Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Diselenide-bond replacement of the external disulfide bond of insulin increases its oligomerization leading to sustained activity.

Seleno-insulin, a class of artificial insulin analogs, in which one of the three disulfide-bonds (S-S's) of wild-type insulin (Ins) is replaced by a diselenide-bond (Se-Se), is attracting attention for its unique chemical and physiological properties that differ from those of Ins. Previously, we pioneered the development of a [C7UA,C7UB] analog of bovine pancreatic insulin (SeIns) as the first example, and demonstrated its high resistance against insulin-degrading enzyme (IDE). In this study, the conditions for the synthesis of SeIns via native chain assembly (NCA) were optimized to attain a maximum yield of 72%, which is comparable to the in vitro folding efficiency for single-chain proinsulin. When the resistance of BPIns to IDE was evaluated in the presence of SeIns, the degradation rate of BPIns became significantly slower than that of BPIns alone. Furthermore, the investigation on the intermolecular association properties of SeIns and BPIns using analytical ultracentrifugation suggested that SeIns readily forms oligomers not only with its own but also with BPIns. The hypoglycemic effect of SeIns on diabetic rats was observed at a dose of 150 µg/300 g rat. The strategy of replacing the solvent-exposed S-S with Se-Se provides new guidance for the design of long-acting insulin formulations.

Structural basis of CXC chemokine receptor 1 ligand binding and activation.

Neutrophil granulocytes play key roles in innate immunity and shaping adaptive immune responses. They are attracted by chemokines to sites of infection and tissue damage, where they kill and phagocytose bacteria. The chemokine CXCL8 (also known as interleukin-8, abbreviated IL-8) and its G-protein-coupled receptors CXCR1 and CXCR2 are crucial elements in this process, and also the development of many cancers. These GPCRs have therefore been the target of many drug development campaigns and structural studies. Here, we solve the structure of CXCR1 complexed with CXCL8 and cognate G-proteins using cryo-EM, showing the detailed interactions between the receptor, the chemokine and Gαi protein. Unlike the closely related CXCR2, CXCR1 strongly prefers to bind CXCL8 in its monomeric form. The model shows that steric clashes would form between dimeric CXCL8 and extracellular loop 2 (ECL2) of CXCR1. Consistently, transplanting ECL2 of CXCR2 onto CXCR1 abolishes the selectivity for the monomeric chemokine. Our model and functional analysis of various CXCR1 mutants will assist efforts in structure-based drug design targeting specific CXC chemokine receptor subtypes.

E. coli production of a multi-disulfide bonded SARS-CoV-2 Omicron BA.5 RBD exhibiting native-like biochemical and biophysical properties.

Low-cost bacterial production of the receptor binding domain (RBD) of the SARS-CoV-2 Omicron spike protein holds significant potential in expediting the development of therapeutics against COVID-19. However, RBD contains eight cysteines forming four disulfide bonds, and expression in E. coli using standard protocols produces insoluble RBD forming non-native disulfide bonds. Here, we expressed RBD in E. coli T7 SHuffle with high aeration, which enhanced disulfide formation in the cytoplasm and reshuffling of non-native disulfide bonds, and at a low temperature of 16°C, which stabilized the native conformation and thus the formation of the native disulfide bonds. The yield of RBD was as high as 3 mg per 200 mL culture. We analyzed the conformational and biophysical properties of our E. coli-expressed RBD. First, the RP-HPLC elution profile indicated a single peak, suggesting that RBD was folded with a single disulfide bond pairing pattern. Next, circular dichroism analysis indicated a secondary structure content very close to that computed from the crystal structure. RBD's thermal denaturation monitored by CD was cooperative, strongly indicating a well-folded protein structure. Moreover, limited proteolysis showed that RBD was nearly as stable as RNase A, and the formation of native disulfide bonds was confirmed by LC-MS analysis. Furthermore, BLI analysis indicated a strong binding of RBD with the hACE2 with a dissociation constant of 0.83 nM, confirming the folded nature of RBD. Altogether, these results demonstrate that our E. coli-expression system can provide a large amount of highly purified RBD with correct disulfide bonds and native-like biochemical and biophysical properties.

Structural insights into the HBV receptor and bile acid transporter NTCP.

Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.

Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3.

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ In ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we "reverse engineered" PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis.

Speeding drug discovery targeting RNAs: An iterative "RNA selection-compounds screening cycle" for exploring RNA-small molecule pairs.

