Assembly of a 20-nm protein cage by Escherichia coli 2-hydroxypentadienoic acid hydratase.

The pentameric Escherichia coli enzyme 2-hydroxypentadienoic acid hydratase assembles to form a 20-nm-diameter particle comprising 60 protein subunits, arranged with 532 symmetry when crystallised at low pH in the presence of phosphate or sulphate ions. The particles form rapidly and are stable in solution during gel filtration at low pH. They are probably formed through trimers of pentamers, which are stabilised by the interaction of two phosphate ions with residues of the N-terminal domains of subunits at the 3-fold axis. Once the particles are formed at high concentrations of phosphate (or sulphate), they remain stable in solution at 20-fold lower concentrations of the anion. Guest molecules can be trapped within the hollow protein shell during assembly. The C-termini of the subunits are freely accessible on the surface of the protein cage and thus are ideal sites for addition of affinity tags or other modifications. These particles offer a convenient model system for studying the assembly of large symmetrical structures and a novel protein nanoparticle for encapsulation and cargo delivery.

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism.

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 A resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the alpha/beta hydrolase fold family, comprising eight beta-strands interconnected by loops and helices. The 2.8 A resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.

The structure of the central stalk in bovine F(1)-ATPase at 2.4 A resolution.

The central stalk in ATP synthase, made of gamma, delta and epsilon subunits in the mitochondrial enzyme, is the key rotary element in the enzyme's catalytic mechanism. The gamma subunit penetrates the catalytic (alpha beta)(3) domain and protrudes beneath it, interacting with a ring of c subunits in the membrane that drives rotation of the stalk during ATP synthesis. In other crystals of F(1)-ATPase, the protrusion was disordered, but with crystals of F(1)-ATPase inhibited with dicyclohexylcarbodiimide, the complete structure was revealed. The delta and epsilon subunits interact with a Rossmann fold in the gamma subunit, forming a foot. In ATP synthase, this foot interacts with the c-ring and couples the transmembrane proton motive force to catalysis in the (alpha beta)(3) domain.

Structure of bovine mitochondrial F(1)-ATPase inhibited by Mg(2+) ADP and aluminium fluoride.

BACKGROUND: The globular domain of the membrane-associated F(1)F(o)-ATP synthase complex can be detached intact as a water-soluble fragment known as F(1)-ATPase. It consists of five different subunits, alpha, beta, gamma, delta and epsilon, assembled with the stoichiometry 3:3:1:1:1. In the crystal structure of bovine F(1)-ATPase determined previously at 2.8 A resolution, the three catalytic beta subunits and the three noncatalytic alpha subunits are arranged alternately around a central alpha-helical coiled coil in the gamma subunit. In the crystals, the catalytic sites have different nucleotide occupancies. One contains the triphosphate form of the nucleotide, the second contains the diphosphate, and the third is unoccupied. Fluoroaluminate complexes have been shown to mimic the transition state in several ATP and GTP hydrolases. In order to understand more about its catalytic mechanism, F(1)-ATPase was inhibited with Mg(2+)ADP and aluminium fluoride and the structure of the inhibited complex was determined by X-ray crystallography. RESULTS: The structure of bovine F(1)-ATPase inhibited with Mg(2+)ADP and aluminium fluoride determined at 2.5 A resolution differs little from the original structure with bound AMP-PNP and ADP. The nucleotide occupancies of the alpha and beta subunits are unchanged except that both aluminium trifluoride and Mg(2+)ADP are bound in the nucleotide-binding site of the beta(DP) subunit. The presence of aluminium fluoride is accompanied by only minor adjustments in the surrounding protein. CONCLUSIONS: The structure appears to mimic a possible transition state. The coordination of the aluminofluoride group has many features in common with other aluminofluoride-NTP hydrolase complexes. Apparently, once nucleotide is bound to the catalytic beta subunit, no additional major structural changes are required for catalysis to occur.

The ATPase inhibitor protein from bovine heart mitochondria: the minimal inhibitory sequence.

The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase. In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield. The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria. Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis. By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47. The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable. At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution. Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase. However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region. The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit. The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase.