Molecular Biophysics
Intrinsically disordered regions that drive phase separation form a robustly distinct protein class
Protein phase separation is thought to be a primary driving force for the formation of membrane-less organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. Among phase-separating (PS) proteins, many have intrinsically disordered regions (IDRs) that are needed for phase separation to occur. Accurate identification of IDRs that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation.Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor
In Helicobacter pylori, the nickel-responsive NikR transcription factor plays a key role in regulating intracellular nickel concentrations, which is an essential process for survival of this pathogen in the acidic human stomach. Nickel binding to H. pylori NikR (HpNikR) allosterically activates DNA binding to target promoters encoding genes involved in nickel homeostasis and acid adaptation, to either activate or repress their transcription. We previously showed that HpNikR adopts an equilibrium between an open conformation and DNA-binding competent cis and trans states.Surface electrostatics dictate RNA-binding protein CAPRIN1 condensate concentration and hydrodynamic properties
Biomolecular condensates concentrate proteins, nucleic acids, and small molecules and play an essential role in many biological processes. Their formation is tuned by a balance between energetically favorable and unfavorable contacts, with charge–charge interactions playing a central role in some systems. The positively charged intrinsically disordered carboxy-terminal region of the RNA-binding protein CAPRIN1 is one such example, phase separating upon addition of negatively charged ATP or high concentrations of sodium chloride (NaCl).Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A
PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to β-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca2+ sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive.Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11
The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat–containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44–Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11–WDR44 complex.Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy
Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin.Might nontransferrin-bound iron in blood plasma and sera be a nonproteinaceous high-molecular-mass FeIII aggregate?
The HFE (Homeostatic Fe regulator) gene is commonly mutated in hereditary hemochromatosis. Blood of (HFE)(−/−) mice and of humans with hemochromatosis contains toxic nontransferrin-bound iron (NTBI) which accumulates in organs. However, the chemical composition of NTBI is uncertain. To investigate, HFE(−/−) mice were fed iron-deficient diets supplemented with increasing amounts of iron, with the expectation that NTBI levels would increase. Blood plasma was filtered to obtain retentate and flow-through solution fractions.Mechanistic insights into poly(C)-binding protein hnRNP K resolving i-motif DNA secondary structures
I-motifs are four-strand noncanonical secondary structures formed by cytosine (C)-rich sequences in living cells. The structural dynamics of i-motifs play essential roles in many cellular processes, such as telomerase inhibition, DNA replication, and transcriptional regulation. In cells, the structural dynamics of the i-motif can be modulated by the interaction of poly(C)-binding proteins (PCBPs), and the interaction is closely related to human health, through modulating the transcription of oncogenes and telomere stability.A lysine residue from an extracellular turret switches the ion preference in a Cav3 T-Type channel from calcium to sodium ions
Cav3 T-type calcium channels from great pond snail Lymnaea stagnalis have a selectivity-filter ring of five acidic residues, EE(D)DD. Splice variants with exons 12b or 12a spanning the extracellular loop between the outer helix IIS5 and membrane-descending pore helix IIP1 (IIS5-P1) in Domain II of the pore module possess calcium selectivity or dominant sodium permeability, respectively. Here, we use AlphaFold2 neural network software to predict that a lysine residue in exon 12a is salt-bridged to the aspartate residue immediately C terminal to the second-domain glutamate in the selectivity filter.DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein
Neutrophil extracellular traps (NETs) are produced through ejection of genomic DNA by neutrophils into extracellular space and serve as a weapon to fight against pathogens. Neutrophil elastase, a serine protease loaded on NETs, attacks and kills pathogens, while extracellular high-mobility-group-box-1 (HMGB1) protein serves as a danger signal to other cells. How the action of these factors is coordinated as part of the innate immune response is not fully understood. In this article, using biochemical and biophysical approaches, we demonstrate that DNA mediates specific proteolysis of HMGB1 by neutrophil elastase and that the proteolytic processing remarkably enhances binding activities of extracellular HMGB1.The ganglioside GM1a functions as a coreceptor/attachment factor for dengue virus during infection
Dengue virus (DENV) is a flavivirus causing an estimated 390 million infections per year around the world. Despite the immense global health and economic impact of this virus, its true receptor(s) for internalization into live cells has not yet been identified, and no successful antivirals or treatments have been isolated to this date. This study aims to improve our understanding of virus entry routes by exploring the sialic acid–based cell surface molecule GM1a and its role in DENV infection. We studied the interaction of the virus with GM1a using fluorescence correlation spectroscopy, fluorescence crosscorrelation spectroscopy, imaging fluorescence correlation spectroscopy, amide hydrogen/deuterium exchange mass spectrometry, and isothermal titration calorimetry.Early stage β-amyloid-membrane interactions modulate lipid dynamics and influence structural interfaces and fibrillation
