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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 88,847 bioRxiv papers from 380,945 authors.

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in category biochemistry

2,865 results found. For more information, click each entry to expand.

1781: Quantitative variations of ADF/cofilin's multiple actions on actin filaments with pH
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Posted to bioRxiv 20 Sep 2018

Quantitative variations of ADF/cofilin's multiple actions on actin filaments with pH
264 downloads biochemistry

Hugo Wioland, Antoine Jégou, Guillaume Romet-Lemonne

Actin Depolymerizing Factor (ADF)/cofilin is the main protein family promoting the disassembly of actin filaments, which is essential for numerous cellular functions. ADF/cofilin proteins disassemble actin filaments through different reactions, as they bind to their sides, sever them, and promote the depolymerization of the resulting ADF/cofilin-saturated filaments. Moreover, the efficiency of ADF/cofilin is known to be very sensitive to pH. ADF/cofilin thus illustrates two challenges in actin biochemistry: separating the different regulatory actions of a single protein, and characterizing them as a function of specific biochemical conditions. Here, we investigate the different reactions of ADF/cofilin on actin filaments, over four different values of pH ranging from pH 6.6 to pH 7.8, using single filament microfluidics techniques. We show that lowering pH reduces the effective filament severing rate by increasing the rate at which filaments become saturated by ADF/cofilin, thereby reducing the number of ADF/cofilin domain boundaries, where severing can occur. The severing rate per domain boundary, however, remains unchanged at different pH values. The ADF/cofilin-decorated filaments (refered to as "cofilactin" filaments) depolymerize from both ends. We show here that, at physiological pH (pH 7.0 to 7.4), the pointed end depolymerization of cofilactin filaments is barely faster than that of bare filaments. In contrast, cofilactin barbed ends undergo an "unstoppable" depolymerization (depolymerizing for minutes despite the presence of free actin monomers and capping protein in solution), throughout our range of pH. We thus show that, at physiological pH, the main contribution of ADF/cofilin to filament depolymerization is at the barbed end.

1782: An Atomistic view of Short-chain Antimicrobial Biomimetic peptides in Action
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Posted to bioRxiv 16 May 2018

An Atomistic view of Short-chain Antimicrobial Biomimetic peptides in Action
263 downloads biochemistry

Jagannath Mondal, Pushpita Ghosh, Xiao Zhu

Amphiphilic β-peptides, which are rationally designed synthetic oligomers, are established biomimetic alternatives of natural antimicrobial peptides. The ability of these biomimetic peptides to form helical amphiphilic conformation using small number of residues provides a greater synthetic advantage over the naturally occurring antimicrobial peptides, which is reflected in more potent antimicrobial activity of β-peptides than its naturally occurring counterparts. Here we address whether the distinct molecular architecture of short-chain and rigid synthetic peptides compared to relatively long and flexible natural antimicrobial peptides translates to a distinct mechanistic action with membrane. By simulating the interaction of membrane with antimicrobial 10-residue β-peptides at diverse range of concentrations we reveal spontaneous insertion of β-peptides in the membrane interface at a low concentration and occurrence of partial water leakage in the membrane at a high concentration. Intriguingly, unlike prototypical natural antimicrobial peptides, the water molecules leaked inside the membrane by these biomimetic peptides do not span entire membrane, as supported by free energy analysis. As a major advancement, this work brings into lights the key distinction in the membrane-activity of short synthetic biomimetic oligomers relative to the natural long-chain antimicrobial peptides.

1783: Base pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar Ribonucleoproteins
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Posted to bioRxiv 06 Mar 2019

Base pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar Ribonucleoproteins
263 downloads biochemistry

Erin Katelyn Kelly, Dominic Philip Czekay, Ute Kothe

H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base-pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide – substrate RNA pairs.

