Heme Trafficking by the Cytochrome C Biogenesis Pathways

Sunday, February 12, 2017 with familial autism spectrum disorder while loss of function results in the Angelman syndrome neurological disorder. BIOGRID recognizes 174 E6APinteractors but only a few have been validated as substrates for ubiquitination. Recently, phosphorylation on E6APT485, an autism related residue, affected RPN10/S5A substrate ubiquitination in cells. The current aim included kinetic analysis of E6AP-catalyzed conjugation of target proteins to provide new insights into the mechanism of substrate ubiquitination. E6AP-dependent Lys48-polyubiquitin chain assembly in the absence of substrate requires two functionally distinct UbcH7~ubiquitin binding sites on the ligase surface and oligomerization. Here, the E6AP ubiquitin-function was analyzed in the presence of RNP10/S5A (regulatory subunit of proteasome 26S) and PRDX1 (antioxidant enzyme). Rates of substrate ubiquitin adduct formation were analyzed under E6AP rate-limiting conditions. Adducts of PRDX1-Ub1 or RPN10/S5AUb1 showed Km values of 851 mM and 0.250.06 mM, respectively; while the kcat values were 0.3 s 1, comparable to 0.5 s 1 observed for polyubiquitin chain assembly without substrate and indicating that the ligase cannot distinguish the lysine nucleophile in Lys48-ubiquitin and Lys-target protein ubiquitination. Analysis of pH-dependent E6AP ligase function inferred a pKa of ~8.4, either in the absence or presence of PRDX1. Removal of the first 250 N-terminal residues reduced ubiquitination of both substrates supporting the presence of previously unrecognized substrate binding domains in this region. A T485D mutation mimicking E6AP phosphorylation, or a D212A mutation, an Angelman syndrome mutation, abrogated substrate ubiquitination, although they retained the polyubiquitin chain assembly function. The results provide new insights of the E6AP ubiquitination mechanism in the presence of target proteins that might explain the deleterious effect of some mutations associated with Angelman syndrome. 328-Pos Board B93 Mechanistic Insights into Ubc13-Catalyzed Ubiquitination Isaiah Sumner1, R. Hunter Wilson1, Walker M. Jones1, Aaron G. Davis1, Serban Zamfir2. 1 Chemistry & Biochemistry, James Madison University, Harrisonburg, VA, USA, 2Chemistry, Virginia Commonwealth University, Richmond, VA, USA. Ubc13 is an E2 enzyme that catalyzes lysine ubiquitination, a type of protein post-translation modification. Ubiquitinating a protein can signal for its degradation and affect its activity. Ubiquitination also plays a role in DNA repair and inflammatory response. Defects in this process are linked to different disorders including cancer, Parkinson’s and Alzheimer’s diseases. The accepted mechanism for Ubc13-catalyzed ubiquitination is a stepwise pathway that proceeds through an oxyanion intermediate. This intermediate is hypothesized to be stabilized by a nearby asparagine residue, which is known as the ‘‘oxyanion hole.’’ However, recent experimental results on mutated Ubc13 have suggested an alternate role for the asparagine. In our study, we use a combination of simulation techniques on the wild-type and mutated Ubc13 to examine its catalytic mechanism. Our calculations indicate that several different intermediates are possible, that water may stabilize the intermediate, and that the asparagine serves to stabilize a random coil near the active site. 329-Pos Board B94 Characterization of the Essential Residues of Cyclooxygenase-1 and 2 Responsible for their Inter-Subunit Communications Upon their Binding to the Corresponding Substrates and Inhibitors Inseok Song. University of Seoul, Seoul, Korea, Republic of. Various conformational changes of a protein upon interaction with its endogenous partner molecules or artificial synthetic compounds imply often an essential role structurally or functionally. Induction triggered by its binding to small ligands can contribute a long-range communication between intraor inter-subunits, which has been exemplified in several model studies. Among these, cyclooxygenases (COX-1 and 2), also known as prostaglandin endoperoxide synthases, display a differential binding pattern between two sequentially identical monomers. COXs catalyze the first committed step in the conversion of arachidonic acid into prostaglandins and thromboxanes. Potential drug-like compounds against COXs have been enormously developed and characterized up to date, which are exploited in this study with an aim to find core regions or residues responsible for their inter-subunit and domain-domain communications. In addition to a careful examination of the COX crystallographic data, extensive analysis of docking experiments with categorized NSAIDs and prediction of hot-spot(s) for proteinprotein interaction were performed, which suggest the theoretical basis for the functionally heterodimeric nature and half-of-the-sites behavior of COXs.

