Protein Protein Interactions Of Glycine Oxidase (Thi O)
Identification of Protein-Protein Interactions of Glycine Oxidase (ThiO) Brandon Turner, Lauren Pioppo December 14, 2011
Abstract: Glycine Oxidase, or ThiO, from Bacillus subtilis catalyzes the FAD dependent oxidation ofglycine to imino-glycine in the thiamin biosynthesis pathway. In this pathway, the mechanism inwhich iminoglycine and ThiS reaches the active site of ThiG remains unknown (Settembre et al2003). Elucidation of this step in the pathway can aid in the development of drugs that targetthiamin biosynthesis in several pathogenic prokaryotes, such as P falciparum, the increasinglyresistant protozoan parasite that causes malaria. Here, we use recombinant His-tagged ORF811from Bacillus muhlenbergius, a close homolog of B. subtilis ThiO, to analyze any protein:proteininteractions among ThiO and other enzymes involved in the passage of iminoglycine to ThiGduring thiamin biosynthesis. A pull-down assay of ORF811 and CFCE of B. muhlenbergius did notreveal any stable protein:protein interactions with ORF811. Additionally, cross-linking studieswere performed to elucidate any transient interactions; a Western blot of these reactionsindicated possible dimer formation of ORF811, but did not reveal any relevant transientinteractions.
Introduction: Many molecular processes throughout the body, in all tissues, involve the interaction ofmultiple proteins. These interactions can range from modification of other proteins, such asphosphorylation or acylation, to the coupling of mechanistic steps by the close physical contactof enzymes in a metabolic pathway. The latter can be accomplished by several methodsincluding the formation of large multi-enzyme complexes, such as pyruvate Dehydrogenase(McMurry, Begley 2005), or unstable transient protein interactions (Krishnamurthy et al 2011).Identifying protein-protein interactions has become a new practice of proteomics (Blagoev et al2003) and can serve to elucidate how complex reactions involving reactive intermediates canprogress at a cellular level. Recently, a new organism named Bacillus muhlenbergius has been identified as a raresoil bacterium inhabiting areas in Pennsylvania near Muhlenberg College, Allentown. Thisorganism is a close relative via genomic sequence of the more ubiquitous B. subtilis. A recentproject has been undertaken by this lab to identify the genomic characteristics of B.muhlenbergius by elucidating the characteristics of several prokaryotic enzymes in hopes ofidentifying enzymes that may be useful for biotechnological advances; one such enzyme isOrf811. Bioinformatic analysis has identified Orf811 as a close homolog of Glycine Oxidase(GO)1 from B. subtilis. GO is commonly referenced as ThiO for its oxidative role thiaminbiosynthesis. This enzyme is a homotetrameric protein that catalyzes the FAD dependentoxidation of glycine to iminoglycine before being shuttled down the anabolic pathway(Settembre et al 2003). However, several key factors in this progression of the pathway remain1 3 GO: Glycine Oxidase. BS : Bissulfosuccinimidyl suberate. EDC: 1-ethyl-3-[dimethylaminopropyl]-carbodiimide.CFCE: Cell-Free Crude Extract. BC: Bait Control. RC: Resin Control
unsolved. The imine product of GO is highly reactive in solvent and is readily hydrolyzed (Mortl2006). This hydrolyzed product is not capable of being used in the pathway as the followingreaction of ThiS requires the imine product to continue. The method by which iminoglycine andThiS reach the active site of ThiG remains to be solved. The solved crystal structure of GO has shown that the enzyme contains a large channelleading to the active site, which is buried within the hydrophobic core. However, the active siteis solvent exposed, indicating that the imine product cannot leave the active site without beinghydrolyzed (Mörtl et al 2004). Several enzymes that produce reactive intermediates have beenshown to contain structural characteristics that allow transfer of the species to the next activesite without exposure to other cellular components or solvent, such as the indole intermediatein tryptophan synthase (Raushel et al 2003). This multifunction enzyme performs both portionsof the reaction with a protein ‘tunnel’ throughout the center to shuttle the reactive indolemolecule to the second active site. Other enzymes that produce reactive species have beenfound to be part of large, multi-enzyme complexes, allowing the close proximity of the enzymesin solvent to facilitate the transfer of the reactive species from one active site to another(McMurry, Begley 2005). Given the reactive nature of iminoglycine and other proteininteractions within the thiamin biosynthesis pathway, such as that between ThiG and ThiS(Settembre et al 2004), it is likely that some form of intermediate shuttling is occurring in orderto prevent hydrolysis prior to reacting with ThiS. In addition, the thiamin biosynthesis pathway has long been known as an antibiotictarget for several pathogenic prokaryotes that are unable to salvage vitamin B1, the mostnotable being P. falciparum, the infections agent responsible for Malaria (Muller et al 2010).Malaria was responsible for roughly 781,000 deaths in 2009 alone and is resistant to otherforms of antibiotic treatment as it is an obligate intracellular parasite (WHO 2011). Identifyingpossible protein-protein interactions within the thiamin biosynthesis pathway can provide anovel drug target for antibiotic research against this increasingly resistant pathogen. In this experiment, we attempted to elucidate the details of this passage of imino-glycine to ThiS by performing a pull-down assay using His-tagged Orf811 and cell free crude
extracts from B. muhlenbergius to identify any stable protein-protein interactions betweenother enzymes in the thiamin synthesis pathway. SDS-PAGE analysis revealed no visibledifference from control samples, indicating that no stable interactions had formed. In anattempt to find any transient interactions between proteins, molecular crosslinkers were usedto identify putative protein-protein interactions involving ThiO. Western blot analysis ofsamples crosslinked by BS3 showed possible dimer formation of ThiO, while EDC crosslinkingshowed no bands. This study is important in identifying interactions between ThiO and otherenzymes in the thiamin synthesis pathway which can serve to explain how this reactionprogresses beyond the oxidation of glycine without loss of product to hydrolysis. Comparison ofthis process in B. muhlenbergius to other prokaryotic organisms can serve to identifyevolutionary characteristics of this new organism and provide drug targets for P. falciparum.