1784: KLK4 inhibition by cyclic and acyclic peptides: structural and dynamical insights into standard-mechanism protease inhibitors
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Posted to bioRxiv 08 Mar 2019

KLK4 inhibition by cyclic and acyclic peptides: structural and dynamical insights into standard-mechanism protease inhibitors
263 downloads biochemistry

Blake T. Riley, Olga Ilyichova, Simon J. de Veer, Joakim E. Swedberg, Emily Wilson, David E Hoke, Jonathan M. Harris, Ashley M. Buckle

Sunflower Trypsin Inhibitor (SFTI-1) is a 14-amino acid serine protease inhibitor. The dual anti-parallel β-sheet arrangement of SFTI-1 is stabilized by a N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favour initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between cleaved and uncleaved inhibitor in the presence of protease. We have determined, at 1.15 Å resolution, the x-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14), and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of altered contact sequence, intramolecular hydrogen bonding network and backbone cyclization, in altering the state of SFTI's scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition, and suggest that modifications on the non-contact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.

1785: A single K+-binding site in the crystal structure of the gastric proton pump
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Posted to bioRxiv 15 Apr 2019

A single K+-binding site in the crystal structure of the gastric proton pump
263 downloads biochemistry

Kenta Yamamoto, Vikas Dubey, Katsumasa Irie, Hanayo Nakanishi, Himanshu Khandelia, Yoshinori Fujiyoshi, Kazuhiro Abe

The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold - the highest cation gradient known in any mammalian tissue - across the membrane.

1786: Bayogenin 3-O-Cellobioside is a novel non-cultivar specific anti-blast metabolite produced in rice in response to Pyricularia oryzae infection
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Posted to bioRxiv 23 May 2019

Bayogenin 3-O-Cellobioside is a novel non-cultivar specific anti-blast metabolite produced in rice in response to Pyricularia oryzae infection
263 downloads biochemistry

Justice Norvienyeku, Lili Lin, Abdul Waheed, Xiaomin Chen, Jiandong Bao, Ammarah Shabbir, Lianyu Lin, Zhenhui Zhong, Wajjiha Batool, Sami Rukaiya Aliyu, Jie Zhou, Guodong Lu, Zonghua Wang

Rice cultivars from japonica and indica lineage possess differential resistance against blast fungus on an account genetic divergence. Whether different rice cultivars also show distinct metabolomic changes in response to P. oryzae, and their role in host resistance, are poorly understood. Here, we examine the responses of six different rice cultivars from japonica and indica lineage challenged with P. oryzae. Both susceptible and resistant rice cultivars expressed several metabolites exclusively during P. oryzae infection, including the saponin Bayogenin 3-O-cellobioside. Bayogenin 3-O-cellobioside level in infected rice directly correlated with their resistant attributes. These findings reveal, for the first time to our knowledge that besides oat, other grass plants including rice produces protective saponins. Our study provides insight into the role of pathogen-mediated metabolomics-reprogramming in host immunity. The correlation between Bayogenin 3-O-Cellobioside levels and blast resistance suggests that engineering saponin expression in cereal crops represents an attractive and sustainable disease control strategy.

1787: The E3 ubiquitin ligase Pib1 regulates effective gluconeogenic shutdown in S. cerevisiae.
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Posted to bioRxiv 17 Jun 2019

The E3 ubiquitin ligase Pib1 regulates effective gluconeogenic shutdown in S. cerevisiae.
262 downloads biochemistry

Vineeth Vengayil, Sunil Laxman

Cells use multiple mechanisms to regulate their metabolic states depending on changes in their nutrient environment. A well-known example is the response of cells to glucose availability. In S. cerevisiae cells growing in glucose-limited medium, the re-availability of glucose leads to the downregulation of gluconeogenesis, the activation of glycolysis, and robust glucose repression. However, our knowledge of the initial mechanisms mediating this glucose-dependent downregulation of the gluconeogenic transcription factors is incomplete. We used the gluconeogenic transcription factor Rds2 as a candidate with which to discover regulators of early events leading to glucose repression. Here, we identify a novel role for the E3 ubiquitin ligase Pib1 in regulating the stability and degradation of Rds2. Glucose addition to glucose-limited cells results in rapid ubiquitination of Rds2, followed by its proteasomal degradation. Through in vivo and in vitro experiments, we establish Pib1 as a ubiquitin E3 ligase that regulates Rds2 ubiquitination and stability. Notably, this Pib1 mediated Rds2 ubiquitination, followed by proteasomal degradation, is specific to the presence of glucose. Pib1 is required for complete glucose repression, and enables cells to optimally grow in competitive environments when glucose becomes re-available. Our results reveal the existence of a Pib1 E3-ubiquitin ligase mediated regulatory program that mediates glucose-repression when glucose availability is restored.