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330-Pos Board B95 A Computational Investigation into the Mechanism of the Histone Acetyltransferase, Gcn5 R. Hunter Wilson, Isaiah Sumner. Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA, USA. Post-translational modifications (PTMs) can have a profound effect on protein structure and function. One such PTM is the acetylation of histone (a protein involved in DNA binding). In this reaction, an enzyme catalyzes the transfer of the acetyl group from acetyl CoA to a free lysine on the histone. This transfer neutralizes the positively charged lysine, which ultimately allows the DNA to be exposed for transcription. In our study, we focus on the acetyltransferase, Gcn5. Details regarding the reaction mechanism used by Gcn5 remain obscured. However, current mechanistic hypotheses suggest that the reaction occurs through a tetrahedral oxyanion intermediate, which is stabilized by a hydrogen bond to a nearby residue, i.e. an oxyanion hole. We utilize molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations in order to further probe possible mechanistic schemes of this reaction. 331-Pos Board B96 Molecular Simulations of Bacterial Lipoprotein Biogenesis Phillip J. Stansfeld. Biochemistry, University of Oxford, Oxford, United Kingdom. Lipoproteins perform critical roles in bacterial physiology, pathogenicity, and antibiotic resistance. Their roles include modulation of the cell envelope structure, signal transduction and transport. Lipoproteins are processed by a pathway of membrane proteins - Sec, Lgt, LspA, Lnt and Lol - which insert, cleave and transport the protein substrate, while affixing lipid moieties to an absolutely conserved cysteine and therefore permitting their tethering to the cell envelope. The recent determination of the three-dimensional protein structures of Lgt and LspA have enlivened lipoprotein research. Furthermore, the latest structures of the Sec translocon with associated signal peptide provide a means to study the initiation point of this pathway and mechanism by which the lipoproteins are inserted into the cell membrane. Structural studies of mature lipoproteins have also recently come to the fore, including the BAM complex, of which four of the five proteins are lipoproteins, and LptDE, an outer membrane barrel with a lipoprotein plug. Both complexes are essential to gram-negative bacteria; respectively, folding the protein-conduits that enable nutrient transport through the outer membrane and assembling the outermost bacterial fortifications. Here we have used a range of molecular simulation, modelling and bioinformatics methods to study this pathway. With initial focus on the first two enzymes of the pathway, Lgt and LspA, our studies have elucidated the mode of binding of signal peptides to both enzymes and interpreted from this the molecular mechanisms involved in the enzymatic reactions. These studies highlight key roles for the most highly conserved residues, whilst also providing a means to inhibit the enzymes, as illustrated by the antibiotic, globomycin, bound to LspA. We have also developed molecular parameters for the cysteine lipid-moeities, and applied these post-translational modifications to simulate the dynamics of both individual lipoproteins and the lipoprotein complexes of BAM and LptDE within the bacterial outer membrane. 332-Pos Board B97 Heme Trafficking by the Cytochrome C Biogenesis Pathways Molly C. Sutherland1, Joel A. Rankin2, Robert G. Kranz1. 1 Washington University in St. Louis, St. Louis, MO, USA, 2Michigan State University, East Lansing, MI, USA. Cytochromes function in electron transport chains to perform critical cellular functions, such as respiration and photosynthesis. Cytochromes c are unique due to their requirement for the covalent attachment of heme via two thioether bonds at a conserved CXXCH motif. Three pathways have been identified for cytochrome c maturation: System I (prokaryotes), System II (prokaryotes) and System III (eukaryotes). System I consists of 8 integral membrane proteins (CcmABCDEFGH), System II is comprised of 2 membrane proteins (CcsBA) and these pathways will be the focus of this presentation. Trafficking of heme from the site of its synthesis (cytoplasm) to the site of attachment to apocytochrome c (periplasm) is critical for cytochrome c biogenesis, yet little direct evidence of heme trafficking exists. The study of heme trafficking has proved elusive in this and most other systems due to tight cellular regulation, as well as the cytotoxic and amphipathic nature of heme. Here, we use key putative heme transporters in the prokaryotic pathways as model systems to develop a novel technique to covalently ‘trap’ heme during the trafficking process. First, the conserved WWD domain, which is predicted to interact with heme in the

