Evolutionary Trace Annotation of Protein Function in the Structural Proteome

By design, structural genomics (SG) solves many structures that cannot be assigned function based on homology to known proteins. Alternative function annotation methods are therefore needed and this study focuses on function prediction with three-dimensional (3D) templates: small structural motifs built of just a few functionally critical residues. Although experimentally proven functional residues are scarce, we show here that Evolutionary Trace (ET) rankings of residue importance are sufficient to build 3D templates, match them, and then assign Gene Ontology (GO) functions in enzymes and non-enzymes alike. In a high-specificity mode, this Evolutionary Trace Annotation (ETA) method covered half (53%) of the 2384 annotated SG protein controls. Three-quarters (76%) of predictions were both correct and complete. The positive predictive value for all GO depths (all-depth PPV) was 84%, and it rose to 94% over GO depths 1-3 (depth 3 PPV). In a high-sensitivity mode, coverage rose significantly (84%), while accuracy fell moderately: 68% of predictions were both correct and complete, all-depth PPV was 75%, and depth 3 PPV was 86%. These data concur with prior mutational experiments showing that ET rank information identifies key functional determinants in proteins. In practice, ETA predicted functions in 42% of 3461 unannotated SG proteins. In 529 cases—including 280 non-enzymes and 21 for metal ion ligands—the expected accuracy is 84% at any GO depth and 94% down to GO depth 3, while for the remaining 931 the expected accuracies are 60% and 71%, respectively. Thus, local structural comparisons of evolutionarily important residues can help decipher protein functions to known reliability levels and without prior assumption on functional mechanisms. ETA is available at http://mammoth.bcm.tmc.edu/eta.     

Structure of the Cytosolic Portion of the Motor Protein Prestin and Functional Role of the STAS Domain in SLC26/SulP Anion Transporters

Prestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases. SLC26 and related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and protein function. This C-terminal portion is mainly composed of a STAS (sulfate transporters and anti-sigma factor antagonist) domain, whose name is due to a remote but significant sequence similarity with bacterial ASA (anti-sigma factor antagonist) proteins. Here we present the crystal structure at 1.57 Å resolution of the cytosolic portion of prestin, the first structure of a SulP transporter STAS domain, and its characterization in solution by heteronuclear multidimensional NMR spectroscopy. Prestin STAS significantly deviates from the related bacterial ASA proteins, especially in the N-terminal region, which—although previously considered merely as a generic linker between the domain and the last transmembrane helix—is indeed fully part of the domain. Hence, unexpectedly, our data reveal that the STAS domain starts immediately after the last transmembrane segment and lies beneath the lipid bilayer. A structure-function analysis suggests that this model can be a general template for most SLC26 and SulP anion transporters and supports the notion that STAS domains are involved in functionally important intramolecular and intermolecular interactions. Mapping of disease-associated or functionally harmful mutations on STAS structure indicates that they can be divided into two categories: those causing significant misfolding of the domain and those altering its interaction properties.     

Binding Hot Spot in the Weak Protein Complex of Physiological Redox Partners Yeast Cytochrome c and Cytochrome c Peroxidase

Transient protein interactions mediate many vital cellular processes such as signal transduction or intermolecular electron transfer. However, due to difficulties associated with their structural characterization, little is known about the principles governing recognition and binding in weak transient protein complexes. In particular, it has not been well established whether binding hot spots, which are frequently found in strong static complexes, also govern transient protein interactions. To address this issue, we have investigated an electron transfer complex of physiological partners from yeast: yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP). Using isothermal titration calorimetry and NMR spectroscopy, we show that Cc R13 is a hot-spot residue, as R13A mutation has a strong destabilizing effect on binding. Furthermore, we employ a double-mutant cycle to illustrate that Cc R13 interacts with CcP Y39. The present results, in combination with those of earlier mutational studies, have enabled us to outline the extent of the energetically important Cc-CcP binding region. Based on our analysis, we propose that binding energy hot spots, which are prevalent in static protein complexes, could also govern transient protein interactions.     

Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the loricariid catfish Pterygoplichthys sp

The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.     

Molecular cloning,characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.