Toward standardization of tau PET imaging corresponding to various tau PET tracers: a multicenter phantom study.

OBJECTIVE: Tau positron emission tomography (PET) imaging is a recently developed non-invasive tool that can detect the density and extension of tau neurofibrillary tangles. Tau PET tracers have been validated to harmonize and accelerate their development and implementation in clinical practice. Whereas standard protocols including injected dose, uptake time, and duration have been determined for tau PET tracers, reconstruction parameters have not been standardized. The present study conducted phantom experiments based on tau pathology to standardize quantitative tau PET imaging parameters and optimize reconstruction conditions of PET scanners at four Japanese sites according to the results of phantom experiments. METHODS: The activity of 4.0 and 2.0 kBq/mL for Hoffman 3D brain and cylindrical phantoms, respectively, was estimated from published studies of brain activity using [18F]flortaucipir, [18F]THK5351, and [18F]MK6240. We developed an original tau-specific volume of interest template for the brain based on pathophysiological tau distribution in the brain defined as Braak stages. We acquired brain and cylindrical phantom images using four PET scanners. Iteration numbers were determined as contrast and recover coefficients (RCs) in gray (GM) and white (WM) matter, and the magnitude of the Gaussian filter was determined from image noise. RESULTS: Contrast and RC converged at ≥ 4 iterations, the error rates of RC for GM and WM were < 15% and 1%, respectively, and noise was < 10% in Gaussian filters of 2-4 mm in images acquired using the four scanners. Optimizing the reconstruction conditions for phantom tau PET images acquired by each scanner improved contrast and image noise. CONCLUSIONS: The phantom activity was comprehensive for first- and second-generation tau PET tracers. The mid-range activity that we determined could be applied to later tau PET tracers. We propose an analytical tau-specific VOI template based on tau pathophysiological changes in patients with AD to standardize tau PET imaging. Phantom images reconstructed under the optimized conditions for tau PET imaging achieved excellent image quality and quantitative accuracy.

Role of Tryptophan 38 in Loading Substrate Chain into the Active-site Tunnel of Cellobiohydrolase I from Trichoderma reesei.

Cellobiohydrolase I from Trichoderma reesei ( Tr Cel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of Tr Cel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of Tr Cel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of Tr Cel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of Tr Cel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake ( k on for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.

Molecular mechanism underlying the selective attack of trehalose lipids on cancer cells as revealed by coarse-grained molecular dynamics simulations.

The present study indicated that the mixed lipid bilayer of dimyristoylphosphatidylcholine (DMPC) and trehalosemonomyristate (TreC14) interacted strongly with the plasma membrane of cancer cells, and not that of normal cells, when the composition of TreC14 was 70%, as revealed by coarse-grained molecular dynamics simulations. These results were consistent with those of previous experimental studies, indicating that DMPC/TreC14 mixed liposomes (DMTreC14) with TreC14 composition at 70% exhibited a strong anti-cancer effect without affecting normal cells. The simulations also revealed that lipids with highly hydrophilic and bulky head groups, such as TreC14, phosphatidylinositol (PI), and phosphatidylserine (PS), showed the tendency to accumulate. This caused both the DMTreC14 and cancer cell membranes to bend into large positive curvatures, resulting in tight contact between them. In contrast, no apparent interaction between the DMTreC14 and normal cell membranes was observed because PI and PS did not exist in the extracellular monolayer of the normal cell membrane.

Clinical practice guidelines for high-resolution breast PET, 2019 edition.

