ResearchPad - molecular-biophysics https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Delineating an extracellular redox-sensitive module in T-type Ca<sup>2+</sup> channels]]> https://www.researchpad.co/article/elastic_article_7283 T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

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<![CDATA[Heterogeneity of proteome dynamics between connective tissue phases of adult tendon]]> https://www.researchpad.co/article/elastic_article_7267 Muscles are anchored to bones through specialized tissues called tendons. Made of bundles of fibers (or fascicles) linked together by an ‘interfascicular’ matrix, healthy tendons are required for organisms to move properly. Yet, these structures are constantly exposed to damage: the interfascicular matrix, in particular, is highly susceptible to injury as it allows the fascicles to slide on each other.

One way to avoid damage could be for the body to continually replace proteins in tendons before they become too impaired. However, the way proteins are renewed in these structures is currently not well understood – indeed, it has long been assumed that almost no protein turnover occurs in tendons. In particular, it is unknown whether proteins in the interfascicular matrix have a higher turn over than those in the fascicles.

To investigate, Choi, Simpson et al. fed rats on water carrying a molecular label that becomes integrated into new proteins. Analysis of individual proteins from the rats’ tendons showed great variation in protein turnover, with some replaced every few days and others only over several years. This suggests that protein turnover is actually an important part of tendon health. In particular, the results show that turnover is higher in the interfascicular matrix, where damage is expected to be more likely.

Protein turnover also plays a part in conditions such as cancer, heart disease and kidney disease. Using approaches like the one developed by Choi, Simpson et al. could help to understand how individual proteins are renewed in a range of diseases, and how to design new treatments.

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<![CDATA[Single-molecule observation of ATP-independent SSB displacement by RecO in <i>Deinococcus radiodurans</i>]]> https://www.researchpad.co/article/N75dd0523-a172-49b7-a20f-e040e1226ee1 Deinococcus radiodurans (DR) survives in the presence of hundreds of double-stranded DNA (dsDNA) breaks by efficiently repairing such breaks. RecO, a protein that is essential for the extreme radioresistance of DR, is one of the major recombination mediator proteins in the RecA-loading process in the RecFOR pathway. However, how RecO participates in the RecA-loading process is still unclear. In this work, we investigated the function of drRecO using single-molecule techniques. We found that drRecO competes with the ssDNA-binding protein (drSSB) for binding to the freely exposed ssDNA, and efficiently displaces drSSB from ssDNA without consuming ATP. drRecO replaces drSSB and dissociates it completely from ssDNA even though drSSB binds to ssDNA approximately 300 times more strongly than drRecO does. We suggest that drRecO facilitates the loading of RecA onto drSSB-coated ssDNA by utilizing a small drSSB-free space on ssDNA that is generated by the fast diffusion of drSSB on ssDNA.

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<![CDATA[Limited dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction]]> https://www.researchpad.co/article/N89b0a066-5932-4aa3-9c28-7c04aeecc210 Stem cells can give rise to many types of specialized cells through a process called differentiation, which is partly regulated by changes in the levels of a protein known as β-catenin. On one hand, a ‘destruction complex’ can keep β-catenin levels low; this complex includes a protein called Axin and an enzyme known as GSK-3, which can tag β-catenin for degradation. On the other hand, when β-catenin levels need to increase, another protein called Dishevelled is activated. By binding to Axin, Dishevelled can bring the destruction complex in contact with other proteins, which leads to the deactivation of GSK-3.

Dishevelled and Axin interact via a region that is similar in the two proteins, called DIX in Dishevelled and DAX in Axin. Studies of DIX and DAX have shown that both regions can form polymers – that is, a high number of similar units can bind together to form larger structures. However, these experiments were at higher concentrations than would be found in the cell. It was thought that, when combined, DIX and DAX might form these long chains together, preventing Axin from carrying out its role in destroying β-catenin. Kan et al. set out to better understand this process by studying how DIX and DAX behave separately, and how they interact.

The proteins were examined using a technique called cryo-electron microscopy, which allows scientists to dissect the structure of large proteins. When there was a high concentration of DIX in the sample, the molecules attached to one another to form long double-stranded helices. Similarly, DAX also formed helices, but these were shorter and only single-stranded. When the two proteins were combined, DAX bound only to the ends of short DIX chains, so that there are not more than four DAX chains attached to each DIX double helix.

To see if this behaviour happens naturally, Kan et al. attached fluorescent tags to Dishevelled proteins and followed them in living cells: this showed that Dishevelled forms smaller chains with fewer than ten molecules. Together these results highlight how Dishevelled binds to Axin to deactivate GSK-3, to prevent the enzyme from promoting β-catenin destruction.

