Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

SUNY Upstate Medical University

The H(+)-translocating F(0)F(1)-ATP synthase of Escherichia coli functions as a rotary motor, coupling the transmembrane movement of protons through F(0) to the synthesis of ATP by F(1). Although the epsilon subunit appears to be tightly associated with the gamma subunit in the central stalk region of the rotor assembly, several studies suggest that the C-terminal domain of epsilon can undergo significant conformational change as part of a regulatory process. Here we use disulfide cross-linking of substituted cysteines on functionally coupled ATP synthase to characterize interactions of epsilon with an F(0) component of the rotor (subunit c) and with an F(1) component of the stator (subunit beta). Oxidation of the engineered F(0)F(1) causes formation of two disulfide bonds, betaD380C-S108C epsilon and epsilonE31C-cQ42C, to give a beta-epsilon-c cross-linked product in high yield. The results demonstrate the ability of epsilon to span the central stalk region from the surface of the membrane (epsilon-c) to the bottom of F(1) (beta-epsilon) and suggest that the conformation detected here is distinct from both the "closed" state seen with isolated epsilon (Uhlin, U., Cox, G. B., and Guss, J. M. (1997) Structure 5, 1219-1230) and the "open" state seen in a complex with a truncated form of the gamma subunit (Rodgers, A. J., and Wilce, M. C. (2000) Nat. Struct. Biol. 7, 1051-1054). The kinetics of beta-epsilon and epsilon-c cross-linking were studied separately using F(0)F(1) containing one or the other matched cysteine pair. The rate of cross-linking at the epsilon/c (rotor/rotor) interface is not influenced by the type of nucleotide added. In contrast, the rate of beta-epsilon cross-linking is fastest under ATP hydrolysis conditions, intermediate with MgADP, and slowest with MgAMP-PNP. This is consistent with a regulatory role for a reversible beta/epsilon (stator/rotor) interaction that blocks rotation and inhibits catalysis. Furthermore, the rate of beta-epsilon cross-linking is much faster than that indicated by previous studies, allowing for the possibility of a rapid response to regulatory signals.

Rotor/Stator interactions of the epsilon subunit in Escherichia coli ATP synthase and implications for enzyme regulation.

Fluorescent probes are essential for the exploration of protein function, detection of molecular interactions, and conformational changes. The nitrilotriacetic acid derivatives of different chromophores were successfully used for site-selective noncovalent fluorescence labeling of histidine-tagged proteins. All of them, however, suffer from the same drawback--loss of the fluorescence upon binding of the nickel ions. Herein we present the solution and solid phase synthesis of water-soluble perylene(dicarboximide) functionalized with a nitrilotriacetic acid moiety (PDI-NTA). The photophysical properties of PDI-NTA revealed an exceptional photostability and fluorescence quantum yield that remained unchanged upon addition of nickel ions. The F1 complex of F0F1-ATP synthase from Escherichia coli, containing three hexahistidine tags, was labeled and the suitability for site-specific labeling of the new chromophore demonstrated using fluorescence correlation spectroscopy.

Exploiting the nitrilotriacetic acid moiety for biolabeling with ultrastable perylene dyes.

ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit ɛ adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F(1) structures.

Structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli in an autoinhibited conformation.

The bacterial ATP synthase (F(O)F(1)) of Escherichia coli has been the prominent model system for genetics, biochemical and more recently single-molecule studies on F-type ATP synthases. With 22 total polypeptide chains (total mass of ∼529 kDa), E. coli F(O)F(1) represents nature's smallest rotary motor, composed of a membrane-embedded proton transporter (F(O)) and a peripheral catalytic complex (F(1)). The ATPase activity of isolated F(1) is fully expressed by the α(3)β(3)γ 'core', whereas single δ and ε subunits are required for structural and functional coupling of E. coli F(1) to F(O). In contrast to mitochondrial F(1)-ATPases that have been determined to atomic resolution, the bacterial homologues have proven very difficult to crystallize. In this paper, we describe a biochemical strategy that led us to improve the crystallogenesis of the E. coli F(1)-ATPase catalytic core. Destabilizing the compact conformation of ε's C-terminal domain with a phosphomimetic mutation (εS65D) dramatically increased crystallization success and reproducibility, yielding crystals of E. coli F(1) that diffract to ∼3.15 Å resolution.

Improved crystallization of Escherichia coli ATP synthase catalytic complex (F1) by introducing a phosphomimetic mutation in subunit ε.

F1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ε, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F1·ε binding and dissociation, we show that formation of the extended, inhibitory conformation of ε (εX) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the εX state, and post-hydrolysis conditions stabilize it. We also show that ε inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ε is responsible for initial binding to F1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ε N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F1-bound ε suggest dynamic conformational and rotational mobility in F1 that is paused near the catalytic dwell position.

F1-ATPase of Escherichia coli: the ε- inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.

Subunit rotation is the mechanochemical intermediate for the catalytic activity of the membrane enzyme FoF1-ATP synthase. smFRET (single-molecule FRET) studies have provided insights into the step sizes of the F1 and Fo motors, internal transient elastic energy storage and controls of the motors. To develop and interpret smFRET experiments, atomic structural information is required. The recent F1 structure of the Escherichia coli enzyme with the ϵ-subunit in an inhibitory conformation initiated a study for real-time monitoring of the conformational changes of ϵ. The present mini-review summarizes smFRET rotation experiments and previews new smFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.

Spotlighting motors and controls of single FoF1-ATP synthase.

The Ess1 prolyl isomerase from Saccharomyces cerevisiae and its human ortholog, Pin1, play critical roles in transcription by regulating RNA polymerase II. In human cells, Pin1 also regulates a variety of signaling proteins and Pin1 misexpression is linked to several human diseases. To gain insight into Ess1/Pin1 function, we carried out a synthetic genetic array screen to identify novel targets of Ess1 in yeast. We identified potential targets of Ess1 in transcription, stress and cell cycle pathways. We focused on the cell cycle regulators Swi6 and Whi5, both of which show highly-regulated nucleo-cytoplasmic shuttling during the cell cycle. Surprisingly, Ess1 did not control their transcription, but instead was necessary for their nuclear localization. Ess1 associated with Swi6 and Whi5 in vivo and bound directly to peptides corresponding to their nuclear localization sequences in vitro. Binding by Ess1 was significant only if the Swi6 and Whi5 peptides were phosphorylated at Ser-Pro motifs, the target sites of cyclin-dependent kinases. Based on these results, we propose a model in which Ess1 induces a conformational switch (cis-trans isomerization) at phospho-Ser-Pro sites within the nuclear targeting sequences of Swi6 and Whi5. This switch would promote nuclear entry and/or retention during late M and G1 phases, and might work by stimulating de-phosphorylation at these sites by the Cdc14 phosphatase. This is the first study to identify targets of Ess1 in yeast other than RNA polymerase II.

Thomas M. Duncan

Department of Biochemistry and Molecular Biology

SUNY Upstate Medical University

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Thomas M. Duncan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology

SUNY Upstate Medical University

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Department of Biochemistry and Molecular Biology