RNA is an emerging target of next-generation drug development. Recently, new small molecules targeting RNAs were discovered by several pharmaceutical companies. Methods have been reported to identify small molecules targeting a specific RNA sequence and structural motif, however, because of diverse sequence and structural motifs potentially present in the druggable functional RNAs, large sets of structure-activity relationships (SARs) information of small molecule - RNA interactions will be required for the acceleration and efficient startup of the discovery programs toward unprecedented RNA targets. Here we describe our iterative RNA selection and compounds screening to accumulate rich information about small molecules - RNA interaction. The RNAs that selectively bind to the initial molecular target, compound 1 from our in-house chemical library (JT-library), was isolated using in vitro selection technique from a hairpin-structured RNA library mimicking precursor microRNA (pre-miRNA). Then, we engineered pre-let-7f-2 to create its mutant that can bind to compound 1 by embedding the in vitro selected RNA motif for compound 1 in the hairpin loop region. The obtained mutant pre-let-7f-2-loop-mt was used as a target for screening 316 analogs of compound 1. A surface plasmon resonance (SPR) -based screening was performed against pre-let-7f-2-loop-mt-immobilized sensor surface and we obtained four compounds that can bind to the RNA. Among these four compounds, three compounds showed higher affinity to pre-let-7f-2-loop-mt than the parental compound 1, which suggests the feasibility of our strategy for gathering the SAR information on small molecule - RNA interactions. We demonstrated only one cycle of RNA selection and compounds screening in the present study, but can continue this cycle with the selected molecule to gain new RNAs and even new RNA motifs and gather much SAR information with improved accuracy.

The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata.

SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac[Formula: see text](2-3)Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc) and its precursor, asialo-GM1 (Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the [Formula: see text]-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.

A Coil-to-Helix Transition Serves as a Binding Motif for hSNF5 and BAF155 Interaction.

Human SNF5 and BAF155 constitute the core subunit of multi-protein SWI/SNF chromatin-remodeling complexes that are required for ATP-dependent nucleosome mobility and transcriptional control. Human SNF5 (hSNF5) utilizes its repeat 1 (RPT1) domain to associate with the SWIRM domain of BAF155. Here, we employed X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and various biophysical methods in order to investigate the detailed binding mechanism between hSNF5 and BAF155. Multi-angle light scattering data clearly indicate that hSNF5171-258 and BAF155SWIRM are both monomeric in solution and they form a heterodimer. NMR data and crystal structure of the hSNF5171-258/BAF155SWIRM complex further reveal a unique binding interface, which involves a coil-to-helix transition upon protein binding. The newly formed αN helix of hSNF5171-258 interacts with the ß2-α1 loop of hSNF5 via hydrogen bonds and it also displays a hydrophobic interaction with BAF155SWIRM. Therefore, the N-terminal region of hSNF5171-258 plays an important role in tumorigenesis and our data will provide a structural clue for the pathogenesis of Rhabdoid tumors and malignant melanomas that originate from mutations in the N-terminal loop region of hSNF5.

Metastable asymmetrical structure of a shaftless V1 motor.

V1-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A3B3 complex and a central DF shaft. The nucleotide-free A3B3 complex of Enterococcus hirae, composed of three identical A1B1 heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A3B3. The nucleotide-free mutant A3B3 complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A3B3 forms a metastable (stressed) asymmetric structure composed of unstable A1B1 conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

Semiautomatic Volumetry of Low Attenuation of Thoracic Aortic Plaques on Curved Planar Reformations Using MDCT Angiographic Data with 0.5 mm Collimation.

To evaluate the relationship of aortic low attenuation plaque volume (LAPV) on multidetector computed tomography (MDCT) with the abdominal aortic aneurysm (AAA), the coronary arterial disease (CAD, ≥50% stenosis), severe (≥90% stenosis) CAD, hypertension, and long-term (≥10 years) hypertension. Curved planar reformations (CPR) of three segments (the ascending, the arch, and the upper descending aorta) of the thoracic aorta were generated with attenuation-dependent color codes to measure LAPV with 0~29 HU and total noncalcified plaque volume (TNPV) with 0~150 HU in 95 patients. Correlation coefficients were employed to assess the impact of each LAPV and TNPV on AAA, CAD, severe CAD, hypertension, and long-term hypertension. Each Mean LAPV/cm and TNPV/cm was statistically greater in the aortic arch than the ascending (p < 0.001 on each) or the proximal descending segment (p < 0.001 on each). LAPV in the aortic arch has moderate correlations with AAA, severe CAD, and long-term hypertension (r = 0.643, 0.639, 0.662, resp.). Plaque volumes in each aortic segment can be measured clinically and the increasing LAPV in the arch may be a significant factor associated with the development of severe atherosclerosis underlying AAA, severe CAD, and long-term hypertension.

Deep Breathing Improves End-Tidal Carbon Dioxide Monitoring of an Oxygen Nasal Cannula-Based Capnometry Device in Subjects Extubated After Abdominal Surgery.