1788: Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry
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Posted to bioRxiv 20 Jun 2018

Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry
262 downloads biochemistry

Joshua S. Sharp, Sandeep K. Misra, Jeffrey J. Persoff, Robert W. Egan, Scot R. Weinberger

Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g. ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultra-fast benchtop method for performing HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond timescale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g. ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be normalized on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

1789: Zinc(II) binding on human wild-type ISCU and Met140 variants modulates Fe-S complex activity
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Posted to bioRxiv 10 Feb 2018

Zinc(II) binding on human wild-type ISCU and Met140 variants modulates Fe-S complex activity
262 downloads biochemistry

Nicholas G Fox, Alain Martelli, Joseph F. Nabhan, Jay Janz, Oktawia Borkowska, Christine Bulawa, Wyatt W. Yue

The human de novo iron-sulfur (Fe-S) assembly complex consists of the cysteine desulfurase NFS1, accessory protein ISD11, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN has been shown to bind the NFS1-ISD11-ISCU complex (SDU), to activate the desulfurase activity and thus Fe-S cluster biosynthesis. Conversely, in the absence of FXN, the NFS1-ISD11 (SD) complex was reported to be inhibited by the binding of recombinant ISCU. Here, we show that recombinant ISCU binds zinc(II) ion, and that the presence of zinc in as-isolated ISCU has impacts on the SDU desulfurase activity as measured by sulfide production. Indeed, the removal of this zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SD alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SD. Recent yeast studies have reported a substitution on the yeast ISCU orthologue Isu, at position Met141 (Met140 in human numbering of precursor protein) to Ile, Leu, Val, or Cys that could bypass the requirement of FXN for Fe-S cluster assembly and cell viability. Using recombinant human proteins, we report no significant differences in the biochemical and biophysical properties observed between wild-type and variants M140I, M140L, and M140V of ISCU. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SD complex. This study therefore identifies an important regulatory role for ISCU-bound zinc in modulation of the human Fe-S assembly system in vitro but no 'FXN bypass' effect on mutations at position Met140 in human ISCU.

1790: A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers
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Posted to bioRxiv 23 Jun 2017

A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers
262 downloads biochemistry

Rahul Chadda, Lucy Cliff, Marley Brimberry, Janice L. Robertson

We previously reported the equilibrium dimerization reaction of the CLC-ec1 Cl-/H+ transporter in 2:1 POPE/POPG membranes (Chadda et al. 2016). This was determined by measuring the probability distributions of subunit capture into extruded liposomes by single-molecule photobleaching analysis across a wide range of subunit/lipid mole fraction densities. In this approach, knowledge of the liposome size distribution is necessary in order to correct the data for random co-capture events and extract the underlying dimerization reaction. For this we used a previously reported cryo-electron microscopy (cryo-EM) measured size distribution of 400 nm extruded liposomes made of E. coli polar lipids (Walden et al. 2007). While the model and data agreed at low densities, we observed systematic inaccuracies at higher densities limiting our ability to extract FDimer in this range. To address this issue, we measured the 400 nm extruded 2:1 POPE/POPG liposome size distribution by cryo-EM and found that there is a small, but significant amount of larger liposomes in the population. Re-analysis of the I201W/I422W (WW) photobleaching data using this distribution shows that the protein is monomeric in the membrane and can serve as an experimental control. Dimer controls were constructed by glutaraldehyde cross-linking of C85A/H234C (WT) or introducing R230C/L249C, which forms a spontaneous disulfide bond. Determination of FDimer based on the experimental controls yields improved fits and no change in the previously reported ΔG° values, providing an alternate model-free approach to measuring CLC-ec1 dimerization in membranes.

