, suggesting it is largely unstructured, and phosphorylation of T133 appears to enable efficient phosphorylation at S80 / T84. Overall our results are consistent with a model in which multisite phosphorylation of GpsB impairs its ability to activate IreK, thereby diminishing signal transduction through the IreK-dependent pathway and modulating phenotypic cephalosporin resistance.

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473-477, 499-502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, contains a single counterion of the protonated Schiff base, E108 in HeR from Thermoplasmatales archaeon SG8-52-1 (TaHeR). Upon light absorption, the M and O intermediates form in HeRs, as well as type-1 microbial rhodopsins, indicating that the proton transfer from the Schiff base leads to the activation of HeRs. The present flash photolysis study of TaHeR in the presence of a pH-sensitive dye showed that TaHeR contains a proton-accepting group (PAG) inside protein. Comprehensive mutation study of TaHeR found the E108D mutant abolishing the M formation, which is not only at pH 8, but also at pH 9 and 10. The lack of M observation does not originate from the short lifetime of the M intermediate in E108D, as FTIR spectroscopy revealed that a red-shifted K-like intermediate is long lived in E108D. It is likely that the K-like intermediate returns to the unphotolyzed state without internal proton transfer in E108D. E108 and D108 are the Schiff base counterions of the wild-type and E108D mutant TaHeR, respectively, whereas small difference in length determine internal proton transfer reaction from the Schiff base. Based on the present finding, we propose that the internal water cluster (four water molecules) constitutes PAG in the M intermediate of TaHeR. In the wild type TaHeR, a protonated water cluster is stabilized by forming a salt bridge with E108. In contrast, slightly shortened counterion (D108) cannot stabilize the protonated water cluster in E108D, and thus impairs internal proton transfer from the Schiff base.

Molecular recognition features (MoRFs) are a commonly occurring type of intrinsically disordered regions (IDRs) that undergo disorder-to-order transition upon binding to partner molecules. We focus on recently characterized and functionally important membrane-binding MoRFs (MemMoRFs). Motivated by the lack of computational tools that predict MemMoRFs, we use a dataset of experimentally annotated MemMoRFs to conceptualize, design, evaluate and release an accurate sequence-based predictor. We rely on state-of-the-art tools that predict residues that possess key characteristics of MemMoRFs, such as intrinsic disorder, disorder-to-order transition and lipid-binding. We identify and combine results from three tools that include flDPnn for the disorder prediction, DisoLipPred for the prediction of disordered lipid-binding regions, and MoRFCHiBiLight for the prediction of disorder-to-order transitioning protein binding regions. Our empirical analysis demonstrates that combining results produced by these three methods generates accurate predictions of MemMoRFs. We also show that use of a smoothing operator produces predictions that closely mimic the number and sizes of the native MemMoRF regions. The resulting CoMemMoRFPred method is available as an easy-to-use webserver at http://biomine.cs.vcu.edu/servers/CoMemMoRFPred/. This tool will aid future studies of MemMoRFs in the context of exploring their abundance, cellular functions, and roles in pathologic phenomena.

Algae encode up to five different types of cryptochrome photoreceptors. So far, relatively little is known about the biological functions of the DASH (Drosophila, Arabidopsis, Synechocystis and Homo)-type cryptochromes. The green alga Chlamydomonas reinhardtii encodes two of them. CRY-DASH1 also called DCRY1 has its maximal absorption peak in the UV-A range. It is localized in the chloroplast and plays an important role in balancing the photosynthetic machinery. Here, we performed a comparative analysis of chloroplast proteins from wild type and a knockout mutant of CRY-DASH1 named cry-dash1mut, using label-free quantitative proteomics as well as immunoblotting. Our results show upregulation of enzymes involved in specific pathways in the mutant including key enzymes of chlorophyll and carotenoid biosynthesis consistent with increased levels of photosynthetic pigments in cry-dash1mut. There is also an increase in certain redox as well as photosystem I and II proteins, including D1. Strikingly, CRY-DASH1 is coregulated in a D1 deletion mutant, where its amount is increased. In contrast, key proteins of the central carbon metabolism, including glycolysis/gluconeogenesis, dark fermentation and the oxidative pentose phosphate pathway are downregulated in cry-dash1mut. Similarly, enzymes of histidine biosynthesis are downregulated in cry-dash1mut leading to a reduction in the amount of free histidine. Yet, transcripts encoding for several of these proteins are at a similar level in the wild type and cry-dash1mut or even opposite. We show that CRY-DASH1 can bind to RNA, taking the psbA RNA encoding D1 as target. These data suggest that CRY-DASH1 regulates plastidial metabolic pathways at the posttranscriptional level.

