The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Deep learning assisted single particle tracking for automated correlation between diffusion and function.

Sub-cellular diffusion in living systems reflects cellular processes and interactions. Recent advances in optical microscopy allow the tracking of this nanoscale diffusion of individual objects with an unprecedented level of precision. However, the agnostic and automated extraction of functional information from the diffusion of molecules and organelles within the sub-cellular environment, is labor-intensive and poses a significant challenge. Here we introduce DeepSPT, a deep learning framework to interpret the diffusional 2D or 3D temporal behavior of objects in a rapid and efficient manner, agnostically. Demonstrating its versatility, we have applied DeepSPT to automated mapping of the early events of viral infections, identifying distinct types of endosomal organelles, and clathrin-coated pits and vesicles with up to 95% accuracy and within seconds instead of weeks. The fact that DeepSPT effectively extracts biological information from diffusion alone illustrates that besides structure, motion encodes function at the molecular and subcellular level.

Deep learning assisted single particle tracking for automated correlation between diffusion and function.

Sub-cellular diffusion in living systems reflects cellular processes and interactions. Recent advances in optical microscopy allow the tracking of this nanoscale diffusion of individual objects with an unprecedented level of precision. However, the agnostic and automated extraction of functional information from the diffusion of molecules and organelles within the sub-cellular environment, is labor-intensive and poses a significant challenge. Here we introduce DeepSPT, a deep learning framework to interpret the diffusional 2D or 3D temporal behavior of objects in a rapid and efficient manner, agnostically. Demonstrating its versatility, we have applied DeepSPT to automated mapping of the early events of viral infections, identifying distinct types of endosomal organelles, and clathrin-coated pits and vesicles with up to 95% accuracy and within seconds instead of weeks. The fact that DeepSPT effectively extracts biological information from diffusion alone indicates that besides structure, motion encodes function at the molecular and subcellular level.

The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both outer-layer proteins from the particle. The other outer-layer protein, VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Conformational changes during the reaction cycle of plasma membrane Ca2+-ATPase in the autoinhibited and activated states.

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity. Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2·Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

FXYD protein isoforms differentially modulate human Na/K pump function.

Tight regulation of the Na/K pump is essential for cellular function because this heteromeric protein builds and maintains the electrochemical gradients for Na+ and K+ that energize electrical signaling and secondary active transport. We studied the regulation of the ubiquitous human α1ß1 pump isoform by five human FXYD proteins normally located in muscle, kidney, and neurons. The function of Na/K pump α1ß1 expressed in Xenopus oocytes with or without FXYD isoforms was evaluated using two-electrode voltage clamp and patch clamp. Through evaluation of the partial reactions in the absence of K+ but presence of Na+ in the external milieu, we demonstrate that each FXYD subunit alters the equilibrium between E1P(3Na) and E2P, the phosphorylated conformations with Na+ occluded and free from Na+, respectively, thereby altering the apparent affinity for Na+. This modification of Na+ interaction shapes the small effects of FXYD proteins on the apparent affinity for external K+ at physiological Na+. FXYD6 distinctively accelerated both the Na+-deocclusion and the pump-turnover rates. All FXYD isoforms altered the apparent affinity for intracellular Na+ in patches, an effect that was observed only in the presence of intracellular K+. Therefore, FXYD proteins alter the selectivity of the pump for intracellular ions, an effect that could be due to the altered equilibrium between E1 and E2, the two major pump conformations, and/or to small changes in ion affinities that are exacerbated when both ions are present. Lastly, we observed a drastic reduction of Na/K pump surface expression when it was coexpressed with FXYD1 or FXYD6, with the former being relieved by injection of PKA's catalytic subunit into the oocyte. Our results indicate that a prominent effect of FXYD1 and FXYD6, and plausibly other FXYDs, is the regulation of Na/K pump trafficking.

E2P-like states of plasma membrane Ca2+­ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues.

The plasma membrane Ca2+­ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeFx), aluminium fluoride (AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·Pi product states. Our results show that the phosphate analogues BeFx, AlFx and MgFx inhibit PMCA Ca2+­ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca2+­ATPase inhibition by AlFx and BeFx depended on Mg2+ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlFx and BeFx but not MgFx apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E → E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA. In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell.

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):502-515, 2018.

Aluminum inhibits the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases by different mechanisms.

Aluminum (Al3+) is involved in the pathophysiology of neurodegenerative disorders. The mechanisms that have been proposed to explain the action of Al3+ toxicity are linked to changes in the cellular calcium homeostasis, placing the transporting calcium pumps as potential targets. The aim of this work was to study the molecular inhibitory mechanism of Al3+ on Ca2+-ATPases such as the plasma membrane and the sarcoplasmic reticulum calcium pumps (PMCA and SERCA, respectively). These P-ATPases transport Ca2+ actively from the cytoplasm towards the extracellular medium and to the sarcoplasmic reticulum, respectively. For this purpose, we performed enzymatic measurements of the effect of Al3+ on purified preparations of PMCA and SERCA. Our results show that Al3+ is an irreversible inhibitor of PMCA and a slowly-reversible inhibitor of SERCA. The binding of Al3+ is affected by Ca2+ in SERCA, though not in PMCA. Al3+ prevents the phosphorylation of SERCA and, conversely, the dephosphorylation of PMCA. The dephosphorylation time courses of the complex formed by PMCA and Al3+ (EPAl) in the presence of ADP or ATP show that EPAl is composed mainly by the conformer E2P. This work shows for the first time a distinct mechanism of Al3+ inhibition that involves different intermediates of the reaction cycle of these two Ca2+-ATPases.