1791: The Ras-like GTPase Rem2 is a potent endogenous inhibitor of calcium/calmodulin-dependent kinase II activity
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Posted to bioRxiv 12 Jun 2017

The Ras-like GTPase Rem2 is a potent endogenous inhibitor of calcium/calmodulin-dependent kinase II activity
262 downloads biochemistry

Leandro Royer, Josiah J. Herzog, Katelyn Kenny, Boriana Tzvetkova, Jesse C. Cochrane, Michael T. Marr, Suzanne Paradis

CaMKII is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue specific manner. For example, in heart muscle, CaMKII regulates Ca2+ homeostasis while in neurons CaMKII regulates activity-dependent dendritic remodeling and Long Term Potentiation (LTP), a biological correlate of learning and memory. Previously, we identified the noncanonical GTPase Rem2 as a critical regulator of dendrite branching and synapse formation in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously undescribed function for the Rem2 protein. To date, only one other endogenous inhibitor of CaMKII has been described: CaMKIIN, which blocks CaMKII activity through binding to the catalytic domain. Our data suggest that Rem2 inhibits CaMKII through a novel mechanism, as inhibition requires the presence of the association domain of CaMKII. Our biochemical finding that Rem2 is a direct, endogenous inhibitor of CaMKII activity, coupled with known functions of Rem2 in neurons, provides a framework which will enable future experiments probing the physiological role of CaMKII inhibition in a cellular context.

1792: Ligand Gaussian accelerated molecular dynamics (LiGaMD): Characterization of ligand binding thermodynamics and kinetics
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Posted to bioRxiv 21 Apr 2020

Ligand Gaussian accelerated molecular dynamics (LiGaMD): Characterization of ligand binding thermodynamics and kinetics
262 downloads biochemistry

Yinglong Miao, Apurba Bhattarai, Jinan Wang

Calculations of ligand binding free energies and kinetic rates are important for drug design. However, such tasks have proven challenging in computational chemistry and biophysics. To address this challenge, we have developed a new computational method "LiGaMD", which selectively boosts the ligand non-bonded interaction potential energy based on the Gaussian accelerated molecular dynamics (GaMD) enhanced sampling technique. Another boost potential could be applied to the remaining potential energy of the entire system in a dual-boost algorithm (LiGaMD_Dual) to facilitate ligand binding. LiGaMD has been demonstrated on host-guest and protein-ligand binding model systems. Repetitive guest binding and unbinding in the β-cyclodextrin host were observed in hundreds-of-nanosecond LiGaMD simulations. The calculated binding free energies of guest molecules with sufficient sampling agreed excellently with experimental data (< 1.0 kcal/mol error). In comparison with previous microsecond-timescale conventional molecular dynamics simulations, accelerations of ligand kinetic rate constants in LiGaMD simulations were properly estimated using Kramers' rate theory. Furthermore, LiGaMD allowed us to capture repetitive dissociation and binding of the benzamidine inhibitor in trypsin within 1 μs simulations. The calculated ligand binding free energy and kinetic rate constants compared well with the experimental data. In summary, LiGaMD provides a promising approach for characterizing ligand binding thermodynamics and kinetics simultaneously, which is expected to facilitate computer-aided drug design. ### Competing Interest Statement The authors have declared no competing interest.

1793: Monitoring the production of high diffraction-quality crystals of two enzymes in real time using in situ dynamic light scattering
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Posted to bioRxiv 06 Jan 2020

Monitoring the production of high diffraction-quality crystals of two enzymes in real time using in situ dynamic light scattering
262 downloads biochemistry

Raphaël de Wijn, Kévin Rollet, Sylvain Engilberge, Alastair G. McEwen, Oliver Hennig, Heike Betat, Mario Mörl, François Riobé, Olivier Maury, Eric Girard, Philippe Bénas, Bernard Lorber, Claude Sauter

The reproducible preparation of well diffracting crystals is a prerequisite for every structural study based on crystallography. An instrument called the XtalController has recently been designed that allows the monitoring of crystallization assays using dynamic light scattering and microscopy, and integrates piezo pumps to alter the composition of the mother liquor during the experiment. We have applied this technology to study the crystallization of two enzymes, the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus and the hen egg white lysozyme in the presence of a synthetic chemical nucleant. We were able to i) detect early nucleation events and ii) drive the crystallization system (through cycles of dissolution/crystallization) towards growth conditions yielding crystals with excellent diffraction properties. This technology opens a way to the rational production of samples for crystallography, ranging from nanocrystals for electron diffraction, microcrystals for serial or conventional X-ray diffraction, to larger crystals for neutron diffraction.

