The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively. For better understanding of this important xenobiotic transporter, we show the xray crystallographic structure of MexA at a resolution of 2.40 Å. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod ␣-helical domain was globular and constructed by a cluster of eight short ␤-sheets. The third domain located distal to the ␣-helical rod was globular and composed of seven short ␤-sheets and one short ␣-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic ␣-barrel domain of OprM via the ␣-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.

The ␥ subunit of the ATP synthase F 1 sector rotates at the center of the ␣ 3 ␤ 3 hexamer during ATP hydrolysis. A gold bead (40 -200 nm diameter) was attached to the ␥ subunit of Escherichia coli F 1 , and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40-and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is ϳ13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F 1 sector rotates much faster than expected from the bulk of ATPase activity, and that ϳ10% of the F 1 molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The ⑀ subunit inhibited rotation and ATPase, whereas ⑀ fused through its carboxyl terminus to cytochrome b 562 showed no effect. The ⑀ subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.The biological energy currency ATP is synthesized by a ubiquitous ATP synthase (F 0 F 1 ) conserved in mitochondria, chloroplasts, and bacteria (see Refs. 1-5, for reviews). Escherichia coli F 0 F 1 has a basic subunit structure consisting of catalytic F 1 (␣ 3 ␤ 3 ␥␦⑀) and transmembrane F 0 (ab 2 c 10 ) sectors with a distinct stoichiometry of eight subunits. The ␣ and ␤ subunits form a catalytic hexamer, ␣ 3 ␤ 3 , the ␥ subunit being located at its center. Ten molecules of the c subunit form a membraneembedded ring for continuous proton transport through its interface with subunit a (6 -8). ATP at a catalytic site in each ␤ subunit is synthesized or hydrolyzed cooperatively, as predicted by the binding change mechanism or rotational catalysis (2), and supported by the crystal structure of the ␣ 3 ␤ 3 ␥ complex (9). The ␥ subunit rotation has been shown biochemically by photobleaching of a probe attached to the carboxyl terminus of the ␥ subunit (10), chemical cross-linking between the ␤ and ␥ subunits (11), cryoelectron microscopy (12), and finally video recording of the ATP-dependent anti-clockwise rotation of an actin filament attached to the ␥ subunit (13-15). Consistent with tight interaction between the ␥ and ⑀ subunits, a filament connected to ⑀ rotated together with the ␥ subunit (16).For proton translocation, rotation of the ␥ subunit is transmitted to the c subunit ring, as shown by rotation of the ␥⑀c 10 complex relative to the ␣ 3 ␤ 3 ab 2 (17). Mechanical rotation of purified F 0 F 1 (17-21) and its membrane-bound form have been shown experimentally (22, 23). The rotation of ␥⑀c 10 was consistent with cross-linking experiments (21, 24 -26). The ␥ subunit and ␥⑀c 10 rotated in F 1 and F 0 F 1 , respectively, ...

A similar approach was taken for mutations in the ␤ subunit key region; consistent with its bulk phase ATPase activities, F 1 with the Ser-174 to Phe substitution (␤S174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120°positions during the stepped revolution. The pause positions were probably not at the "ATP waiting" dwell but at the "ATP hydrolysis/product release" dwell, since the ATP concentration used for the assay was ϳ30-fold higher than the K m value for ATP. A ␤Gly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of ␤S174F. The revertant (␤G149A/␤S174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant ␤G149A/␤S174L. These results indicate that the domain between ␤-sheet 4 (␤Ser-174) and P-loop (␤Gly-149) is important to drive rotation.A ubiquitous ATP synthase (FoF 1 ) synthesizes ATP coupled with an electrochemical proton gradient formed by a respiratory chain (for reviews, see Refs 1-5). FoF 1 consists of a catalytic sector, F 1 (␣ 3 ␤ 3 ␥␦⑀), and a membrane-embedded proton pathway, Fo (ab 2 c 10 ), and can reversibly transport protons coupled with ATP hydrolysis. The ␣ and ␤ subunits form a catalytic hexamer (␣ 3 ␤ 3 ), the central space of which is occupied by the ␥ subunit ␣-helices. ATP is synthesized or hydrolyzed cooperatively at a catalytic site in each ␤ subunit as the binding change mechanism predicts (2). The ␥ subunit rotation in ␣ 3 ␤ 3 has been supported by biochemical studies (1, 6, 7), a crystal structure of the ␣ 3 ␤ 3 ␥ complex (8), and video recorded using an actin filament as a probe (9, 10). Consistent with ATP-dependent proton translocation, a ␥⑀c 10 complex rotated relative to the ␣ 3 ␤ 3 ␦ab 2 in the purified FoF 1 (11-14) or its membrane-bound form (15, 16). The rotation of FoF 1 in liposomes has been revealed by means of single molecule fluorescence resonance energy transfer (17).Counterclockwise rotation of the ␥ subunit has been studied more recently with probes giving low viscous drag such as colloidal gold (14,18,19). The three 120°steps in one revolution of Bacillus F 1 were first observed using an actin filament (20), and later the single 120°step was further subdivided into two substeps (80°and 40°) using gold beads that allow finer observation and analysis (14,19,21). The substeps with larger displacement angles (80°) and smaller substeps (40°) are assigned to ATP binding and hydrolysis/product release steps, respectively (19,21,22). We have observed that the rotation speed of beads attached to the Escherichia coli ␥ subunit varied, reflecting stochastic fluctuations (18,23). Although the average speeds were dependent on the diameter of beads, 40-and 60-nm diameter beads rotated with essentially the same rate (18), suggesting that their rotation speeds were close to that of...