Breast positron emission tomography (PET) has had insurance coverage when performed with conventional whole-body PET in Japan since 2013. Together with whole-body PET, accurate examination of breast cancer and diagnosis of metastatic disease are possible, and are expected to contribute significantly to its treatment planning. To facilitate a safer, smoother, and more appropriate examination, the Japanese Society of Nuclear Medicine published the first edition of practice guidelines for high-resolution breast PET in 2013. Subsequently, new types of breast PET have been developed and their clinical usefulness clarified. Therefore, the guidelines for breast PET were revised in 2019. This article updates readers as to what is new in the second edition. This edition supports two different types of breast PET depending on the placement of the detector: the opposite-type (positron emission mammography; PEM) and the ring-shaped type (dedicated breast PET; dbPET), providing an overview of these scanners and appropriate imaging methods, their clinical applications, and future prospects. The name "dedicated breast PET" from the first edition is widely used to refer to ring-shaped type breast PET. In this edition, "breast PET" has been defined as a term that refers to both opposite- and ring-shaped devices. Up-to-date breast PET practice guidelines would help provide useful information for evidence-based breast imaging.

Crystalline chitin hydrolase is a burnt-bridge Brownian motor.

Motor proteins are essential units of life and are well-designed nanomachines working under thermal fluctuations. These proteins control moving direction by consuming chemical energy or by dissipating electrochemical potentials. Chitinase A from bacterium Serratia marcescens (SmChiA) processively moves along crystalline chitin by hydrolysis of a single polymer chain to soluble chitobiose. Recently, we directly observed the stepping motions of SmChiA labeled with a gold nanoparticle by dark-field scattering imaging to investigate the moving mechanism. Time constants analysis revealed that SmChiA moves back and forth along the chain freely, because forward and backward states have a similar free energy level. The similar probabilities of forward-step events (83.5%=69.3%+14.2%) from distributions of step sizes and chain-hydrolysis (86.3%=(1/2.9)/(1/2.9+1/18.3)×100) calculated from the ratios of time constants of hydrolysis and the backward step indicated that SmChiA moves forward as a result of shortening of the chain by a chitobiose unit, which stabilizes the backward state. Furthermore, X-ray crystal structures of sliding intermediate and molecular dynamics simulations showed that SmChiA slides forward and backward under thermal fluctuation without large conformational changes of the protein. Our results demonstrate that SmChiA is a burnt-bridge Brownian ratchet motor.

CD8+ regulatory T cells are critical in prevention of autoimmune-mediated diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic ß-cells are destroyed. Intestinal helminths can cause asymptomatic chronic and immunosuppressive infections and suppress disease in rodent models of T1D. However, the underlying regulatory mechanisms for this protection are unclear. Here, we report that CD8+ regulatory T (Treg) cells prevent the onset of streptozotocin -induced diabetes by a rodent intestinal nematode. Trehalose derived from nematodes affects the intestinal microbiota and increases the abundance of Ruminococcus spp., resulting in the induction of CD8+ Treg cells. Furthermore, trehalose has therapeutic effects on both streptozotocin-induced diabetes and in the NOD mouse model of T1D. In addition, compared with healthy volunteers, patients with T1D have fewer CD8+ Treg cells, and the abundance of intestinal Ruminococcus positively correlates with the number of CD8+ Treg cells in humans.

Group 3 LEA Protein Model Peptides Suppress Heat-Induced Lysozyme Aggregation. Elucidation of the Underlying Mechanism Using Coarse-Grained Molecular Simulations.

We investigated experimentally whether a short peptide, PvLEA-22, which consists of two tandem repeats of an 11-mer motif of Group 3 late embryogenesis abundant proteins, has a chaperone-like function for denatured proteins. Lysozyme was selected as a target protein. Turbidity measurements indicated that the peptide suppresses the heat-induced aggregation of lysozyme when added at a molar ratio of PvLEA-22/lysozyme >40. Circular dichroism and differential scanning calorimetry measurements confirmed that the lysozyme was denatured on heating but spontaneously refolded on subsequent cooling in the presence of the peptide. As a result, up to 80% of the native catalytic activity of lysozyme was preserved. Similar chaperone-like activity was also observed for a peptide with the same amino acid composition as PvLEA-22 but whose sequence is scrambled. To elucidate the underlying mechanism of the chaperone function of these peptides, we performed coarse-grained molecular dynamics simulations. This revealed that a denatured lysozyme molecule is shielded by several peptide molecules in aqueous solution, which acts as a physical barrier, reducing the opportunities for collision between denatured proteins. An important finding was that a peptide bound to the denatured protein is very rapidly replaced by another; due to such rapid exchange, peptide-protein contact time is very short, that is, on the order of ∼200 ns. Therefore, the peptide does not constrain the behavior of the denatured protein, which can refold freely.