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periplasmic space and is found in both System I and System II pathways, was used to develop the heme trapping technique. Heme was covalently trapped in the WWD domains of CcmC (System I) and CcsA (System II), providing the first direct evidence of how heme is trafficked to the periplasm in cytochrome c biogenesis. Currently, this trapping approach is being used on other integral membrane proteins in the pathway to capture heme intermediates and delineate the exact paths for trafficking in vivo. We envision that this approach will be applicable to other heme transporters and trafficking pathways from prokaryotes through eukaryotes. 333-Pos Board B98 Designed Enzymes and the Driving Forces Behind Interdomain Electron Transfer Mia C. Brown, Kelly Greenland, Lei Zhang, Ronald L. Koder. City College of New York, New York, NY, USA. Synthetic biology and biodesign approaches to redox active enzymes will require the construction of artificial electron transport chains, particularly chains which can interconvert between one- and two-electron chemistry. To both demonstrate this capability and explore the engineering parameters necessary for rapid and efficient electron transport through artificial electron transport chains, we have constructed a natural protein-designed protein chimera in which the diflavin domain of P-450 BM3 is connected to a de novo designed, heme binding four helix bundle. This single chain protein contains one FMN, one FAD, and two heme cofactors. This chimera reacts with NADPH, taking in its two electrons at the FAD cofactor, breaking them into single electrons at the FMN cofactor, and then transferring them into the artificial heme domain. We have tested three different heme analogues with varying mid-point potentials to examine the effect of driving forces on interdomain electron transfer rates. Finally, as our heme-binding domain is capable of binding oxygen in the reduced state, I will present some results using this construct as an artificial nitric oxide dioxygenase, which can perform NADPH-driven catalysis. 334-Pos Board B99 Factors Governing Autooxidation of Human Hemoglobin Andres S. Benitez Cardenas1, John S. Olson2. 1 Biosciences, Rice University, Houston, TX, USA, 2Biosciences, Rice University, HOUSTON, TX, USA. Determining mechanisms for the autooxidation of hemoglobin is required for understanding and treating unstable hemoglobinopathies and for developing more stable hemoglobin based O2 carriers. Previous studies suggested significant differences in autooxidation rates of a and b subunits. We used an azide reaction assay to measure the concentrations of ferric a and b chains at different time points during autooxidation. Our results showed no differences between the subunits. To obtain more accurate time courses for autooxidation, we deconvoluted observed spectra into the decay of HbO2, metHb appearance, hemichrome generation, and increases in turbidity due to hemin loss and apoprotein precipitation. The time courses for HbO2 decay at high concentrations (R 100mM heme) accelerate implying cooperative autooxidation, where as at low concentrations (% 10uM) the time courses are biphasic. These results suggest that the biphasic time courses at low hemoglobin are due to differences between tetramers and dimers. We have also measured autooxidation rates for a recombinant hemoglobin, rHb0.1, that contains a genetically crosslinked di-a subunit. This hemoglobin shows a monophasic time course for autooxidation at both high and low protein concentrations, and the azide binding assay showed equal amounts of ferric a and b subunits. We have also examined recombinant mutant hemoglobins to examine the structural factors that govern autooxidation. Increased rates of autooxidation were found for rHb Providence, rHb Bethesda, rHb Presbyterian, and rHb Kirklareli. We have also confirmed that the rate of autooxidation shows a bell-shaped dependence on oxygen concentration and increases markedly as the pH is decreased. Supported by NIH Grant P01 HL110900 and by Grant C-0612 from the Robert A. Welch Foundation. 335-Pos Board B100 Assessing the Spectroscopic Properties and Enzyme Activity of Fluorescent Caspase Substrates Gena Lenti, Nicholas Tassone, Srirajkumar Ranganathan, Caitlin Karver, Cathrine A. Southern. Chemistry, DePaul University, Chicago, IL, USA. Inflammatory caspases (caspase-1, 4 and 5 in humans and caspase-11 in mice) are cysteine-dependent, aspartate-specific proteases implicated in inflammatory, autoimmune and autoinflammatory disorders. To date, assays seeking to test the activity of caspases-1, 5 and 11 have all used Ac-WEHD-AMC as their fluorogenic substrate. To explore the possibility that alternative fluorogenic peptides may exhibit enhanced assay properties, we have designed, synthesized, and characterized several novel fluorogenic peptides containing

