How release of phosphate from mammalian F1-ATPase generates a rotary substep.

The rotation of the central stalk of F1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F1-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the α(E)β(E)-catalytic interface, whereas in F1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the β(E)-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the β(E)-subunit disrupts interactions in the α(E)β(E-)catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the α(E)-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°-30°

The affinity purification and characterization of ATP synthase complexes from mitochondria.

The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.This work was funded by the intramural programme of the Medical Research Council (MRC). T.J.C was the recipient of an MRC graduate studentship

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF₁.

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α(DP)- and β(DP)-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β(E)-subunits. The nucleotide binding site in β(E)-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.Support for this work was provided by the Medical Research Council, UK, including a PhD studentship (to G.C.R.) and a Career Training Fellowship (to J.V.B.), by the European Drug Initiative in Channels and Transporters (EDICT; to J.E.W.), and by a grant from NIH no. R01GM66223 to D.M.M

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases

Relational knowledge leadership and local economic development

This paper concerns the role of spatial leadership in the development of the knowledge-based economy. It is argued within academic and practitioner circles that leadership of knowledge networks requires a particular non-hierarchical style that is required to establish an ambience conducive to networking and knowledge sharing across boundaries. In this paper, we explore this hypothesis at both theoretical and empirical levels. Theoretically, we propose a conceptualization of relational knowledge leadership, which is ‘nomadic’ in its capacity to travel across multiple scales and cross sectoral, thematic and geographical boundaries. We have operationalized this type of relational knowledge leadership along four key features, derived from literatures on regional learning, organizational leadership and place leadership. Two empirical case studies are then presented, one from Birmingham in the UK and one from Eindhoven in the Netherlands, exploring how these features are expressed on the sub-national level. Also conclusions are drawn regarding the status of relational knowledge leadership. It is argued that the concept of relational knowledge leadership as viewed through our analytical lens does accord with the experience of leadership in the two cases presented. The cases also show that this style of leadership is confronted with three types of tensions that play through knowledge networking. Furthermore, it is argued that the cases exhibit this style of leadership to different degrees, reflecting their different cultural and political context