A novel ring-shaped reaction pathway with interconvertible intermediates in chitinase A as revealed by QM/MM simulation combined with a one-dimensional projection technique.

Substrate-assisted catalysis (SAC), a mechanism of chitin hydrolysis by chitinases belonging to the glycoside hydrolase family 18 (GH18), has been studied experimentally and theoretically for several decades. However, the detailed reaction mechanism in chitinase A (ChiA) remains unclear at the atomic level. In this study, we investigated glycosylation, the first step of SAC, of ChiA obtained from Serratia marcescens (SmChiA), using QM/MM simulations combined with a one-dimensional projection (ODP) technique, which enabled us to explore the multi-dimensional free energy surface efficiently. The results showed that the reaction proceeds via a novel ring-shaped concerted reaction pathway with interconvertible intermediates, viz. oxazolinium ion and oxazoline, which have not been fully identified in previous studies. We also compared this chitin hydrolysis mechanism in SmChiA with that in SmChiB reported previously. The computational protocol developed in this study could also be applicable for elucidating complicated reaction mechanisms in other enzymes.

Cationic Copolymer-Chaperoned 2D-3D Reversible Conversion of Lipid Membranes.

Nanosheets have thicknesses on the order of nanometers and planar dimensions in the micrometer range. Nanomaterials that are capable of converting reversibly between 2D nanosheets and 3D structures in response to specific triggers can enable construction of nanodevices. Supra-molecular lipid nanosheets and their triggered conversions to 3D structures including vesicles and cups are reported. They are produced from lipid vesicles upon addition of amphiphilic peptides and cationic copolymers that act as peptide chaperones. By regulation of the chaperoning activity of the copolymer, 2D to 3D conversions are reversibly triggered, allowing tuning of lipid bilayer structures and functionalities.

Development of an ATP force field for coarse-grained simulation of ATPases and its application to the maltose transporter.

The biological functions of ATPases, such as myosin, kinesin, and ABC transporter, are due to large conformational motions driven by energy obtained from ATP. Elucidation of the mechanisms underlying these ATP-driven movements is one of the greatest challenges in computational chemistry. It has been shown that the MARTINI coarse-grained method is a promising tool for the investigation of large conformational motions in various proteins. However, this method has not yet been applied to ATPases because of the lack of a force field for the ATP molecule. Here, we developed force field parameters for the ATP molecule and conducted simulations using these parameters for the subunits (MalK2 ) and the full-length structure (MalFGK2 -E) of a maltose transporter. It was found for both targets that the dimerization of the nucleotide binding domains (NBDs) is induced upon ATP binding. Moreover, for the full-length transporter, the conformational transition from the pre-translocation state to the outward-facing state was observed and was accompanied by an initial transport motion of the substrate. It is expected that coarse-grained simulations utilizing the parameters for the ATP molecule developed here will serve as a powerful tool for investigating other ATPases as well. © 2019 Wiley Periodicals, Inc.

Identification of a novel strong promoter from the anhydrobiotic midge, Polypedilum vanderplanki, with conserved function in various insect cell lines.

Larvae of the African midge Polypedilum vanderplanki (Diptera: Chironomidae) show a form of extreme desiccation tolerance known as anhydrobiosis. The cell line Pv11 was recently established from the species, and these cells can also survive under desiccated conditions, and proliferate normally after rehydration. Here we report the identification of a new promoter, 121, which has strong constitutive transcriptional activity in Pv11 cells and promotes effective expression of exogenous genes. Using a luciferase reporter assay, this strong transcriptional activity was shown to be conserved in cell lines from various insect species, including S2 (Drosophila melanogaster, Diptera), SaPe-4 (Sarcophaga peregrina, Diptera), Sf9 (Spodoptera frugiperda, Lepidoptera) and Tc81 (Tribolium castaneum, Coleoptera) cells. In conjunction with an appropriate selection maker gene, the 121 promoter was able to confer zeocin resistance on SaPe-4 cells and allowed the establishment of stable SaPe-4 cell lines expressing the fluorescent protein AcGFP1; this is the first report of heterologous gene expression in this cell line. These results show the 121 promoter to be a versatile tool for exogenous gene expression in a wide range of insect cell lines, particularly useful to those from non-model insect species.