1794: A DNA G-quadruplex/i-motif hybrid
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Posted to bioRxiv 15 Aug 2019

A DNA G-quadruplex/i-motif hybrid
262 downloads biochemistry

Betty Chu, Daoning Zhang, Paul J. Paukstelis

DNA can form many structures beyond the canonical Watson-Crick double helix. It is now clear that noncanonical structures are present in genomic DNA and have biological functions. G-rich G-quadruplexes and C-rich i-motifs are the most well-characterized noncanonical DNA motifs that have been detected in vivo with either proscribed or postulated biological roles. Because of their independent sequence requirements, these structures have largely been considered distinct types of quadruplexes. Here, we describe the crystal structure of the DNA oligonucleotide, d(CCAGGCTGCAA), that self-associates to form a quadruplex structure containing two central antiparallel G-tetrads and six i-motif C-C+ base pairs. Solution studies suggest a robust structural motif capable of assembling as a tetramer of individual strands or as a dimer when composed of tandem repeats. This hybrid structure highlights the growing structural diversity of DNA and suggests that biological systems may harbor many functionally important non-duplex structures.

1795: A conserved D/E-P motif in the nucleotide binding domain of plant ABCB/PGP-type ABC transporters defines their auxin transport capacity
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Posted to bioRxiv 09 May 2020

A conserved D/E-P motif in the nucleotide binding domain of plant ABCB/PGP-type ABC transporters defines their auxin transport capacity
261 downloads biochemistry

Pengchao Hao, Jian Xia, Jie Liu, Martin diDonato, Konrad Pakula, Aurélien Bailly, Michal Jasinski, Markus Geisler

Auxin transport activity of ABCB1 was suggested to be regulated by physical interaction with the FKBP42/Twisted Dwarf1 (TWD1), a bona fide peptidylprolyl cis-trans isomerase (PPIase), but all attempts to demonstrate such a PPIase activity on TWD1 have failed so far. By using a structure-based approach we have identified a series of surface-exposed proline residues in the C- terminal nucleotide binding fold and linker of Arabidopsis ABCB1 that do not alter ABCB1 protein stability or location but its catalytic transport activity. P1.008 was uncovered as part of a conserved signature D/E-P motif that seems to be specific for Auxin-Transporting ABCBs, we now refer to as ATAs. Beside the proline, also mutation of the acidic moiety prior to the proline abolishes auxin transport activity by ABCB1. So far, all higher plant ABCBs for that auxin transport was safely proven carry this conserved motif underlining its diagnostic potential. Introduction of this D/E-P motif into malate importer, ABCB14, increases both its malate and its background auxin transport activity, suggesting that this motif has an impact on transport capacity. The D/E- P1.008 motif is also important for ABCB1-TWD1 interaction and activation of ABCB1-mediated auxin transport by TWD1, supporting a scenario in that TWD1 acts as an activator of ABCB1 transport activity by means of its PPIase. In summary, our data imply a dual function for TWD1 acting as an ABCB co-chaperone required for ABCB biogenesis and as a putative activator of ABCB-mediated auxin transport by cis-trans isomerization of peptidyl-prolyl bonds. ### Competing Interest Statement The authors have declared no competing interest.

