Protein Phosphorylation and Prevention of Cytochrome Oxidase Inhibition by ATP: Coupled Mechanisms of Energy Metabolism Regulation

SummaryRapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage

Multidimensional mutual information methods for the analysis of covariation in multiple sequence alignments

Several methods are available for the detection of covarying positions from a multiple sequence alignment (MSA). If the MSA contains a large number of sequences, information about the proximities between residues derived from covariation maps can be sufficient to predict a protein fold. If the structure is already known, information on the covarying positions can be valuable to understand the protein mechanism. In this study we have sought to determine whether a multivariate extension of traditional mutual information (MI) can be an additional tool to study covariation. The performance of two multidimensional MI (mdMI) methods, designed to remove the effect of ternary/quaternary interdependencies, was tested with a set of 9 MSAs each containing <400 sequences, and was shown to be comparable to that of methods based on maximum entropy/pseudolikelyhood statistical models of protein sequences. However, while all the methods tested detected a similar number of covarying pairs among the residues separated by < 8 {\AA} in the reference X-ray structures, there was on average less than 65% overlap between the top scoring pairs detected by methods that are based on different principles. We have also attempted to identify whether the difference in performance among methods is due to different efficiency in removing covariation originating from chains of structural contacts. We found that the reason why methods that derive partial correlation between the columns of a MSA provide a better recognition of close contacts is not because they remove chaining effects, but because they filter out the correlation between distant residues that originates from general fitness constraints. In contrast we found that true chaining effects are expression of real physical perturbations that propagate inside proteins, and therefore are not removed by the derivation of partial correlation between variables.Comment: 21 pages, 4 figures, 1 table, supporting information containing 2 additional figures is included at the end of the manuscrip

Preliminary X-ray analysis of a new crystal form of the Escherichia coli KDO8P synthase

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the biosynthesis of an essential component of the lipopolysaccharide of all Gram-negative bacteria. The structure and mechanism of KDO8P synthase are being actively studied as this enzyme represents an important target for antibiotic therapy. The structure of the Escherichia coli KDO8P synthase in cubic crystals (space group I23) has recently been determined and the enzyme shown to be a tetramer of identical subunits. However, this information is challenged by biochemical studies, which suggest that the enzyme behaves in solution as a homotrimer. Here, the preparation and preliminary X-ray analysis of monoclinic crystals of KDO8P synthase are reported. The crystals belong to space group P21, with unit-cell parameters a≃ 50, b≃ 140, c≃ 74 Å, β≃ 105°. The structure of KDO8P synthase in the monoclinic crystal form was determined by molecular replacement, using as a search model one of the subunits of the enzyme in the cubic crystals. A tetramer of KDO8P synthase with 222 local symmetry is also present in the asymmetric unit of the P21 crystals, with a solvent content of 43%. The observation that the same quaternary structure of KDO8P synthase is observed in two different crystal forms belonging to distinct crystal systems (monoclinic and cubic) suggests that a tetramer is the native form of the enzyme

Structure and mechanism of the iron‐sulfur flavoprotein phthalate dioxygenase reductase

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154520/1/fsb2009014006.pd

Cloning and Sequence Analysis of the Structural Gene for the bc 1 - Type Rieske Iron-Sulfur Protein from Thermus thermophilus HB8

The structural gene encoding the Rieske iron-sulfur protein from Thermus thermophilus HB8 has been cloned and sequenced. The gene encodes a protein of 209 amino acids that begins with a hydrophilic N-terminus followed by a stretch of 21 hydrophobic amino acids that could serve as a transmembrane helix. The remainder of the protein has a hydrophobicity pattern typical of a water-soluble protein. A phylogenetic analysis of 26 Rieske proteins that are part of bc 1 or b 6 f complexes shows that they fall into three major groups: eubacterial and mitochondrial, cyanobacterial and plastid, and five highly divergent outliers, including that of Thermus . Although the overall homology with other Rieske proteins is very low, the C-terminal half of the Thermus protein contains the signature sequence CTHLGC-(13X)-CPCH that most likely provides the ligands of the [2Fe-2S] cluster. It is proposed that this region of the protein represents a small domain that folds independently and that the encoding DNA sequence may have been transferred during evolution to several unrelated genes to provide the cluster attachment site to proteins of different origin. The role of individual residues in this domain of the Thermus protein is discussed vis-a-vis the three-dimensional structure of the bovine protein (Iwata et al ., 1996 Structure 4 , 567–579).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44801/1/10863_2004_Article_409077.pd

First observation of quantum interference in the process phi -> KS KL ->pi+pi-pi+pi-: a test of quantum mechanics and CPT symmetry

We present the first observation of quantum interference in the process phi -> KS KL ->pi+pi-pi+pi-. This analysis is based on data collected with the KLOE detector at the e^+e^- collider DAFNE in 2001--2002 for an integrated luminosity of about 380pb^-1. Fits to the distribution of Delta t, the difference between the two kaon decay times, allow tests of the validity of quantum mechanics and CPT symmetry. No deviations from the expectations of quantum mechanics and CPT symmetry have been observed. New or improved limits on various decoherence and CPT violation parameters have been obtainedComment: submitted to Physics Letter B one number changed old:gamma=(1.1+2.9-2.4)10^-21 GeV new:(1.3+2.8-2.4)10^-21GeV corrected typo

Measurement of the Branching Ratio of the KL -> pi+pi- decay with the KLOE Detector

We present a measurement of the branching ratio of the CP violating decay KL->pi+pi- performed by the KLOE experiment at the phi factory DAFNE. We use 328 pb-1 of data collected in 2001 and 2002, corresponding to ~ 150 million tagged KL mesons. We find BR(KL->pi+pi-) = $(1.963 +/- 0.012 +/- 0.017)x 10^-3. This branching ratio measurement is fully inclusive of final-state radiation. Using the above result, we determine the modulus of the amplitude ratio |\eta_{+-}| to be (2.219 +/- 0.013)x 10^{-3} and |\epsilon| to be (2.216 +/- 0.013)x 10^{-3}.Comment: Subitted to Phys. Lett.