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40 °C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties.

The APOBEC3 family of human cytidine deaminases is involved in various cellular processes, including the innate and acquired immune system, mostly through inducing C-to-U in single-stranded DNA and/or RNA mutations. Although recent studies have examined RNA editing by APOBEC3A (A3A), its intracellular target specificity are not fully characterized. To address this gap, we performed in-depth analysis of cellular RNA editing using our recently developed sensitive cell-based fluorescence assay. Our findings demonstrate that A3A and an A3A-loop1-containing APOBEC3B (A3B) chimera are capable of RNA editing. We observed that A3A prefers to edit specific RNA substrates which are not efficiently deaminated by other APOBEC members. The editing efficiency of A3A is influenced by the RNA sequence contexts and distinct stem-loop secondary structures. Based on the identified RNA specificity features, we predicted potential A3A-editing targets in the encoding region of cellular mRNAs and discovered novel RNA transcripts that are extensively edited by A3A. Furthermore, we found a trend of increased synonymous mutations at the sites for more efficient A3A-editing, indicating evolutionary adaptation to the higher editing rate by A3A. Our results shed light on the intracellular RNA editing properties of A3A and provide insights into new RNA targets and potential impact of A3A-mediated RNA editing.

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Viruses are believed to be the obligate intracellular parasites that only carry genes essential for infecting and hijacking the host cell machinery. However, a recently discovered group of viruses belonging to the phylum nucleocytovirocota, also known as the nucleo-cytoplasmic large DNA viruses (NCLDVs), possess a number of genes that code for proteins predicted to be involved in metabolism, and DNA replication, and repair. In the present study, first, using proteomics of viral particles, we show that several proteins required for the completion of the DNA base excision repair (BER) pathway are packaged within the virions of Mimivirus as well as related viruses while they are absent from the virions of Marseillevirus and Kurlavirus that are NCLDVs with smaller genomes. We have thoroughly characterized three putative base excision repair enzymes from Mimivirus, a prototype NCLDV and successfully reconstituted the BER pathway using the purified recombinant proteins. The mimiviral uracil-DNA glycosylase (mvUDG) excises uracil from both ssDNA and dsDNA, a novel finding contrary to earlier studies. The putative AP-endonuclease (mvAPE) specifically cleaves at the abasic site created by the glycosylase while also exhibiting the 3'-5' exonuclease activity. The Mimivirus polymerase X protein (mvPolX) can bind to gapped DNA substrates and perform single nucleotide gap-filling followed by downstream strand displacement. Furthermore, we show that when reconstituted in vitro, mvUDG, mvAPE, and mvPolX function cohesively to repair a uracil-containing DNA predominantly by long patch BER and together, may participate in the BER pathway during the early phase of Mimivirus life-cycle.

Identification of key sequence, expression and function related features of nucleic acid-sensing host proteins is of fundamental importance to understand the dynamics of pathogen-specific host responses. To meet this objective, we considered toll-like receptors (TLRs), a representative class of membrane-bound sensor proteins, from 17 vertebrate species covering mammals, birds, reptiles, amphibians, and fishes in this comparative study. We identified the molecular signatures of host TLRs that are responsible for sensing pathogen nucleic acids or other pathogen-associated molecular patterns (PAMPs), and potentially play important roles in host defence mechanism. Interestingly, our findings reveal that such host-specific features are directly related to the strand (single or double) specificity of nucleic acid from pathogens. However, during host-pathogen interactions, such features were unable to explain the pathogenic PAMP (i.e., DNA, RNA or other) selectivity, suggesting a more complex mechanism. Using these features, we developed a number of machine learning models, of which Random Forest achieved a high performance (94.57% accuracy) to predict strand specificity of TLRs from protein-derived features. We applied the trained model to propose strand specificity of some previously uncharacterized distinct fish-specific novel TLRs (TLR18, TLR23, TLR24, TLR25, TLR27).