BACKGROUND: Capnometry detects hypoventilation earlier than pulse oximetry while supplemental oxygen is being administered. We compared the end-tidal CO2 (PETCO2 ) measured using a newly developed oxygen nasal cannula with a CO2-sampling port and the PaCO2 in extubated subjects after abdominal surgery. We also investigated whether the difference between PaCO2 and PETCO2 is affected by resting, by spontaneous breathing with the mouth consciously closed, and by deep breathing with the mouth closed. METHODS: Adult post-abdominal surgery subjects admitted to the ICU were enrolled. After extubation, oxygen was supplied at 4 L/min using a capnometry-type oxygen cannula. The breathing frequency, PETCO2 , and PaCO2 were measured after 30 min of oxygen supplementation. PETCO2 was continuously measured during rest, during breathing with the mouth consciously closed, and during deep breathing with the mouth closed. The difference between PETCO2 and PaCO2 during various breathing patterns was analyzed using the Bland-Altman method. RESULTS: Twenty subjects were included. The bias ± SD (limits of agreement) for breathing frequency measured by capnometry compared with those obtained by direct measurement was 0.4 ± 3.6 (-6.7 to 7.4). In PETCO2 compared with PaCO2 , the biases (limits of agreement) were 14.8 ± 8.2 (-1.3 to 30.9) at rest, 10.2 ± 6.4 (-2.3 to 22.7) with the mouth closed, and 7.7 ± 5.6 (-3.2 to 18.6) for deep breathing with the mouth closed. PETCO2 determined using the capnometry device yielded unreliable and widely ranging values under various breathing patterns. However, deep breathing with the mouth closed decreased the difference between PETCO2 and PaCO2 , as compared with other breathing patterns. CONCLUSIONS: PETCO2 measurements under deep breathing with mouth closed with a capnometry-type oxygen cannula improved the prediction of the absolute value of PaCO2 in extubated post-abdominal surgical subjects without respiratory dysfunction.

Crystal structures of the ATP-binding and ADP-release dwells of the V1 rotary motor.

V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 µM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model.

[The Use of Arginine Vasopressin and PhosphodiesteraseIII Inhibitor for Circulatory Shock after the Resection of a Massive Adrenal Pheochromocytoma].

A 72-year-old man developed hypertensive crisis five month previously, and was diagnosed with massive adrenal pheochromocytoma, with a diameter of 14.5 cm. Preoperative echocardiography revealed normal cardiac function. The open abdominal surgery was performed under general anesthesia. During manipulation of the tumor he developed hypertension and tachycardia. Severe hypotension (50/25 mmHg) and mild bradycardia (70 beats x min(-1)) followed the resection of the tumor. In addition to volume replacement noradrenaline and adrenaline were administered, but the systolic blood pressure rose only to 60-70 mmHg. In order to treat vasodilatory shock, we started to administer arginine vasopressin infusion at 0.03 units x min(-1). His systolic blood pressure rose to 90 mmHg. The patient was transferred to the intensive care unit (ICU) postoperatively. The echocardiography revealed diffuse hypokinetic cardiac function. In order to treat cardiogenic shock, we started to administer olprinone (phosphodiesteraseIII inhibitor, infusion of 0.1 µg x kg(-1) x min(-1)). On postoperative day 2, circulatory shock improved and the patient was discharged from the ICU. In conclusion, circulatory shock after the resection of a massive pheochromocytoma was due to the down regulation of α and ß adrenergic receptors. The non-adrenergic vasoconstrictor and inotrope were useful for this situation.

X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.

Binding interactions of the peripheral stalk subunit isoforms from human V-ATPase.

The mammalian peripheral stalk subunits of the vacuolar-type H(+)-ATPases (V-ATPases) possess several isoforms (C1, C2, E1, E2, G1, G2, G3, a1, a2, a3, and a4), which may play significant role in regulating ATPase assembly and disassembly in different tissues. To better understand the structure and function of V-ATPase, we expressed and purified several isoforms of the human V-ATPase peripheral stalk: E1G1, E1G2, E1G3, E2G1, E2G2, E2G3, C1, C2, H, a1NT, and a2NT. Here, we investigated and characterized the isoforms of the peripheral stalk region of human V-ATPase with respect to their affinity and kinetics in different combination. We found that different isoforms interacted in a similar manner with the isoforms of other subunits. The differences in binding affinities among isoforms were minor from our in vitro studies. However, such minor differences from the binding interaction among isoforms might provide valuable information for the future structural-functional studies of this holoenzyme.

Evaluation of an oxygen mask-based capnometry device in subjects extubated after abdominal surgery.

