PaperPlayer biorxiv biophysics

Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.21.306704v1?rss=1

Authors: Dienerowitz, M., Howard, J., Quinn, S. D., Dienerowitz, F., Leake, M. C.

Abstract:

Single-molecule Forster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap's inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. Here we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with 1 ms time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.22.298109v1?rss=1

Authors: Shang, J., Kojetin, D.

Abstract:

Ligands bind to an occluded orthosteric pocket within the nuclear receptor (NR) ligand-binding domain (LBD), but experimentally it remains unclear whether ligand binding proceeds through induced fit or conformational selection mechanisms. Using NMR spectroscopy lineshape analysis, we show that ligand binding to the peroxisome proliferator-activated receptor gamma (PPAR{gamma}) LBD involves a two-step induced fit mechanism including an initial fast step followed by slow conformational change. Surface plasmon resonance and isothermal titration calorimetry heat capacity analysis support the fast kinetic binding step and the conformational change after binding step. The putative initial ligand binding pose is suggested in several crystal structures of PPAR{gamma} LBD where a ligand is bound to a surface pore formed by helix 3, the {beta}-sheet, and the {Omega} loop; one of several ligand entry sites suggested in previous targeted and unbiased molecular simulations. These findings, when considered with a recent NMR study showing the activation function-2 (AF-2) helix 12 exchanges in and out of the orthosteric pocket in apo/ligand-free PPAR{gamma}, suggest an activation mechanism whereby agonist binding occurs through an initial encounter complex with the LBD followed by transition of the ligand into the orthosteric pocket concomitant with a conformational change resulting in a solvent-exposed active helix 12 conformation.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.22.306647v1?rss=1

Authors: Wilkinson, M. E., Fica, S. M., Galej, W. P., Nagai, K.

Abstract:

The catalytic spliceosome exists in equilibrium between the branching (B*/C) and exon ligation (C*/P) conformations. Here we present the electron cryo-microscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 [A] resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. In the Ci conformation, the exon-ligation factors Prp18 and Slu7 are already bound before ATP hydrolysis by Prp16, which destabilises the branching conformation. Biochemical assays suggest these pre-bound factors prime C complex for conversion to C* by Prp16. A complete model of the Prp19-complex (NTC) shows how branching factors Yju2 and Isy1 bind the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain associated with the C* complex to promote exon ligation. Our results explain how Prp16 action modulates dynamic binding of step-specific factors to alternatively stabilise the C or C* conformation and establish equilibrium of the catalytic spliceosome.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.22.309146v1?rss=1

Authors: Banigan, E. J., Mirny, L. A.

Abstract:

Compaction of chromosomes is essential for reliable transmission of genetic information. Experiments suggest that this ~1000-fold compaction is driven by condensin complexes that extrude chromatin loops, i.e., progressively collect chromatin fiber from one or both sides of the complex to form a growing loop. Theory indicates that symmetric two-sided loop extrusion can achieve such compaction, but recent single-molecule studies observed diverse dynamics of condensins that perform one-sided, symmetric two-sided, and asymmetric two-sided extrusion. We use simulations and theory to determine how these molecular properties lead to chromosome compaction. High compaction can be achieved if even a small fraction of condensins have two essential properties: a long residence time and the ability to perform two-sided (not necessarily symmetric) extrusion. In mixtures of condensins I and II, coupling of two-sided extrusion and stable chromatin binding by condensin II promotes compaction. These results provide missing connections between single-molecule observations and chromosome-scale organization.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.22.308627v1?rss=1

Authors: Muthuramalingam, M., Whittier, S., Lovell, S., Battaile, K. P., Tachiyama, S., Johnson, D. K., Picking, W. L., Picking, W. D.

Abstract:

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject effector proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common overall architecture in all systems studied thus far, comprising three major components - the cytoplasmic sorting platform, the envelope-spanning basal body and the external needle with controlling tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two- hybrid analysis. The functional need for Arg96 in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.21.307322v1?rss=1

Authors: Miller, N. T., Vaughn, M. D., Burnap, R. L.

Abstract:

Cyclic electron flow (CEF) around Photosystem I is vital to balancing the photosynthetic energy budget of cyanobacteria and other photosynthetic organisms. The coupling of CEF to proton pumping has long been hypothesized to occur, providing proton motive force (PMF) for the synthesis of ATP with no net cost to [NADPH]. This is thought to occur largely through the activity of NDH-1 complexes, of which cyanobacteria have four with different activities. While a much work has been done to understand the steady-state PMF in both the light and dark, and fluorescent probes have been developed to observe these fluxes in vivo, little has been done to understand the kinetics of these fluxes, particularly with regard to NDH-1 complexes. To monitor the kinetics of proton pumping in Synechocystis sp. PCC 6803, the pH sensitive dye Acridine Orange was used alongside a suite of inhibitors in order to observe light-dependent proton pumping. The assay was demonstrated to measure photosynthetically driven proton pumping and used to measure the rates of proton pumping unimpeded by dark {Delta}pH. Here, the cyanobacterial NDH-1 complexes are shown to pump a sizable portion of proton flux when CEF-driven and LEF-driven proton pumping rates are observed and compared in mutants lacking some or all NDH-1 complexes. It is also demonstrated that PSII and LEF are responsible for the bulk of light induced proton pumping, though CEF and NDH-1 are capable of generating ~40% of the proton pumping rate when LEF is inactivated.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.21.307439v1?rss=1

Authors: Wang, X., Zhang, S., Qiao, S., Yu, J., Zeng, J., Shan, S., Lan, J., Tian, L., Zhang, L.

Abstract:

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt this receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. Through structure and sequence comparisons, we identified critical residues in the RBD that underlie the different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes and propose that N-linked glycans serve as conformational control elements of the RBD. These results collectively indicate that strong RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.18.303644v1?rss=1

Authors: Li, A., Stroik, D. R., Yuen, S. L., Kleinboehl, E., Cornea, R. L., Thomas, D. D.

Abstract:

The Ca-ATPase isoform 2a (SERCA2a) re-sequesters cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. A central factor in heart failure is abnormally high cytosolic [Ca2+], resulting in pathophysiology and decreased cardiac performance. Therefore, augmentation of the SERCA2a Ca2+ transport activity is a promising therapeutic approach. A novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing the protein phospholamban (PLB) from its inhibitory site on SERCA2a. In the present study, we have developed several cell-based FRET biosensor systems for time-resolved FRET (TR-FRET) measurements of the protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the putative SERCA2a binding site, we transiently transfected DWORF into a stable cell line expressing SERCA2a labeled with green fluorescent protein (GFP, the FRET donor) and PLB labeled with red fluorescent protein (RFP, the FRET acceptor). We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Functional analysis demonstrates that DWORF activates SERCA in both the presence and absence of PLB. Furthermore, using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues that are necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.21.305797v1?rss=1

Authors: Kondapuram, M., Frieg, B., Yueksel, S., Schwabe, T., Sattler, C., Lelle, M., Schweinitz, A., Schmauder, R., Benndorf, K., Gohlke, H., Kusch, J.

Abstract:

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics simulations to show that residue K464 of the C-linker is essential for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that both cAMP and K464E induce a rotation of the intracellular domain relative to the channel pore, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. The adopted poses are in excellent agreement with structural results.

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Link to bioRxiv paper:

http://biorxiv.org/cgi/content/short/2020.09.21.306357v1?rss=1

Authors: Ma, J., Su, D., Huang, X., Liang, Y., Ma, Y., Liang, P., Zheng, S.

Abstract:

Less than a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 22 million people worldwide with a death toll approaching 1 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Angstrom and 2.6 Angstrom respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine.

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