Molecular interactions between β-amyloid (Aβ) peptide and membranes contribute to the neuronal toxicity of Aβ and the pathology of Alzheimer’s disease. Neuronal plasma membranes serve as biologically relevant environments for the Aβ aggregation process as well as affect the structural polymorphisms of Aβ aggregates. However, the nature of these interactions is unknown. Here, we utilized solid-state NMR spectroscopy to explore the site-specific interactions between Aβ peptides and lipids in synaptic plasma membranes at the membrane-associated nucleation stage.The N-terminal disease–associated R5L Tau mutation increases microtubule shrinkage rate due to disruption of microtubule-bound Tau patches
Regulation of the neuronal microtubule cytoskeleton is achieved through the coordination of microtubule-associated proteins (MAPs). MAP-Tau, the most abundant MAP in the axon, functions to modulate motor motility, participate in signaling cascades, as well as directly mediate microtubule dynamics. Tau misregulation is associated with a class of neurodegenerative diseases, known as tauopathies, including progressive supranuclear palsy, Pick's disease, and Alzheimer's disease. Many disease-associated mutations in Tau are found in the C-terminal microtubule-binding domain.N-terminal acetylation and arginylation of actin determines the architecture and assembly rate of linear and branched actin networks
The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis, and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and cofilin, recent work also implicates posttranslational acetylation or arginylation of the actin N terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood.The heme-regulated inhibitor kinase requires dimerization for heme-sensing activity
The heme-regulated inhibitor (HRI) is a heme-sensing kinase that regulates mRNA translation in erythroid cells. In heme deficiency, HRI is activated to phosphorylate eukaryotic initiation factor 2α and halt production of globins, thus avoiding accumulation of heme-free globin chains. HRI is inhibited by heme via binding to one or two heme-binding domains within the HRI N-terminal and kinase domains. HRI has recently been found to inhibit fetal hemoglobin (HbF) production in adult erythroid cells.Densely methylated DNA traps Methyl-CpG–binding domain protein 2 but permits free diffusion by Methyl-CpG–binding domain protein 3
The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands).The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction
The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered.Phenol sensing in nature is modulated via a conformational switch governed by dynamic allostery
The NtrC family of proteins senses external stimuli and accordingly stimulates stress and virulence pathways via activation of associated σ54-dependent RNA polymerases. However, the structural determinants that mediate this activation are not well understood. Here, we establish using computational, structural, biochemical, and biophysical studies that MopR, an NtrC protein, harbors a dynamic bidirectional electrostatic network that connects the phenol pocket to two distal regions, namely the “G-hinge” and the “allosteric linker.” While the G-hinge influences the entry of phenol into the pocket, the allosteric linker passes the signal to the downstream ATPase domain.Specific phosphorylation of microtubule-associated protein 2c by extracellular signal–regulated kinase reduces interactions at its Pro-rich regions
Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal–regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies.Autoinhibition of the GEF activity of cytoskeletal regulatory protein Trio is disrupted in neurodevelopmental disorder-related genetic variants
TRIO encodes a cytoskeletal regulatory protein with three catalytic domains—two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain—as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders.Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin
Sodium-pumping rhodopsins (NaRs) are membrane transporters that utilize light energy to pump Na+ across the cellular membrane. Within the NaRs, the retinal Schiff base chromophore absorbs light, and a photochemically induced transient state, referred to as the “O intermediate”, performs both the uptake and release of Na+. However, the structure of the O intermediate remains unclear. Here, we used time-resolved cryo-Raman spectroscopy under preresonance conditions to study the structure of the retinal chromophore in the O intermediate of an NaR from the bacterium Indibacter alkaliphilus.The nematode serotonin-gated chloride channel MOD-1: A novel target for anthelmintic therapy
Anthelmintics are used to treat human and veterinary parasitic diseases and to reduce crop and livestock production loss associated with parasitosis. The free-living nematode Caenorhabditis elegans, a model system for anthelmintic drug discovery, has a serotonin (5-HT)-gated chloride channel, MOD-1, which belongs to the Cys-loop receptor family and modulates locomotory and behavioral functions. Since MOD-1 is unique to nematodes, it is emerging as an attractive anthelmintic drug target, but details of MOD-1 function are unclear.An intrinsically disordered transcription activation domain increases the DNA binding affinity and reduces the specificity of NFκB p50/RelA
Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD’s influence on NFκB–DNA interactions.Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction
TransMEMbrane 16A (TMEM16A) is a Ca2+-activated Cl− channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca2+, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P2 head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels.Intermolecular functional coupling between phosphoinositides and the potassium channel KcsA
Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K+ channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology. It has been reported that KcsA activity is regulated by membrane phospholipids, such as phosphatidylglycerol.
