Takashi Higuchi

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H+ or Na+ across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H+-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1–3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

High-precision comparison of the antiproton-to-proton charge-to-mass ratio

Invariance under the charge, parity, time-reversal (CPT) transformation is one of the fundamental symmetries of the standard model of particle physics. This CPT invariance implies that the fundamental properties of antiparticles and their matter-conjugates are identical, apart from signs. There is a deep link between CPT invariance and Lorentz symmetry—that is, the laws of nature seem to be invariant under the symmetry transformation of spacetime—although it is model dependent. A number of high-precision CPT and Lorentz invariance tests—using a co-magnetometer, a torsion pendulum and a maser, among others—have been performed, but only a few direct high-precision CPT tests that compare the fundamental properties of matter and antimatter are available. Here we report high-precision cyclotron frequency comparisons of a single antiproton and a negatively charged hydrogen ion (H−) carried out in a Penning trap system. From 13,000 frequency measurements we compare the charge-to-mass ratio for the antiproton to that for the proton and obtain . The measurements were performed at cyclotron frequencies of 29.6 megahertz, so our result shows that the CPT theorem holds at the atto-electronvolt scale. Our precision of 69 parts per trillion exceeds the energy resolution of previous antiproton-to-proton mass comparisons as well as the respective figure of merit of the standard model extension by a factor of four. In addition, we give a limit on sidereal variations in the measured ratio of <720 parts per trillion. By following the arguments of ref. 11, our result can be interpreted as a stringent test of the weak equivalence principle of general relativity using baryonic antimatter, and it sets a new limit on the gravitational anomaly parameter of < 8.7 × 10−7.

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature 427:36–44; Zimmer J, et al. (2008) Nature 455:936–943; Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107:17182–17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid’s contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal/(mol·Å2), the solvation parameter for transfer from translocon to bilayer is 6–10 cal/(mol·Å2), pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the translocon.

Crystal structure of the cytosolic domain of the cation diffusion facilitator family protein

Divalent cations are essential to all living organisms. For example, Zn is involved in enzymatic catalysis and protein structural stabilization.1 However, the free cytosolic Zn concentration is regulated below the nanomolar level in bacterial cells, because an excess of Zn is toxic.2 Hence, living organisms have systems to export heavy metal ions. Cation diffusion facilitator (CDF) proteins are one group of heavy metal ion efflux transporters.3 This family of proteins is ubiquitously distributed in all three phylogenetic domains, and is present in the bacterial cell membrane, the vacuolar membranes in both plants and yeast, and the Golgi apparatus of animals.4 Furthermore, prokaryotic CDF proteins are known to facilitate the transport of various divalent ions, including Zn, Co, Mn, Fe, Cd, and Ni,5–9 to maintain the homeostasis of these cations. Recently, some structures of prokaryotic CDF proteins were reported. First, the crystal structure of the fulllength YiiP from Escherichia coli was determined at 3.8 Å resolution.10 This dimeric structure presents an outwardfacing Y-shaped structure with the transmembrane domains splayed apart with each protomer binding four zinc ions. Subsequently, both the Zn-bound and apoform structures of the cytosolic domain of Thermus thermophilus CzrB were solved at 1.8 and 1.7 Å resolutions, respectively.11 The Zn-bound dimeric structure of the cytosolic domain of CzrB resembles that of YiiP. In the Zn-bound form of the dimeric CzrB cytosolic domain, the protomers associate with each other via Zn binding. In contrast, in the apo form of the CzrB cytosolic domain, the protomers are splayed apart. This conformational rearrangement is considered to be important for the regulation of the transport activity. Here, we have solved the crystal structure of the cytosolic domain of Thermotoga maritima (TM0876206-306) CDF, and discussed the structural differences between TM0876206-306 and CzrB.

A parts-per-billion measurement of the antiproton magnetic moment

Precise comparisons of the fundamental properties of matter–antimatter conjugates provide sensitive tests of charge–parity–time (CPT) invariance, which is an important symmetry that rests on basic assumptions of the standard model of particle physics. Experiments on mesons, leptons and baryons have compared different properties of matter–antimatter conjugates with fractional uncertainties at the parts-per-billion level or better. One specific quantity, however, has so far only been known to a fractional uncertainty at the parts-per-million level: the magnetic moment of the antiproton, . The extraordinary difficulty in measuring with high precision is caused by its intrinsic smallness; for example, it is 660 times smaller than the magnetic moment of the positron. Here we report a high-precision measurement of in units of the nuclear magneton μN with a fractional precision of 1.5 parts per billion (68% confidence level). We use a two-particle spectroscopy method in an advanced cryogenic multi-Penning trap system. Our result  = −2.7928473441(42)μN (where the number in parentheses represents the 68% confidence interval on the last digits of the value) improves the precision of the previous best measurement by a factor of approximately 350. The measured value is consistent with the proton magnetic moment, μp = 2.792847350(9)μN, and is in agreement with CPT invariance. Consequently, this measurement constrains the magnitude of certain CPT-violating effects to below 1.8 × 10−24 gigaelectronvolts, and a possible splitting of the proton–antiproton magnetic moments by CPT-odd dimension-five interactions to below 6 × 10−12 Bohr magnetons.