A LEA model peptide protects the function of a red fluorescent protein in the dry state.

We tested whether a short model peptide derived from a group 3 late embryogenesis abundant (G3LEA) protein is able to maintain the fluorescence activity of a red fluorescent protein, mKate2, in the dry state. The fluorescence intensity of mKate2 alone decreased gradually through repeated dehydration-rehydration treatments. However, in the presence of the LEA model peptide, the peak intensity was maintained almost perfectly during such stress treatments, which implies that the three dimensional structure of the active site of mKate2 was protected even under severe desiccation conditions. For comparison, similar experiments were performed with other additives such as a native G3LEA protein, trehalose and BSA, all of whose protective abilities were lower than that of the LEA model peptide.

Replica exchange molecular dynamics simulation study on the mechanism of desiccation-induced structuralization of an intrinsically disordered peptide as a model of LEA proteins.

Group 3 late embryogenesis abundant (G3LEA) proteins, which act as a well-characterized desiccation protectant in anhydrobiotic organisms, are structurally disordered in solution, but they acquire a predominantly α-helical structure during drying. Thus, G3LEA proteins are now accepted as intrinsically disordered proteins (IDPs). Their functional regions involve characteristic 11-mer repeating motifs. In the present study, to elucidate the origin of the IDP property of G3LEA proteins, we applied replica exchange molecular dynamics (REMD) simulation to a model peptide composed of two tandem repeats of an 11-mer motif and its counterpart peptide whose amino acid sequence was randomized with the same amino acid composition as that of the 11-mer motif. REMD simulations were performed for a single α-helical chain of each peptide and its double-bundled strand in a wide water content ranging from 5 to 78.3 wt%. In the latter case, we tested different types of arrangement: 1) the dipole moments of the two helices were parallel or anti-parallel and 2) due to the amphiphilic nature of the α-helix of the 11-mer motif, two types of the side-to-side contact were tested: hydrophilic-hydrophilic facing or hydrophobic-hydrophobic facing. Here, we revealed that the single chain alone exhibits no IDP-like properties, even if it involves the 11-mer motif, and the hydrophilic interaction of the two chains leads to the formation of a left-handed α-helical coiled coil in the dry state. These results support the cytoskeleton hypothesis that has been proposed as a mechanism by which G3LEA proteins work as a desiccation protectant.

Physicochemical Aspects of the Biological Functions of Trehalose and Group 3 LEA Proteins as Desiccation Protectants.

In this review, we first focus on the mechanism by which the larva of the sleeping chironomid, Polypedilum vanderplanki, survives an extremely dehydrated state and describe how trehalose and probably late embryogenesis abundant (LEA) proteins work as desiccation protectants. Second, we summarize the solid-state and solution properties of trehalose and discuss why trehalose works better than other disaccharides as a desiccation protectant. Third, we describe the structure and function of two model peptides based on group 3 LEA proteins after a short introduction of native LEA proteins themselves. Finally, we present our conclusions and a perspective on the application of trehalose and LEA model peptides to the long-term storage of biological materials.

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin.

Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s-1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.

Multicenter study of quantitative PET system harmonization using NIST-traceable 68Ge/68Ga cross-calibration kit.

