Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex.

Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E2. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5.

Mechanism of inhibition of the V-type molecular motor by tributyltin chloride.

Tributyltin chloride (TBT-Cl) is an endocrine disruptor found in many animal species, and it is also known to be an inhibitor for the V-ATPases that are emerging as potential targets in the treatment of diseases such as osteoporosis and cancer. We demonstrated by using biochemical and single-molecular imaging techniques that TBT-Cl arrests an elementary step for rotary catalysis of the V(1) motor domain. In the presence of TBT-Cl, the consecutive rotation of V(1) paused for a long duration ( approximately 0.5 s), even at saturated ATP concentrations, and the pausing positions were localized at 120 degrees intervals. Analysis of both the pausing time and moving time revealed that TBT-Cl has little effect on the binding affinity for ATP, but, rather, it arrests the catalytic event(s). This is the first report to demonstrate that an inhibitor arrests an elementary step for rotary catalysis of a V-type ATP-driven rotary motor.

Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase.

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.

Structure of a central stalk subunit F of prokaryotic V-type ATPase/synthase from Thermus thermophilus.

The crystal structure of subunit F of vacuole-type ATPase/synthase (prokaryotic V-ATPase) was determined to of 2.2 A resolution. The subunit reveals unexpected structural similarity to the response regulator proteins that include the Escherichia coli chemotaxis response regulator CheY. The structure was successfully placed into the low-resolution EM structure of the prokaryotic holo-V-ATPase at a location indicated by the results of crosslinking experiments. The crystal structure, together with the single-molecule analysis using fluorescence resonance energy transfer, showed that the subunit F exhibits two conformations, a 'retracted' form in the absence and an 'extended' form in the presence of ATP. Our results postulated that the subunit F is a regulatory subunit in the V-ATPase.

The F subunit of Thermus thermophilus V1-ATPase promotes ATPase activity but is not necessary for rotation.

V(1)-ATPase from the thermophilic bacterium Thermus thermophilus is a molecular rotary motor with a subunit composition of A(3)B(3)DF, and its central rotor is composed of the D and F subunits. To determine the role of the F subunit, we generated an A(3)B(3)D subcomplex and compared it with A(3)B(3)DF. The ATP hydrolyzing activity of A(3)B(3)D (V(max) = 20 s(-1)) was lower than that of A(3)B(3)DF (V(max) = 31 s(-1)) and was more susceptible to MgADP inhibition during ATP hydrolysis. A(3)B(3)D was able to bind the F subunit to form A(3)B(3)DF. The C-terminally truncated F((Delta85-106)) subunit was also bound to A(3)B(3)D, but the F((Delta69-106)) subunit was not, indicating the importance of residues 69-84 of the F subunit for association with A(3)B(3)D. The ATPase activity of A(3)B(3)DF((Delta85-106)) (V(max) = 24 s(-1)) was intermediate between that of A(3)B(3)D and A(3)B(3)DF. A single molecule experiment showed the rotation of the D subunit in A(3)B(3)D, implying that the F subunit is a dispensable component for rotation itself. Thus, the F subunit binds peripherally to the D subunit, but promotes V(1)-ATPase catalysis.

Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase.

The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-A resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V(1) (soluble) and the V(o) (membrane bound) domains. Subunit C, attached to the V(o) domain, seems to have a socket like function in attaching the central-stalk subunits of the V(1) domain. This architecture seems essential for the reversible association/dissociation of the V(1) and the V(o) domains, unique for V-ATPase activity regulation.