Structural basis for amino acid export by DMT superfamily transporter YddG

The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins.

Double-trap measurement of the proton magnetic moment at 0.3 parts per billion precision.

Nailing down the proton magnetic moment Fundamental physical laws are believed to remain the same if subjected to three simultaneous transformations: flipping the sign of electric charge, taking a mirror image, and running time backward. To test this charge, parity, and time-reversal (CPT) symmetry, it is desirable to know the fundamental properties of particles such as the proton to high precision. Schneider et al. used a double ion trap to determine the magnetic moment of a single trapped proton to a precision of 0.3 parts per billion. Comparatively precise measurements of the same quantity in the antiproton are now needed for a rigorous test of CPT symmetry. Science, this issue p. 1081 An optimized double–Penning trap technique improves the precision measurement of the proton magnetic moment by a factor of 11. Precise knowledge of the fundamental properties of the proton is essential for our understanding of atomic structure as well as for precise tests of fundamental symmetries. We report on a direct high-precision measurement of the magnetic moment μp of the proton in units of the nuclear magneton μN. The result, μp = 2.79284734462 (±0.00000000082) μN, has a fractional precision of 0.3 parts per billion, improves the previous best measurement by a factor of 11, and is consistent with the currently accepted value. This was achieved with the use of an optimized double–Penning trap technique. Provided a similar measurement of the antiproton magnetic moment can be performed, this result will enable a test of the fundamental symmetry between matter and antimatter in the baryonic sector at the 10−10 level.

Highly-sensitive superconducting circuits at ~700 kHz with tunable quality factors for image-current detection of single trapped antiprotons

We developed highly sensitive image-current detection systems based on superconducting toroidal coils and ultra-low noise amplifiers for non-destructive measurements of the axial frequencies (550-800 kHz) of single antiprotons stored in a cryogenic multi-Penning-trap system. The unloaded superconducting tuned circuits show quality factors of up to 500 000, which corresponds to a factor of 10 improvement compared to our previously used solenoidal designs. Connected to ultra-low noise amplifiers and the trap system, signal-to-noise-ratios of 30 dB at quality factors of >20 000 are achieved. In addition, we have developed a superconducting switch which allows continuous tuning of the detectors quality factor and to sensitively tune the particle-detector interaction. This allowed us to improve frequency resolution at constant averaging time, which is crucial for single antiproton spin-transition spectroscopy experiments, as well as improved measurements of the proton-to-antiproton charge-to-mass ratio.

Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

Significance Membrane proteins are central players in all cells, and their structure and function are under intense study. However, we still lack a detailed understanding of the process whereby they are integrated into biological membranes. Most membrane proteins are integrated cotranslationally into the membrane bilayer. Although the energetics that drive membrane protein integration are known in outline, detailed studies are difficult because the naturally occurring amino acids represent only a limited set of side-chain chemistries. Here we use synthetic, nonproteinogenic amino acids engineered into a transmembrane segment to systematically probe the energetics of membrane insertion in a way not possible with the set of natural amino acids. Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of “snorkeling” of charged amino acids toward the lipid–water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔGapp. These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells.

Precise Measurement of Muonium HFS at J-PARC MUSE

Hiroyuki A. Torii1 on behalf of MuSEUM Collaboration∗. H. A. Torii1, M. Aoki2, Y. Fukao3, Y. Higashi1, T. Higuchi1, H. Iinuma3, Y. Ikedo3, K. Ishida4, M. Iwasaki4, R. Kadono3, O. Kamigaito4, S. Kanda5, D. Kawall6, N. Kawamura3, A. Koda3, K. M. Kojima3, K. Kubo7, Y. Matsuda1, T. Mibe3, Y. Miyake3, T. Mizutani1, K. Nagamine4, K. Nishiyama3, T. Ogitsu3, R. Okubo3, N. Saito3, K. Sasaki3, K. Shimomura3, P. Strasser3, M. Sugano3, M. Tajima1, K. S. Tanaka1,4 D. Tomono4†, E. Torikai8, A. Toyoda3, K. Ueno3, Y. Ueno1, A. Yamamoto3, and M. Yoshida3. 1Graduate School of Arts and Sciences, University of Tokyo; 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan 2Osaka University; Toyonaka, Osaka 560-0043, Japan 3KEK; 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan 4RIKEN; 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 5Department of Physics, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 6University of Massachusetts Amherst; MA 01003-9337, USA 7International Christian University (ICU); Mitaka, Tokyo 181-8585, Japan 8University of Yamanashi; Kofu, Yamanashi 400-8511, Japan †Current affiliation: Kyoto University; Kyoto 606-8501, Japan ∗The collaboration name MuSEUM stands for “Muonium Spectroscopy Experiment Using Microwave.” E-mail: torii@radphys4.c.u-tokyo.ac.jp