coumarin derivatives. The coumarin derivatives were incorporated into peptides with various amino acid residues: WEHDA, WEHD, LEVD, LEHD as either a side chain of a non-natural amino acid, or at the C-terminus. The fluorescence quantum yields of these peptides were obtained, allowing the viability of these substrates to enhance the signal to noise ratio in caspase enzyme assays to be assessed. Biochemical assays were then carried out to determine if the signal to noise ratio indicated by the fluorescence quantum yield results correlated with caspase activity and could be applied to inhibitor screening assays. 336-Pos Board B101 Single Molecule Enzymology with Outer Membrane Protein G Bach G. Pham. University of Massachusetts - Amherst, Amherst, MA, USA. We have developed a nanopore sensor based on Outer membrane protein G (OmpG) to study enzyme kinetics at the single molecule level. OmpG is a b -barrel porin with seven flexible loops that we had previously exploited in protein sensing. A recognition peptide sequence was inserted into one of OmpG’s loops allowing it to undergo enzymatic reaction. Caspase 7 (casp-7), a protease implicated in apoptosis, was chosen as the target enzyme to interact and chemically alter the OmpG sensor. Currently, we can unambiguously detect the enzyme-substrate complex of casp-7 and OmpG, as well as the OmpG cleavage product in real time. We found that the key to the robust efficiency of casp-7 activity (and many other caspases) is that substrate is always cleaved once bound to casp-7. Casp-7 cleavage is essentially an irreversible reaction (k-1 = 0; k2 = 8.3 s 1). In addition, we manipulated the pH and observe the effects on k2 that support the catalytic mechanism of cysteine proteases. Our results allow us to probe more closely the catalytic mechanism of an enzyme which cannot be probed using conventional ensemble assays. Thus, we can use our single molecule OmpG enzymology platform to a variety of other enzyme targets in a similar fashion. 337-Pos Board B102 Protein Semi-Synthesis to Characterize Phospho-Regulation of Human UNG2 Brian P. Weiser, James T. Stivers, Philip A. Cole. Pharmacology & Molecular Sciences, Johns Hopkins University, Baltimore, MD, USA. The human nuclear Uracil DNA Glycosylase (hUNG2) initiates the base excision repair pathway that removes uracil from genomic DNA. The catalytic domain of hUNG2 is preceded by a disordered N-terminus that contains numerous sites for post-translational modifications. We hypothesized that certain modifications might affect hUNG2 activity and/or its interactions with its protein binding partner, Proliferating Cell Nuclear Antigen (PCNA), which is thought to bind hUNG2 residues 4-11. Using protein semisynthesis, we prepared full-length hUNG2 with a phosphorylation at either Thr6 or Tyr8, and these constructs were purified to >95%. The uracil excision activity of phospho-hUNG2 proteins was comparable to unmodified hUNG2 when using a 19mer duplex DNA substrate. However, fluorescence anisotropy measurements showed that the phospho-hUNG2s had >10-fold weaker affinities for free PCNA. The PCNA affinity of a synthetic peptide that corresponds to UNG2 residues 1-19 was similar to that of full-length hUNG2. Our data indicates that the N-terminal 11 residues of hUNG2 are necessary and sufficient for high-affinity binding to PCNA, and that phosphorylation within this motif disrupts binding. How phosphorylation affects other aspects of hUNG2 activity is now being explored. 338-Pos Board B103 Voltage Dependent Phosphatase Activity is Enhanced by Intracellular Acidification Angeliki Mavrantoni, Kirstin Hobiger, Dominik Oliver, Christian R. Halaszovich. Neurophysiology, University Marburg, Marburg, Germany. Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4) P2/PI(3,4,5)P3-3-phosphatases. For non-mammalian VSPs, this activity is regulated by membrane voltage via a voltage sensor domain (VSD). For mammalian VSPs the VSD seems insensitive to voltage changes yet still essential for control of the phosphatase activity. Under physiological conditions, the non-mammalian VSPs strongly deplete PI(4,5)P2 in a voltage dependent manner. The physiological regulator of mammalian VSP activity remains elusive. VSPs are suggested to play a role in fertilization and development, where changes in intracellular pH are known to occur. They are found to be expressed in tissues like kidney, stomach, sperm, and ovary which are known to undergo such pH changes. Therefore, we speculated that intracellular pH might modulate VSP activity. To test this hypothesis we performed whole-cell patch-clamp experiments in CHO cells expressing diverse VSPs and fluorescent PI(4,5)P2 reported