1796: Elucidating the roles of Alzheimer disease-associated proteases and the signal-peptide peptidase-like 3 (SPPL3) in the shedding of glycosyltransferases
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Posted to bioRxiv 08 May 2018

Elucidating the roles of Alzheimer disease-associated proteases and the signal-peptide peptidase-like 3 (SPPL3) in the shedding of glycosyltransferases
261 downloads biochemistry

Assou El-Battari, Sylvie Mathieu, Romain Sigaud, Maëlle Prorok-Hamon, L’Houcine Ouafik, Charlotte Jeanneau

The Golgi resident glycosyltransferases (GTs) are membrane-bound glycoproteins but are frequently found as soluble proteins in biological fluids where their function remains largely unknown. Previous studies have established that the release of these proteins involved Alzheimer disease-associated proteases such as β-secretases (BACE1 and BACE2) and the intramembrane-cleaving aspartyl proteases Presenilins 1 and 2. Recent studies have involved another intramembrane-cleaving enzyme, the signal peptide peptidese-like-3 (SPPL3). Except for the latter, the two former studies mostly addressed particular cases of GTs, namely ST6Gal-I (BACEs) or GnT-V (Presenilins). Therefore the question still remains as which of these secretases is truly responsible for the cleavage and secretion of GTs. We herein combined the 3 proteases in a single study with respect to their abilities to release 3 families of GTs encompassing three N-acetylglucosaminyltransferases, two fucosyltransferases and two sialyltransferases. Green fluorescent protein (gfp)-fused versions of these GTs were virally transduced in mouse embryonic fibroblasts devoid of BACEs, Presenilins or SPPL3. We found that neither BACE nor Presenilins are involved in the shedding of these glycosyltransferases, while SPPL3 was involved in the cleavage and release of some but not all GTs. Notably, the γ-secretase inhibitor DFK-167 was the only molecule capable of significantly decreasing glycosyltransferase secretion, suggesting the involvement of γ-secretase(s), yet different from Presenilins but comprising SPPL3 among other proteases still to be identified. Using confocal microscopy, we show that SPPL3 selectivity towards GTs relays not only on sequence specificity but also depends on how GTs distribute in the cell with respect SPPL3 during their cycling within and outside the Golgi.

1797: Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements
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Posted to bioRxiv 02 Nov 2019

Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements
261 downloads biochemistry

Rory Henderson, Maolin Lu, Ye Zhou, Zekun Mu, Robert Parks, Qifeng Han, Allen L. Hsu, Elizabeth Carter, Scott C Blanchard, RJ Edwards, Kevin Wiehe, Kevin O. Saunders, Mario J Borgnia, Alberto Bartesaghi, Walther Mothes, Barton F Haynes, Priyamvada Acharya, S. Munir Alam

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, we introduced Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis performed on the soluble ectodomain BG505 SOSIP Env trimers, cell-surface expressed BG505 full-length trimers and single-molecule Forster Resonance Energy Transfer (smFRET) performed on the full-length virion-bound Env confirmed that these mutations prevented CD4-induced transitions of the HIV-1 Env. Structural analysis by single-particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins revealed rearrangements in the gp120 topological layer contacts with gp41. Specifically, a conserved tryptophan at position 571 (W571) was displaced from its typical pocket at the interface of gp120 topological layers 1 and 2 by lysine 567, disrupting key gp120- gp41 contacts and rendering the Env insensitive to CD4 binding. Vector based analysis of closed Env SOSIP structures revealed the newly designed trimers exhibited a quaternary structure distinct from that typical of SOSIPs and residing near a cluster of Env trimers bound to vaccine-induced fusion peptide-directed antibodies (vFP Mabs). These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.

1798: Molecular Architecture of the Bardet-Biedl Syndrome Protein 2-7-9 Subcomplex
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Posted to bioRxiv 11 Jul 2019

Molecular Architecture of the Bardet-Biedl Syndrome Protein 2-7-9 Subcomplex
261 downloads biochemistry

W. Grant Ludlam, Takuma Aoba, Jorge Cuéllar, M. Teresa Bueno-Carrasco, Aman Makaju, James D. Moody, Sarah Franklin, José M. Valpuesta, Barry M. Willardson

