Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins.

Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray ionization-MS (ESI-MS) of an intact protein generates a profile defining the native covalent state of the gene product and its heterogeneity. Here we apply ESI-MS technology with accuracy exceeding 0.01% to a hydrophobic membrane protein with 12-transmembrane alpha-helices, the full-length lactose permease from Escherichia coli. Furthermore, ESI-MS is used to titrate reactive thiols with N-ethylmaleimide. Treatment of the native protein solubilized in detergent micelles reveals only two reactive thiols, and both are protected by a substrate analog.

Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1.

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.

Crystal structure of the BTB domain from PLZF.

The BTB domain (also known as the POZ domain) is an evolutionarily conserved protein-protein interaction motif found at the N terminus of 5-10% of C2H2-type zinc-finger transcription factors, as well as in some actin-associated proteins bearing the kelch motif. Many BTB proteins are transcriptional regulators that mediate gene expression through the control of chromatin conformation. In the human promyelocytic leukemia zinc finger (PLZF) protein, the BTB domain has transcriptional repression activity, directs the protein to a nuclear punctate pattern, and interacts with components of the histone deacetylase complex. The association of the PLZF BTB domain with the histone deacetylase complex provides a mechanism of linking the transcription factor with enzymatic activities that regulate chromatin conformation. The crystal structure of the BTB domain of PLZF was determined at 1.9 A resolution and reveals a tightly intertwined dimer with an extensive hydrophobic interface. Approximately one-quarter of the monomer surface area is involved in the dimer intermolecular contact. These features are typical of obligate homodimers, and we expect the full-length PLZF protein to exist as a branched transcription factor with two C-terminal DNA-binding regions. A surface-exposed groove lined with conserved amino acids is formed at the dimer interface, suggestive of a peptide-binding site. This groove may represent the site of interaction of the PLZF BTB domain with nuclear corepressors or other nuclear proteins.

The crystal structure of enoyl-CoA hydratase complexed with octanoyl-CoA reveals the structural adaptations required for binding of a long chain fatty acid-CoA molecule.

The structure of the hexameric rat mitochondrial enoyl-Coenzyme A (CoA) hydratase, co-crystallised with the inhibitor octanoyl-CoA, has been refined at a resolution of 2.4 A. Enoyl-CoA hydratase catalyses the hydration of 2,3-unsaturated enoyl-CoA thioesters. In the crystal structure only four of the six active sites of the hexamer in the asymmetric unit are occupied with a ligand molecule, showing an unliganded and a liganded active site within the same crystal form. While the protein assembly and fold is identical to the previously solved acetoacetyl-CoA complex, differences are observed close to the fatty acid binding pocket due to the different nature of the ligands. The fatty acid tail of octanoyl-CoA is bound in an extended conformation. This is possible because a high B-factor loop, which separates in the acetoacetyl-CoA complex the binding pocket of the acetoacetyl-CoA fatty acid tail from the intertrimer space, has moved aside to allow binding of the longer octanoyl-CoA moiety. The movement of this loop opens a tunnel which traverses the complete subunit from the solvent space to the intertrimer space. The conformation of the catalytic residues is identical, in both structures as well as in the liganded and the unliganded active sites. In the unliganded active sites a water molecules is bound between the two catalytic glutamate, residues Glu144 and Glu164. After superposition of a liganded active site on an unliganded active site this water molecule is close to the carbon centre that becomes hydroxylated in the hydratase reaction. These findings support the idea that the active site is rigid and that the catalytic residues and the water molecule, as seen in the unliganded active site, are pre-positioned for very efficient catalysis.

The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism.

The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the conversion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon atoms. This reaction is the last step of the beta-oxidation pathway. The crystal structure of unliganded peroxisomal thiolase of the yeast Saccharomyces cerevisiae has been refined at 1.8 A resolution. An unusual feature of this structure is the presence of two helices, completely buried in the dimer and sandwiched between two beta-sheets. The analysis of the structure shows that the sequences of these helices are not hydrophobic, but generate two amphipathic helices. The helix in the N-terminal domain exposes the polar side-chains to a cavity at the dimer interface, filled with structured water molecules. The central helix in the C-terminal domain exposes its polar residues to an interior polar pocket. The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism. From previous studies it is known that Cys125, His375 and Cys403 are important catalytic residues. In the proposed model the acetoacetyl group fits near the two catalytic cysteine residues, such that the oxygen atoms point towards the protein interior. The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 A. The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond between SG(Cys125) and C3(acetoacetyl-CoA) of the intermediate complex of the two-step reaction. The CoA moiety is proposed to bind in a groove on the surface of the protein molecule. Most of the interactions of the CoA molecule are with atoms of the loop domain. The three phosphate groups of the CoA moiety are predicted to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric thiolases.

Crystal structure of enoyl-coenzyme A (CoA) hydratase at 2.5 angstroms resolution: a spiral fold defines the CoA-binding pocket.

The crystal structure of rat liver mitochondrial enoyl-coenzyme A (CoA) hydratase complexed with the potent inhibitor acetoacetyl-CoA has been refined at 2.5 angstroms resolution. This enzyme catalyses the reversible addition of water to alpha,beta-unsaturated enoyl-CoA thioesters, with nearly diffusion-controlled reaction rates for the best substrates. Enoyl-CoA hydratase is a hexamer of six identical subunits of 161 kDa molecular mass for the complex. The hexamer is a dimer of trimers. The monomer is folded into a right-handed spiral of four turns, followed by two small domains which are involved in trimerization. Each turn of the spiral consists of two beta-strands and an alpha-helix. The mechanism for the hydratase/dehydratase reaction follows a syn-stereochemistry, a preference that is opposite to the nonenzymatic reaction. The active-site architecture agrees with this stereochemistry. It confirms the importance of Glu164 as the catalytic acid for providing the alpha-proton during the hydratase reaction. It also shows the importance of Glu144 as the catalytic base for the activation of a water molecule in the hydratase reaction. The comparison of an unliganded and a liganded active site within the same crystal form shows a water molecule in the unliganded subunit. This water molecule is bound between the two catalytic glutamates and could serve as the activated water during catalysis.