Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with Alexa Fluor 568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on their subcellular localization.

pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.

Exploring the Retinal Binding Cavity of Archaerhodopsin-3 by Replacing the Retinal Chromophore With a Dimethyl Phenylated Derivative

Rhodopsins act as photoreceptors with their chromophore retinal (vitamin-A aldehyde) and they regulate light-dependent biological functions. Archaerhodopsin-3 (AR3) is an outward proton pump that has been widely utilized as a tool for optogenetics, a method for controlling cellular activity by light. To characterize the retinal binding cavity of AR3, we synthesized a dimethyl phenylated retinal derivative, (2E,4E,6E,8E)-9-(2,6-Dimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenal (DMP-retinal). QM/MM calculations suggested that DMP-retinal can be incorporated into the opsin of AR3 (archaeopsin-3, AO3). Thus, we introduced DMP-retinal into AO3 to obtain the non-natural holoprotein (AO3-DMP) and compared some molecular properties with those of AO3 with the natural A1-retinal (AO3-A1) or AR3. Light-induced pH change measurements revealed that AO3-DMP maintained slow outward proton pumping. Noteworthy, AO3-DMP had several significant changes in its molecular properties compared with AO3-A1 as follows; 1) spectroscopic measurements revealed that the absorption maximum was shifted from 556 to 508 nm and QM/MM calculations showed that the blue-shift was due to the significant increase in the HOMO-LUMO energy gap of the chromophore with the contribution of some residues around the chromophore, 2) time-resolved spectroscopic measurements revealed the photocycling rate was significantly decreased, and 3) kinetical spectroscopic measurements revealed the sensitivity of the chromophore binding Schiff base to attack by hydroxylamine was significantly increased. The QM/MM calculations show that a cavity space is present at the aromatic ring moiety in the AO3-DMP structure whereas it is absent at the corresponding β-ionone ring moiety in the AO3-A1 structure. We discuss these alterations of the difference in interaction between the natural A1-retinal and the DMP-retinal with binding cavity residues.

Retinal Vibrations in Bacteriorhodopsin are Mechanically Harmonic but Electrically Anharmonic: Evidence From Overtone and Combination Bands

Fundamental vibrations of the chromophore in the membrane protein bacteriorhodopsin (BR), a protonated Schiff base retinal, have been studied for decades, both by resonance Raman and by infrared (IR) difference spectroscopy. Such studies started comparing vibrational changes between the initial BR state (all-trans retinal) and the K intermediate (13-cis retinal), being later extended to the rest of intermediates. They contributed to our understanding of the proton-pumping mechanism of BR by exploiting the sensitivity of fundamental vibrational transitions of the retinal to its conformation. Here, we report on new bands in the 2,500 to 1,800 cm−1 region of the K-BR difference FT-IR spectrum. We show that the bands between 2,500 and 2,300 cm−1 originate from overtone and combination transitions from C-C stretches of the retinal. We assigned bands below 2,300 cm−1 to the combination of retinal C-C stretches with methyl rocks and with hydrogen-out-of-plane vibrations. Remarkably, experimental C-C overtone bands appeared at roughly twice the wavenumber of their fundamentals, with anharmonic mechanical constants ≤3.5 cm−1, and in some cases of ∼1 cm−1. Comparison of combination and fundamental bands indicates that most of the mechanical coupling constants are also very small. Despite the mechanical quasi-harmonicity of the C-C stretches, the area of their overtone bands was only ∼50 to ∼100 times smaller than of their fundamental bands. We concluded that electrical anharmonicity, the second mechanism giving intensity to overtone bands, must be particularly high for the retinal C-C stretches. We corroborated the assignments of negative bands in the K-BR difference FT-IR spectrum by ab initio anharmonic vibrational calculations of all-trans retinal in BR using a quantum-mechanics/molecular mechanics approach, reproducing reasonably well the small experimental anharmonic and coupling mechanical constants. Yet, and in spite accounting for both mechanical and electrical anharmonicities, the intensity of overtone C-C transitions was underestimated by a factor of 4–20, indicating room for improvement in state-of-the-art anharmonic vibrational calculations. The relatively intense overtone and combination bands of the retinal might open the possibility to detect retinal conformational changes too subtle to significantly affect fundamental transitions but leaving a footprint in overtone and combination transitions.

The evolutionary conserved TLDc domain defines a new class of (H+)V-ATPase interacting proteins

We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved “TLDc” domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.

Structure and activation mechanism of the hexameric plasma membrane H+-ATPase

The S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.

Directional Proton Conductance in Bacteriorhodopsin Is Driven by 2 Concentration Gradient, Not Affinity Gradient

It is widely spread that microorganisms can harvest energy from sun light to establish electrochemical potential across cell membrane by pumping protons outward. Light driven proton pumping against a transmembrane gradient entails exquisite electronic and conformational reconfiguration at fs to ms time scales. However, transient molecular events along the photocycle of bacteriorhodopsin are difficult to comprehend from noisy electron density maps obtained from multiple experiments when the intermediate populations coexist and evolve as a function of 13 decades of time. Here I report an in-depth meta-analysis of the recent time-resolved datasets collected by several consortiums. This analysis deconvolutes the observed mixtures, thus substantially improves the quality of the electron density maps, and provides a clear visualization of the isolated intermediates from I to M. The primary photoproducts revealed here suggest a proton transfer uphill against 15 pH units is accomplished by the same physics that governs the tablecloth trick. While the Schiff base is displaced at the beginning of the photoisomerization within ~30 fs, the proton stays due to its inertia. This affinity-independent early deprotonation builds up a steep proton concentration gradient that drives the directional proton conductance toward the extracellular medium. This mechanism fundamentally deviates from the widely adopted assumption based on equilibrium processes driven by light-induced changes of proton affinities. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable.

The Evolutionary Conserved TLDc Domain Defines a New Class of (H+)V-ATPase Interacting Proteins

We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved “TLDc” domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, the purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, the point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs present upstream of the TLDc domain in Ncoa7 and Tldc2 enhancedshow a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.

Plasmodium falciparum vacuolar pyrophosphatase 1 for ring stage development and its transition to trophozoite

It is widely accepted that glycolysis alone is sufficient to support the energy demand of intraerythrocytic malaria parasites when they grow inside RBCs. However, here we show that the metabolic by-product pyrophosphate (PPi) is a critical energy source for ring stage development and the transition from the ring to trophozoite stage. During early phases of the asexual lifecycle, the parasite utilizes PfVP1 (Plasmodium falciparum vacuolar pyrophosphatase 1), an ancient PPi-driven proton pump, to pump protons across the parasite plasma membrane to maintain the membrane potential and cytosolic pH. Conditional deletion of PfVP1 leads to delayed ring stage development and a complete blockage of the ring to trophozoite transition, which can be partially rescued by Arabidopsis thaliana vacuolar pyrophosphatase 1, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae. Proton-pumping pyrophosphatases are absent in humans and animals, which highlights the possibility of developing highly selective VP1 inhibitors against the malaria parasite.