Quantifying prion disease penetrance using large population control cohorts.

More than 100,000 genetic variants are reported to cause Mendelian disease in humans, but the penetrance-the probability that a carrier of the purported disease-causing genotype will indeed develop the disease-is generally unknown. We assess the impact of variants in the prion protein gene (PRNP) on the risk of prion disease by analyzing 16,025 prion disease cases, 60,706 population control exomes, and 531,575 individuals genotyped by 23andMe Inc. We show that missense variants in PRNP previously reported to be pathogenic are at least 30 times more common in the population than expected on the basis of genetic prion disease prevalence. Although some of this excess can be attributed to benign variants falsely assigned as pathogenic, other variants have genuine effects on disease susceptibility but confer lifetime risks ranging from <0.1 to ~100%. We also show that truncating variants in PRNP have position-dependent effects, with true loss-of-function alleles found in healthy older individuals, a finding that supports the safety of therapeutic suppression of prion protein expression.

Functional conformations for pyruvate carboxylase during catalysis explored by cryoelectron microscopy.

The tetrameric enzyme pyruvate carboxylase (PC), a biotin-dependent carboxylase, produces oxaloacetate by two consecutive reactions that take place in distant active sites. Previous crystal structures revealed two different configurations for PC tetramers, the so-called symmetric and asymmetric, which were understood as characteristic molecular architectures for PC from different organisms. We have analyzed PC samples from Staphylococcus aureus while the enzyme generates oxaloacetate, expecting PC tetramers to display the conformational landscape relevant for its functioning. Using cryoelectron microscopy (cryo-EM) and sorting techniques, we detect previously defined symmetric and asymmetric architectures, demonstrating that PC maps both arrangements by large conformational changes. Furthermore, we observe that each configuration is coupled to one of the two consecutive enzymatic reactions. The findings describe the structural transitions relevant for the allosteric control of the multifunctional PC and demonstrate that by cryo-EM and classification, we can characterize freely working macromolecules.

Characterizing the importance of the biotin carboxylase domain dimer for Staphylococcus aureus pyruvate carboxylase catalysis.

Biotin carboxylase (BC) is a conserved component among biotin-dependent carboxylases and catalyzes the MgATP-dependent carboxylation of biotin, using bicarbonate as the CO2 donor. Studies with Escherichia coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of the tetrameric pyruvate carboxylase (PC) holoenzyme. We have introduced site-specific mutations into the BC domain dimer interface of Staphylococcus aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation experiments. The K442E mutant and the ScBC chimera disrupted the BC dimer and were catalytically inactive, while the F403A mutant and the EcBC chimera were still tetrameric and retained catalytic activity. The R54E mutant was also tetrameric but was catalytically inactive. Crystal structures of the R54E, F403A, and K442E mutants showed that they were tetrameric in the crystal, with conformational changes near the mutation site as well as in the tetramer organization. We have also produced the isolated BC domain of SaPC. In contrast to E. coli BC, the SaPC BC domain is monomeric in solution and catalytically inactive.

Mechanism for the inhibition of the carboxyltransferase domain of acetyl-coenzyme A carboxylase by pinoxaden.

Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and have been targeted for drug development against obesity, diabetes, and other diseases. The carboxyltransferase (CT) domain of this enzyme is the site of action for three different classes of herbicides, as represented by haloxyfop, tepraloxydim, and pinoxaden. Our earlier studies have demonstrated that haloxyfop and tepraloxydim bind in the CT active site at the interface of its dimer. However, the two compounds probe distinct regions of the dimer interface, sharing primarily only two common anchoring points of interaction with the enzyme. We report here the crystal structure of the CT domain of yeast ACC in complex with pinoxaden at 2.8-Å resolution. Despite their chemical diversity, pinoxaden has a similar binding mode as tepraloxydim and requires a small conformational change in the dimer interface for binding. Crystal structures of the CT domain in complex with all three classes of herbicides confirm the importance of the two anchoring points for herbicide binding. The structures also provide a foundation for understanding the molecular basis of the herbicide resistance mutations and cross resistance among the herbicides, as well as for the design and development of new inhibitors against plant and human ACCs.

Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase.

Pyruvate carboxylase (PC) is a conserved multifunctional enzyme linked to important metabolic diseases. PC homotetramer is arranged in two layers with two opposing monomers per layer. Cryo-EM explores the conformational variability of PC in the presence of different substrates. The results demonstrate that the biotin-carboxyl carrier protein (BCCP) domain localizes near the biotin carboxylase (BC) domain of its own monomer and travels to the carboxyltransferase (CT) domain of the opposite monomer. All density maps show noticeable conformational differences between layers, mainly for the BCCP and BC domains. This asymmetry may be indicative of a coordination mechanism where monomers from different layers catalyze the BC and CT reactions consecutively. A conformational change of the PC tetramerization (PT) domain suggests a new functional role in communication. A long-range communication pathway between subunits in different layers, via interacting PT-PT and BC-BC domains, may be responsible for the cooperativity of PC from Staphylococcus aureus.

A symmetrical tetramer for S. aureus pyruvate carboxylase in complex with coenzyme A.

Pyruvate carboxylase (PC) is a conserved metabolic enzyme with important cellular functions. We report crystallographic and cryo-electron microscopy (EM) studies of Staphylococcus aureus PC (SaPC) in complex with acetyl-CoA, an allosteric activator, and mutagenesis, biochemical, and structural studies of the biotin binding site of its carboxyltransferase (CT) domain. The disease-causing A610T mutation abolishes catalytic activity by blocking biotin binding to the CT active site, and Thr908 might play a catalytic role in the CT reaction. The crystal structure of SaPC in complex with CoA reveals a symmetrical tetramer, with one CoA molecule bound to each monomer, and cryo-EM studies confirm the symmetrical nature of the tetramer. These observations are in sharp contrast to the highly asymmetrical tetramer of Rhizobium etli PC in complex with ethyl-CoA. Our structural information suggests that acetyl-CoA promotes a conformation for the dimer of the biotin carboxylase domain of PC that might be catalytically more competent.

Crystal structure of biotin carboxylase in complex with substrates and implications for its catalytic mechanism.

Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 A resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu(296) is the general base that extracts the proton from bicarbonate, and Arg(338) is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-A shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1' atom of biotin, providing strong evidence that the phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1' atom. The structural observations are supported by mutagenesis and kinetic studies. Overall, this first structure of BC in complex with substrates offers unprecedented insights into the molecular mechanism for the catalysis by this family of enzymes.