PURPOSE: The present study aimed to define the errors in SUV and demonstrate the feasibility of SUV harmonization among contemporary PET/CT scanners using a novel National Institute of Standards and Technology (NIST)-traceable 68Ge/68Ga source as the reference standard. METHODS: We used 68Ge/68Ga dose calibrator and PET sources made with same batch of 68Ge/68Ga embedded in epoxy that is traceable to the NIST standard. Bias in the amount of radioactivity and the radioactive concentrations measured by the dose calibrators and PET/CT scanners, respectively, was determined at five Japanese sites. We adjusted optimal dial setting of the dose calibrators and PET reconstruction parameters to close the actual amount of radioactivity and the radioactive concentration, respectively, of the NIST-traceable 68Ge/68Ga sources to harmonize SUV. Errors in SUV before and after harmonization were then calculated at each site. RESULTS: The average bias in the amount of radioactivity and the radioactive concentrations measured by dose calibrator and PET scanner was -4.94% and -12.22%, respectively, before, and -0.14% and -4.81%, respectively, after harmonization. Corresponding averaged errors in SUV measured under clinical conditions were underestimated by 7.66%, but improved by -4.70% under optimal conditions. CONCLUSION: Our proposed method using an NIST-traceable 68Ge/68Ga source identified bias in values obtained using dose calibrators and PET scanners, and reduced SUV variability to within 5% across different models of PET scanners at five sites. Our protocol using a standard source has considerable potential for harmonizing the SUV when contemporary PET scanners are involved in multicenter studies.

Multicentre analysis of PET SUV using vendor-neutral software: the Japanese Harmonization Technology (J-Hart) study.

BACKGROUND: Recent developments in hardware and software for PET technologies have resulted in wide variations in basic performance. Multicentre studies require a standard imaging protocol and SUV harmonization to reduce inter- and intra-scanner variability in the SUV. The Japanese standardised uptake value (SUV) Harmonization Technology (J-Hart) study aimed to determine the applicability of vendor-neutral software on the SUV derived from positron emission tomography (PET) images. The effects of SUV harmonization were evaluated based on the reproducibility of several scanners and the repeatability of an individual scanner. Images were acquired from 12 PET scanners at nine institutions. PET images were acquired over a period of 30 min from a National Electrical Manufacturers Association (NEMA) International Electrotechnical Commission (IEC) body phantom containing six spheres of different diameters and an 18F solution with a background activity of 2.65 kBq/mL and a sphere-to-background ratio of 4. The images were reconstructed to determine parameters for harmonization and to evaluate reproducibility. PET images with 2-min acquisition × 15 contiguous frames were reconstructed to evaluate repeatability. Various Gaussian filters (GFs) with full-width at half maximum (FWHM) values ranging from 1 to 15 mm in 1-mm increments were also applied using vendor-neutral software. The SUVmax of spheres was compared with the reference range proposed by the Japanese Society of Nuclear Medicine (JSNM) and the digital reference object (DRO) of the NEMA phantom. The coefficient of variation (CV) of the SUVmax determined using 12 PET scanners (CVrepro) was measured to evaluate reproducibility. The CV of the SUVmax determined from 15 frames (CVrepeat) per PET scanner was measured to determine repeatability. RESULTS: Three PET scanners did not require an additional GF for harmonization, whereas the other nine required additional FWHM values of GF ranging from 5 to 9 mm. The pre- and post-harmonization CVrepro of six spheres were (means ± SD) 9.45% ± 4.69% (range, 3.83-15.3%) and 6.05% ± 3.61% (range, 2.30-10.7%), respectively. Harmonization significantly improved reproducibility of PET SUVmax (P = 0.0055). The pre- and post-harmonization CVrepeat of nine scanners were (means ± SD) 6.59% ± 1.29% (range, 5.00-8.98%) and 4.88% ± 1.64% (range, 2.65-6.72%), respectively. Harmonization also significantly improved the repeatability of PET SUVmax (P < 0.0001). CONCLUSIONS: Harmonizing SUV using vendor-neutral software produced SUVmax for 12 scanners that fell within the JSNM reference range of a NEMA body phantom and improved SUVmax reproducibility and repeatability.