Mutations in the genes that encode β-catenin or its regulators are associated with cancer. Ultimately, a better understanding of how β-catenin is regulated could help to identify new opportunities for drug development.

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<![CDATA[Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs]]> https://www.researchpad.co/article/N58103102-565b-4494-8b69-a2dcfc1a57fa Cation-chloride-cotransporters (CCCs) catalyze transport of Cl- with K+ and/or Na+across cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the Mus musculus K+-Cl- cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K+ and Cl- ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.

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<![CDATA[A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter]]> https://www.researchpad.co/article/N31301349-16ac-43e0-9228-476ce24b03ef ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

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<![CDATA[Top-down machine learning approach for high-throughput single-molecule analysis]]> https://www.researchpad.co/article/N957aad02-2c00-4587-a7f5-2b73aea07b8d During a chemical or biological process, a molecule may transition through a series of states, many of which are rare or short-lived. Advances in technology have made it easier to detect these states by gathering large amounts of data on individual molecules. However, the increasing size of these datasets has put a strain on the algorithms and software used to identify different molecular states.

Now, White et al. have developed a new algorithm called DISC which overcomes this technical limitation. Unlike most other algorithms, DISC requires minimal input from the user and uses a new method to group the data into categories that represent distinct molecular states. Although this new approach produces a similar end-result, it reaches this conclusion much faster than more commonly used algorithms.

To test the effectiveness of the algorithm, White et al. studied how individual molecules of a chemical known as cAMP bind to parts of proteins called cyclic nucleotide binding domains (or CNDBs for short). A fluorescent tag was attached to single molecules of cAMP and data were collected on the behavior of each molecule. Previous evidence suggested that when four CNDBs join together to form a so-called tetramer complex, this affects the binding of cAMP. Using the DISC system, White et al. showed that individual cAMP molecules interact with all four domains in a similar way, suggesting that the binding of cAMP is not impacted by the formation of a tetramer complex.

Analyzing this data took DISC less than 20 minutes compared to existing algorithms which took anywhere between four hours and two weeks to complete. The enhanced speed of the DISC algorithm could make it easier to analyze much larger datasets from other techniques in addition to fluorescence. This means that a greater number of states can be sampled, providing a deeper insight into the inner workings of biological and chemical processes.

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<![CDATA[A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery]]> https://www.researchpad.co/article/Nc90ed1fd-0433-47f8-99bd-914130e4e6c6

The ClpXP degradation machine consists of a hexameric AAA+ unfoldase (ClpX) and a pair of heptameric serine protease rings (ClpP) that unfold, translocate, and subsequently degrade client proteins. ClpXP is an important target for drug development against infectious diseases. Although structures are available for isolated ClpX and ClpP rings, it remains unknown how symmetry mismatched ClpX and ClpP work in tandem for processive substrate translocation into the ClpP proteolytic chamber. Here, we present cryo-EM structures of the substrate-bound ClpXP complex from Neisseria meningitidis at 2.3 to 3.3 Å resolution. The structures allow development of a model in which the sequential hydrolysis of ATP is coupled to motions of ClpX loops that lead to directional substrate translocation and ClpX rotation relative to ClpP. Our data add to the growing body of evidence that AAA+ molecular machines generate translocating forces by a common mechanism.

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<![CDATA[Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate]]> https://www.researchpad.co/article/N274f4952-2de8-48eb-9cdf-d1239cceea30

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

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<![CDATA[The Cryo-EM structure of pannexin 1 reveals unique motifs for ion selection and inhibition]]> https://www.researchpad.co/article/N3ac6bf2e-abc4-4337-8c3b-6350abdf198b

Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1.

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<![CDATA[Taking a close look at a large-pore channel]]> https://www.researchpad.co/article/Ne1f3b2bd-883f-41cc-b649-888e9e881f84

The structure of pannexin 1, a channel protein with a large pore, has been determined for the first time.

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<![CDATA[A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment]]> https://www.researchpad.co/article/Nbe9a7b76-8c01-489a-b41d-c82050dff1cb

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

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<![CDATA[The guide sRNA sequence determines the activity level of box C/D RNPs]]> https://www.researchpad.co/article/N32fdb9c1-8366-4e0a-92c1-866d17aeaba5

2’-O-rRNA methylation, which is essential in eukaryotes and archaea, is catalysed by the Box C/D RNP complex in an RNA-guided manner. Despite the conservation of the methylation sites, the abundance of site-specific modifications shows variability across species and tissues, suggesting that rRNA methylation may provide a means of controlling gene expression. As all Box C/D RNPs are thought to adopt a similar structure, it remains unclear how the methylation efficiency is regulated. Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate–guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles.