BACKGROUND: For early detection of respiratory and hemodynamic changes during anesthesia, continuous end-tidal carbon dioxide concentration (PETCO2) is monitored by capnometry. However, the accuracy of CO2 monitoring during spontaneous breathing in extubated patients remains undetermined. Therefore, we aimed to compare P(ETCO2) measured by capnometry using an oxygen mask with a carbon dioxide sampling port (capnometry-type oxygen mask) and P(CO2) in extubated subjects who had undergone abdominal surgery. Furthermore, we investigated whether spontaneous deep breathing affected dissociation between P(aCO2) and P(ETCO2). METHODS: Adult post-abdominal surgery subjects admitted to the ICU were enrolled in this study. After extubation, oxygen was supplied at 6 L/min using the capnometry-type oxygen mask. After 30 min of oxygen supply, P(aCO2) blood gas analysis was performed, and P(ETCO2) was measured under resting and deep-breathing conditions. For both resting and deep-breathing conditions, the correlation between P(aCO2) and P(ETCO2) was analyzed. Furthermore, bias, precision, and limits of agreement were calculated using the Bland-Altman method. RESULTS: Twenty-five subjects (15 men, 10 women) with a mean age of 62 y (interquartile range of 57-76 y) and body mass index of 20-24 kg/m(2) were studied. The correlation (r) between P(aCO2) and P(ETCO2) under resting and deep-breathing conditions was 0.50 and 0.56, respectively. Compared with P(aCO2), the bias and limits of agreement were -12.6 (-20.6 to -4.6) for resting P(ETCO2) and -9.1 (-16.0 to -2.1) for deep-breathing P(ETCO2). The association between P(aCO2) and deep-breathing P(ETCO2) was significantly smaller compared with resting P(ETCO2) (P = .002). CONCLUSIONS: It is possible to measure the P(ETCO2) under varying breathing conditions with the capnometry-type oxygen mask in subjects receiving oxygen supplementation after extubation following upper abdominal surgery to determine whether they are properly ventilating. (ClinicalTrials.gov registration UMIN000011925.).

Loose binding of the DF axis with the A3B3 complex stimulates the initial activity of Enterococcus hirae V1-ATPase.

Vacuolar ATPases (V-ATPases) function as proton pumps in various cellular membrane systems. The hydrophilic V1 portion of the V-ATPase is a rotary motor, in which a central-axis DF complex rotates inside a hexagonally arranged catalytic A3B3 complex by using ATP hydrolysis energy. We have previously reported crystal structures of Enterococcushirae V-ATPase A3B3 and A3B3DF (V1) complexes; the result suggested that the DF axis induces structural changes in the A3B3 complex through extensive protein-protein interactions. In this study, we mutated 10 residues at the interface between A3B3 and DF complexes and examined the ATPase activities of the mutated V1 complexes as well as the binding affinities between the mutated A3B3 and DF complexes. Surprisingly, several V1 mutants showed higher initial ATPase activities than wild-type V1-ATPase, whereas these mutated A3B3 and DF complexes showed decreased binding affinities for each other. However, the high ATP hydrolysis activities of the mutants decreased faster over time than the activity of the wild-type V1 complex, suggesting that the mutants were unstable in the reaction because the mutant A3B3 and DF complexes bound each other more weakly. These findings suggest that strong interaction between the DF complex and A3B3 complex lowers ATPase activity, but also that the tight binding is responsible for the stable ATPase activity of the complex.

Biochemical and biophysical properties of interactions between subunits of the peripheral stalk region of human V-ATPase.

Peripheral stalk subunits of eukaryotic or mammalian vacuolar ATPases (V-ATPases) play key roles in regulating its assembly and disassembly. In a previous study, we purified several subunits and their isoforms of the peripheral stalk region of Homo sapiens (human) V-ATPase; such as C1, E1G1, H, and the N-terminal cytoplasmic region of V(o), a1. Here, we investigated the in vitro binding interactions of the subunits at the stalk region and measured their specific affinities. Surface plasmon resonance experiments revealed that the subunit C1 binds the E1G1 heterodimer with both high and low affinities (2.8 nM and 1.9 µM, respectively). In addition, an E1G1-H complex can be formed with high affinity (48 nM), whereas affinities of other subunit pairs appeared to be low (∼0.21-3.0 µM). The putative ternary complex of C1-H-E1G1 was not much strong on co-incubation of these subunits, indicating that the two strong complexes of C1-E1G1 and H-E1G1 in cooperation with many other weak interactions may be sufficiently strong enough to withstand the torque of rotation during catalysis. We observed a partially stable quaternary complex (consisting of E1G1, C1, a1(NT), and H subunits) resulting from discrete peripheral subunit interactions stabilizing the complex through their intrinsic affinities. No binding was observed in the absence of E1G1 (using only H, C1, and a1(NT)); therefore, it is likely that, in vivo, the E1G1 heterodimer has a significant role in the initiation of subunit assembly. Multiple interactions of variable affinity in the stalk region may be important to the mechanism of reversible dissociation of the intact V-ATPase.