Ion channels have historically been viewed as distinct from secondary active transporters. However, the recent discovery that the CLC ‘chloride channel’ family is made up of both channels and active transporters has led to the hypothesis that the ion-transport mechanisms of these two types of membrane proteins may be similar. Here we use single-channel analysis to demonstrate that ClC-0 channel gating (opening and closing) involves the transmembrane movement of protons. This result indicates that ClC-0 is a ‘broken’ Cl−/H+ antiporter in which one of the conformational states has become leaky for chloride ions. This finding clarifies the evolutionary relationship between the channels and transporters and conveys that similar mechanisms and analogous protein movements are used by both.

The P4 protein of bacteriophage 12 is a hexameric molecular motor closely related to superfamily 4 helicases. P4 converts chemical energy from ATP hydrolysis into mechanical work, to translocate single-stranded RNA into a viral capsid. The molecular basis of mechanochemical coupling, i.e. how small ϳ1 Å changes in the ATP-binding site are amplified into nanometer scale motion along the nucleic acid, is not understood at the atomic level. Here we study in atomic detail the mechanochemical coupling using structural and biochemical analyses of P4 mutants. We show that a conserved region, consisting of superfamily 4 helicase motifs H3 and H4 and loop L2, constitutes the moving lever of the motor. The lever tip encompasses an RNAbinding site that moves along the mechanical reaction coordinate. The lever is flanked by ␥-phosphate sensors (Asn-234 and Ser-252) that report the nucleotide state of neighboring subunits and control the lever position. Insertion of an arginine finger (Arg-279) into the neighboring catalytic site is concomitant with lever movement and commences ATP hydrolysis. This ensures cooperative sequential hydrolysis that is tightly coupled to mechanical motion. Given the structural conservation, the mutated residues may play similar roles in other hexameric helicases and related molecular motors.Helicases are molecular motors that unwind doublestranded nucleic acids using the energy of NTP hydrolysis. Helicases have been divided into four superfamilies (SF1-SF4) 8 based on the sequence identity among the conserved helicase motifs (1). Helicases belonging to SF1 and SF2 (e.g. RecBCD and RecQ) function as monomers and provide simple model systems for studying DNA translocation and strand separation. An inchworming mechanism of translocation and unwinding was proposed on the basis of extensive structural and biochemical data (2, 3).Members of the SF3 and SF4 function as hexameric rings that encircle their polynucleotide substrates (DNA or RNA) and translocate along one strand. A recent landmark structural study on human papilloma virus helicase E1 (SF3 helicase) demonstrated that DNA is bound to conserved ␤-hairpins on multiple E1 subunits (4). A correlation between the positions of E1 ␤-hairpin and the conformations of the corresponding ATPbinding site provided evidence for sequential hydrolysis and translocation by an "escort" mechanism (4). Another important study on Rho transcription terminator (SF5 helicase (5)) has shown that RNA binds to multiple sites (Q loop, R loop, and P loop) in a cleft between two neighboring Rho protomers (6). Rho helicase also utilizes a sequential hydrolysis scheme (7); however, the translocation along RNA was proposed to proceed by a directed hand-off mechanism mediated by local conformational changes within the Q loops. A well known SF4 helicase, gp4 from bacteriophage T7 (T7 gp4), also employs a sequential ATP hydrolysis (8 -12). A set of gp4 x-ray structures suggested that ATP hydrolysis triggers sequential subunit rotations, which were proposed to effect nucleic acid...

Molecular motors undergo cyclical conformational changes and convert chemical energy into mechanical work. The conformational dynamics of a viral packaging motor, the hexameric helicase P4 of dsRNA bacteriophage phi8, was visualized by hydrogen-deuterium exchange and high-resolution mass spectrometry. Concerted changes of exchange kinetics revealed a cooperative unit that dynamically links ATP-binding sites and the central RNA-binding channel. The cooperative unit is compatible with a structure-based model in which translocation is mediated by a swiveling helix. Deuterium labeling also revealed the transition state associated with RNA loading, which proceeds via opening of the hexameric ring. The loading mechanism is similar to that of other hexameric helicases. Hydrogen-deuterium exchange provides an important link between time-resolved spectroscopic observations and high-resolution structural snapshots of molecular machines.

P4 is a hexameric ATPase that serves as the RNA packaging motor in double-stranded RNA bacteriophages from the Cystoviridae family. P4 shares sequence and structural similarities with hexameric helicases. A structure-based mechanism for mechano-chemical coupling has recently been proposed for P4 from bacteriophage 12. However, coordination of ATP hydrolysis among the subunits and coupling with RNA translocation remains elusive. Here we present detailed kinetic study of nucleotide binding, hydrolysis, and product release by 12 P4 in the presence of different RNA and DNA substrates. Whereas binding affinities for ATP and ADP are not affected by RNA binding, the hydrolysis step is accelerated and the apparent cooperativity is increased. No nucleotide binding cooperativity is observed. We propose a stochastic-sequential cooperativity model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. The translocation step appears coupled to hydrolysis, which is coordinated among three neighboring subunits. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive.Genomes of many viruses are encapsulated through NTPdriven packaging motor into preformed capsids. This process requires a portal complex that operates as the molecular motor and converts chemical energy into mechanical work. Doublestranded RNA bacteriophages from the Cystoviridae family (6-14) package their ssRNA 1 genomic precursors using a hexameric portal complex, the packaging NTPase P4 (1). P4 proteins share sequence and structural similarities with hexameric helicases and some of them possess helicase activity (2, 3). The P4 hexamer is also used as a passive pore for the exit of nascent transcripts from the viral core (4).A power stroke mechanism was proposed on the basis of P4 structures encompassing the key states of the catalytic cycle (3). Nucleotide exchange and hydrolysis was shown to induce concerted structural changes in two regions, namely the P-loop and the L2 loop-␣6 helix segment. The P-loop (Walker A or H1a motif in hexameric helicases) interacts with ␣ and ␤ phosphates of the nucleotide bound within the catalytic site. The L2 loop (H3 motif) is directly connected to the ␣6-helix (H4 motif) and binds to RNA. In the pre-hydrolysis state (P4-MgAMP-CPP complex) the P-loop is in a "relaxed" configuration and the L2 loop/␣6 helix is in an "up" configuration. After hydrolysis (P4-MgADP complex) the P-loop is in the "strained" configuration and the L2 loop/␣6 helix is in the "down" configuration. In the absence of any nucleotide the P-loop is in the relaxed configuration and the L2 loop/␣6 helix can swivel between the up and down configurations. Thus, it was proposed that the binding of ATP locks the L2 loop/␣6 helix in the up configuration, where it engages the ssRNA. Upon ATP hydrolysis, the nucleotide bind...