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<![CDATA[Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme]]> https://www.researchpad.co/article/N98045833-8cd0-44a3-b20d-435809bf7fc2

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

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<![CDATA[Genetic inactivation of mTORC1 or mTORC2 in neurons reveals distinct functions in glutamatergic synaptic transmission]]> https://www.researchpad.co/article/N6540914b-bab8-4f5b-9cd2-89aafe1878f7

Although mTOR signaling is known as a broad regulator of cell growth and proliferation, in neurons it regulates synaptic transmission, which is thought to be a major mechanism through which altered mTOR signaling leads to neurological disease. Although previous studies have delineated postsynaptic roles for mTOR, whether it regulates presynaptic function is largely unknown. Moreover, the mTOR kinase operates in two complexes, mTORC1 and mTORC2, suggesting that mTOR’s role in synaptic transmission may be complex-specific. To better understand their roles in synaptic transmission, we genetically inactivated mTORC1 or mTORC2 in cultured mouse glutamatergic hippocampal neurons. Inactivation of either complex reduced neuron growth and evoked EPSCs (eEPSCs), however, the effects of mTORC1 on eEPSCs were postsynaptic and the effects of mTORC2 were presynaptic. Despite postsynaptic inhibition of evoked release, mTORC1 inactivation enhanced spontaneous vesicle fusion and replenishment, suggesting that mTORC1 and mTORC2 differentially modulate postsynaptic responsiveness and presynaptic release to optimize glutamatergic synaptic transmission.

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<![CDATA[Human RPA activates BLM’s bidirectional DNA unwinding from a nick]]> https://www.researchpad.co/article/N7674358b-1ebb-49b1-aef5-d137271f733a

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

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<![CDATA[An asymmetric sheath controls flagellar supercoiling and motility in the leptospira spirochete]]> https://www.researchpad.co/article/N0d9031a7-c6e8-445d-83ab-2755592426c8

Spirochete bacteria, including important pathogens, exhibit a distinctive means of swimming via undulations of the entire cell. Motility is powered by the rotation of supercoiled 'endoflagella' that wrap around the cell body, confined within the periplasmic space. To investigate the structural basis of flagellar supercoiling, which is critical for motility, we determined the structure of native flagellar filaments from the spirochete Leptospira by integrating high-resolution cryo-electron tomography and X-ray crystallography. We show that these filaments are coated by a highly asymmetric, multi-component sheath layer, contrasting with flagellin-only homopolymers previously observed in exoflagellated bacteria. Distinct sheath proteins localize to the filament inner and outer curvatures to define the supercoiling geometry, explaining a key functional attribute of this spirochete flagellum.

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<![CDATA[Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations]]> https://www.researchpad.co/article/N1bb6834f-9ba5-4e2a-bf53-38581a0dc83e

Direct activation of the human phospholipase C-γ isozymes (PLC-γ1, -γ2) by tyrosine phosphorylation is fundamental to the control of diverse biological processes, including chemotaxis, platelet aggregation, and adaptive immunity. In turn, aberrant activation of PLC-γ1 and PLC-γ2 is implicated in inflammation, autoimmunity, and cancer. Although structures of isolated domains from PLC-γ isozymes are available, these structures are insufficient to define how release of basal autoinhibition is coupled to phosphorylation-dependent enzyme activation. Here, we describe the first high-resolution structure of a full-length PLC-γ isozyme and use it to underpin a detailed model of their membrane-dependent regulation. Notably, an interlinked set of regulatory domains integrates basal autoinhibition, tyrosine kinase engagement, and additional scaffolding functions with the phosphorylation-dependent, allosteric control of phospholipase activation. The model also explains why mutant forms of the PLC-γ isozymes found in several cancers have a wide spectrum of activities, and highlights how these activities are tuned during disease.

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<![CDATA[Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry]]> https://www.researchpad.co/article/N52b716fe-215b-4a30-98b6-6504dcb2e7c4

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

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<![CDATA[Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones]]> https://www.researchpad.co/article/Na4a94f1d-a35d-46c7-95d9-26faa13ff4fa

Hsp70 molecular chaperones are abundant ATP-dependent nanomachines that actively reshape non-native, misfolded proteins and assist a wide variety of essential cellular processes. Here, we combine complementary theoretical approaches to elucidate the structural and thermodynamic details of the chaperone-induced expansion of a substrate protein, with a particular emphasis on the critical role played by ATP hydrolysis. We first determine the conformational free-energy cost of the substrate expansion due to the binding of multiple chaperones using coarse-grained molecular simulations. We then exploit this result to implement a non-equilibrium rate model which estimates the degree of expansion as a function of the free energy provided by ATP hydrolysis. Our results are in quantitative agreement with recent single-molecule FRET experiments and highlight the stark non-equilibrium nature of the process, showing that Hsp70s are optimized to effectively convert chemical energy into mechanical work close to physiological conditions.

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