Molecular dynamics simulation study on the structural instability of the most common cystic fibrosis-associated mutant ΔF508-CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that belongs to the ATP binding cassette protein superfamily. Deletion of phenylalanine at position 508 (ΔF508) is the most common CF-associated mutation and is present in nearly 90% of CF patients. Currently, atomistic level studies are insufficient for understanding the mechanism by which the deletion of a single amino acid causes greatly reduced folding as well as trafficking and gating defects. To clarify this mechanism, we first constructed an atomic model of the inward-facing ΔF508-CFTR and performed allatom molecular dynamics (MD) simulations of the protein in a membrane environment. All of the computational methodologies used are based on those developed in our previous study for wild-type CFTR. Two important findings were obtained. First, consistent with several previous computational results, the deletion of F508 causes a disruption of a hydrophobic cluster located at the interface between the nucleotide binding domain 1 (NBD1) and intracellular loop 4 (ICL4). This exerts unfavorable influences on the correlated motion between ICLs and transmembrane domains (TMDs), likely resulting in gating defects. Second, the F508 deletion affected the NBD1-NBD2 interface via allosteric communication originating from the correlated motion between NBDs and ICLs. As a result, several unusual inter-residue interactions are caused at the NBD1-NBD2 interface. In other words, their correct dimerization is impaired. This study provided insight into the atomic-level details of structural and dynamics changes caused by the ΔF508 mutation and thus provides good insight for drug design.

The mechanism of nucleotide-binding domain dimerization in the intact maltose transporter as studied by all-atom molecular dynamics simulations.

The Escherichia coli maltose transporter MalFGK2 -E belongs to the protein superfamily of ATP-binding cassette (ABC) transporters. This protein is composed of heterodimeric transmembrane domains (TMDs) MalF and MalG, and the homodimeric nucleotide-binding domains (NBDs) MalK2 . In addition to the TMDs and NBDs, the periplasmic maltose binding protein MalE captures maltose and shuttle it to the transporter. In this study, we performed all-atom molecular dynamics (MD) simulations on the maltose transporter and found that both the binding of MalE to the periplasmic side of the TMDs and binding of ATP to the MalK2 are necessary to facilitate the conformational change from the inward-facing state to the occluded state, in which MalK2 is completely dimerized. MalE binding suppressed the fluctuation of the TMDs and MalF periplasmic region (MalF-P2), and thus prevented the incorrect arrangement of the MalF C-terminal (TM8) helix. Without MalE binding, the MalF TM8 helix showed a tendency to intrude into the substrate translocation pathway, hindering the closure of the MalK2 . This observation is consistent with previous mutagenesis experimental results on MalF and provides a new point of view regarding the understanding of the substrate translocation mechanism of the maltose transporter.

Probing native metal ion association sites through quenching of fluorophores in the nucleotide-binding domains of the ABC transporter MsbA.

ATP-binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells. Binding of ATP to the nucleotide-binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane. The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs. Here, we labeled the ABC transporter MsbA, a homodimeric flippase from Escherichia coli, with a fluorescent probe, Alexa532, within the NBDs. ATP application elicited collisional quenching, whereas no quenching was observed after the addition of ATP analogs or ATP hydrolysis inhibitors. The Alexa532-conjugated MsbA variants exhibited transition metal ion Förster resonance energy transfer (tmFRET) after the addition of Ni2+, and ATP decreased this Ni2+-mediated FRET of the NBDs. Structure modeling developed from crystallographic data and examination of tmFRET measurements of MsbA variants in the absence of ATP revealed the presence of metal ion-associated pockets (MiAPs) in the NBDs. Three histidines were predicted to participate in chelating Ni2+ in the two possible MiAPs. Performing histidine-substitution experiments with the NBDs showed that the dissociation constant for Ni2+ of MiAP2 was smaller than that of MiAP1. The structural allocation of the MiAPs was further supported by showing that the addition of Cu2+ resulted in higher quenching than Ni2+ Taken together, the present study showed that the NBDs contain two native binding sites for metal ions and ATP addition affects the Ni2+-binding activity of the MiAPs.