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1. Intrinsically disordered electronegative clusters improve stability and binding specificity of RNA-binding proteins

   Zaharias, S. ; Zhang, Z. ; Davis, K. ; Fargason, T. ; Cashman, D. ; Yu, T. ; Zhang, J. ( July 2021 , Journal of biological chemistry)

   Musier-Forsyth, Karin (Ed.)

   RNA-binding proteins play crucial roles in various cellular functions, and contain abundant disordered protein regions. The disordered regions in RNA-binding proteins are rich in repetitive sequences, such as poly-K/R, poly-N/Q, poly-A, and poly-G residues. Our bioinformatic analysis identified a largely neglected repetitive sequence family we define as electronegative clusters (ENCs) that contain acidic residues and/or phosphorylation sites. The abundance and length of ENCs exceed other known repetitive sequences. Despite their abundance, the functions of ENCs in RNA-binding proteins are still elusive. To investigate the impacts of ENCs on protein stability, RNA-binding affinity, and specificity, we selected one RNA-binding protein, the ribosomal biogenesis factor 15 (Nop15) as a model. We found that the Nop15 ENC increases protein stability and inhibits nonspecific RNA binding, but minimally interferes with specific RNA binding. To investigate the effect of ENCs on sequence specificity of RNA binding, we grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3 (SRSF3). Using RNA Bind-n-Seq, we found that the engineered ENC inhibits disparate RNA motifs differently, instead of weakening all RNA motifs to the same extent. The motif site directly involved in electrostatic interaction is more susceptible to the ENC inhibition. These results suggest that one of functions of ENCs is to regulate RNA binding via electrostatic interaction. This is consistent with our finding that ENCs are also overrepresented in DNA-binding proteins, while underrepresented in halophiles, in which nonspecific nucleic acid binding is inhibited by high concentrations of salts. 

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2. Are granulins copper sequestering proteins?

   https://doi.org/10.1002/prot.26031  

   Bhopatkar, Anukool A. ; Rangachari, Vijayaraghavan ( December 2020 , Proteins: Structure, Function, and Bioinformatics)

   Granulins (GRN 1‐7) are short (~6 kDa), cysteine‐rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These peptides, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Previously we showed that GRN‐3 and GRN‐5 are fully disordered in the reduced form implicating redox sensitive attributes to the proteins. Redox‐based modulations are often carried out by metalloproteins in mitigating oxidative stress and maintaining metal‐homeostasis within cells. To probe whether GRNs play a role in metal sequestration, we tested the metal binding propensity of the reduced forms of GRNs −3 and − 5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu and no other divalent metal cations, with a greater specificity for Cu(I). Binding of Cu did not result in a disorder‐to‐order structural transition but partly triggered the multimerization of GRNs via uncoordinated cystines at both pH conditions. Overall, the results indicate that GRNs −3 and − 5 have surprisingly strong affinity for Cu in the pM range, comparable to other known copper sequestering proteins. The results also hint at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu‐induced ROS cytotoxicity in cells. Together, this report uncovers metal‐coordinating property of GRNs for the first time, which may have profound significance in their structure and pathophysiological functions.

    

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3. Bridging DNA contacts allow Dps from E. coli to condense DNA

   https://doi.org/10.1093/nar/gkae223  

   Shahu, Sneha ; Vtyurina, Natalia ; Das, Moumita ; Meyer, Anne S. ; Ganji, Mahipal ; Abbondanzieri, Elio A. ( April 2024 , Nucleic Acids Research)

   The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps–DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

    

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4. Directed Evolution of a Picomolar Affinity High Specificity Antibody Targeting Phosphorylated Tau

   https://doi.org/10.1074/jbc.RA118.003557  

   Li, Dan ; Wang, Lei ; Maziuk, Brandon F. ; Yao, Xudong ; Wolozin, Benjamin ; Cho, Yong Ku ( June 2018 , Journal of Biological Chemistry)

   Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTM-targeting antibodies have reported frequent non-specific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phospho-threonine 231 (pT231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinity-matured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved > 20-fold over that of the wild-type antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity towards the non-phosphorylated target site, but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phospho-tau antibody. In cell- and tissue-imaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTM-specific antibodies, and highlight the importance of assessing the specificity in the antibody engineering process. 

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5. Coacervation of poly-electrolytes in the presence of lipid bilayers: mutual alteration of structure and morphology

   https://doi.org/10.1039/D2SC02013K  

   Mondal, Sayantan ; Cui, Qiang ( July 2022 , Chemical Science)

   Many intrinsically disordered peptides have been shown to undergo liquid–liquid phase separation and form complex coacervates, which play various regulatory roles in the cell. Recent experimental studies found that such phase separation processes may also occur at the lipid membrane surface and help organize biomolecules during signaling events; in some cases, phase separation of proteins at the membrane surface was also observed to lead to significant remodeling of the membrane morphology. The molecular mechanisms that govern the interactions between complex coacervates and lipid membranes and the impacts of such interactions on their structure and morphology, however, remain unclear. Here we study the coacervation of poly-glutamate (E 30 ) and poly-lysine (K 30 ) in the presence of lipid bilayers of different compositions. We carry out explicit-solvent coarse-grained molecular dynamics simulations by using the MARTINI (v3.0) force-field. We find that more than 20% anionic lipids are required for the coacervate to form stable contact with the bilayer. Upon wetting, the coacervate induces negative curvature to the bilayer and facilitates local lipid demixing, without any peptide insertion. The magnitude of negative curvature, extent of lipid demixing, and asphericity of the coacervate increase with the concentration of anionic lipids. Overall, we observe a decrease in the number of contacts among the polyelectrolytes as the droplet spreads over the bilayer. Therefore, unlike previous suggestions, interactions among polyelectrolytes do not constitute a driving force for the membrane bending upon wetting by the coacervate. Rather, analysis of interaction energy components suggests that bending of the membrane is favored by enhanced interactions between polyelectrolytes with lipids as well as with counterions. Kinetic studies reveal that, at the studied polyelectrolyte concentrations, the coacervate formation precedes bilayer wetting. 

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