Bardet-Biedl syndrome (BBS) is a genetic disease caused by mutations that disrupt the function of the BBSome, an eight-subunit complex that plays an important role in transport of proteins in primary cilia. To better understand the molecular basis of the disease, we analyzed the structure of a BBSome subcomplex consisting of three homologous BBS proteins (BBS2, BBS7, and BBS9) by an integrative structural modeling approach using electron microscopy and chemical crosslinking coupled with mass spectrometry. The resulting molecular model revealed an overall structure that resembles a flattened triangle. Within the structure, BBS2 and BBS7 form a tight dimer based on a coiled-coil interaction, and BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-linked mutation of BBS2 (R632P) is located in the α-helical domain at the interface between BBS2 and BBS9, and binding experiments showed that this mutation disrupted the interaction of BBS2 with BBS9. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing a molecular explanation for BBS in patients harboring this mutation.

1799: Dscam homophilic specificity is generated by high order cis-multimers coupled with trans self-binding of variable Ig1 in Chelicerata
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Posted to bioRxiv 16 Dec 2019

Dscam homophilic specificity is generated by high order cis-multimers coupled with trans self-binding of variable Ig1 in Chelicerata
260 downloads biochemistry

Fengyan Zhou, Guozheng Cao, Songjun Dai, Guo Li, Hao Li, Zhu Ding, Shouqing Hou, Bingbing Xu, Wendong You, Feng Shi, Xiaofeng Yang, Yongfeng Jin

By alternative splicing, Drosophila Down syndrome cell adhesion molecule (Dscam1) encodes tens of thousands of proteins required for establishing neural circuits, while Chelicerata encodes a family of ~ 100 shortened Dscam (sDscam) isoforms via alternative promoters. We report that Dscam isoforms interact promiscuously in cis to generate a vast repertoire of combinatorial homophilic recognition specificities in Chelicerata. Specifically, sDscams formed high order cis-multimers without isoform specificity involving the membrane-proximal fibronectin type III (FNIII) 1-3 and transmembrane (TM) domains and associated specifically in trans via antiparallel self-binding of the first variable immunoglobulin (Ig1) domain. We propose that such sDscam combinatorial homophilic specificity is sufficient to provide each neuron with a unique identity for self/non-self discrimination. In many respects, our results amazingly mirror those reported for the structurally unrelated vertebrate protocadherins (Pcdh) rather than for the closely related fly Dscam1. Thus, our findings blur the distinction between the neuronal self-avoidance of invertebrates and vertebrates and provide insight into the basic principles and evolution of metazoan self-avoidance and self/non-self discrimination.

1800: Dextranol: A better lyoprotectant
more details view paper

Posted to bioRxiv 07 Dec 2018

Dextranol: A better lyoprotectant
260 downloads biochemistry

Bryan J Jones, Advitiya Mahajan, Alptekin Aksan

Dextranol, a reduced dextran, prevents damage to stored dry protein samples that unmodified dextran would otherwise cause. Lyoprotectants like the polysaccharide dextran are critical for preserving dried protein samples by forming rigid a glass that protects entrapped protein molecules. Stably dried proteins are important for maintaining critical information in clinical samples like blood serum. However, we found that dextran reacts with serum proteins during storage, producing high-molecular weight Amadori-product conjugates. These conjugates appeared in a matter of days or weeks when stored at elevated temperatures (37° or 45°C), but also appeared on a timescale of months when stored at room temperature. We synthesized a less reactive dextranol by reducing dextran’s anomeric carbon from an aldehyde to an alcohol. Serum samples dried in a dextranol-based matrix protected the serum proteins from forming high-molecular weight conjugates. The levels of four cancer-related serum biomarkers (prostate specific antigen, neuropiln-1, osteopontin, and metalloproteinase 7) decreased, as measured by immunoassay, when serum samples were stored for one to two weeks in dextran-based matrix. Switching to a dextran-based lyoprotection matrix slightly reduced the damage to osteopontin and completely stopped any detectable damage during storage in the other three biomarkers when for a period of two weeks at 45°C. Dextranol offers a small and easy modification to dextran that significantly improves the molecule’s function as a lyoprotectant by eliminating the potential for damaging protein-polysacharide conjugation.

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