Materials and MethodsChemicals and Equipment All reaction kits were purchased from Thermo Scientific. Chemicals were purchased fromQiagen, except for imidazole and Triton X-100 (ACROS), TRIS and 10% Tween 20 (BioRad), andGelCode Stain and Coomassie Plus Protein Assay Reagent (Thermo Scientific). All high speed(>10,000g) centrifugation was performed in the Sorvall RC6 High Speed Centrifuge, while slowerspeeds were performed in the Eppendorf 5430. Cell Lysis was performed using a BeadBeater(BioSpec).Bioinformatics Analysis A Bioinformatic analysis of Orf811 was performed using ExPASy to obtain a partialprotein sequence from cDNA. The Basic Alignment Search Tool (BLASTP) was used to identifypotential homologs of Orf811 using the translated sequence. GenBank was then used toidentify the genomic context of GO.Preparation of Bacillus muhlenbergius Cells Two cultures of B. muhlenbergius cells were grown in 1 liter of minimal media lackingthiamin (5x M9 salts, Difco casamino acids, 20% glucose) at 30 C for about 48 hours. Cells wereharvested by centrifugation at 10,000xg for 30 min. Cell pellets were lysed by BeadBeating onice (12 cycles of beating for 15 seconds, followed by 45 seconds of rest) and centrifuged at15,000xg to obtain CFCE. The concentration of the crude extract was determined using aBradford assay. Bovine serum albumin (BSA) was used for calibration.Pull-Down Assay: A pull-down assay of B. muhlenbergius ORF811 was performed using the ProFoundPull-Down PolyHis Protein:Protein Interaction Kit. Poly-His tagged ORF811 (3.5 mg/ml), agenerous gift from Dr. Keri Colabroy, Muhlenberg College, was used as bait protein, andprepared cell-free crude extracts of B. muhlenbergius were used as a source of prey proteins.
Orf811 and CFCE were combined prior to use in the pull-down to a final protein concentrationof 2.35 mg/ml. This combined protein solution was also used for both cross-linking studies. Thepull-down was performed according to the protocol provided by Thermo Scientific. Briefly,three 1.0 mL columns were prepared [(Bait Control (+Orf811, -CFCE), Resin Control (-Orf811,+CFCE), Sample (+Orf811, -CFCE)] and treated with the corresponding proteins. Columns wereincubated at 4°C for 1 hour following the addition of Orf811 and an additional hour afterwashing the column using the provided wash solution and treatment with CFCE. All flowthrough was collected for analysis. Bound Orf811 was eluted from the columns using ImidazoleElution Buffer (From kit).Cross-Linking Studies Cell-free crude extracts of B. muhlenbergius and purified poly-His tagged ORF811 werebuffer exchanged into 20 mM potassium phosphate monobasic buffer (pH 7.8) using an Econo-Pac 10 DG gel filtration column. This mixture was cross-linked with bissulfosuccinimidylsuberate (BS3) following the protocol provided by Thermo Scientific. Cell-free crude extracts ofB. muhlenbergius and purified poly-His tagged ORF811 were buffer exchanged into 0.1 M MESbuffer (2-[N-morpholino]ethane sulfonic acid, pH 4.94) using an Econo-Pac 10 DG gel filtrationcolumn. This mixture was cross-linked with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC) following the protocol provided by Thermo Scientific. Briefly, 10mg EDC was added to 1 mL of ultra-pure (miliQ) H2O to prepare thecrosslinking solution. 100 uL of EDC solution was added to a 1 mL sample of combined proteinsolution and incubated at room temperature for two hours. The reaction was desalted by use ofAmicron 4.0 mL Ultra Filtration with addition of MES buffer, centrifuged 3x at 7100xg for 10min. For BS3, a 12.5 mM solution was prepared in a 50-molar excess to protein concentrationby adding 277 uL of 20 mM Potassium Phosphate buffer to 2 mg of BS 3. 100 uL of BS3 solutionwas added to 500 uL of combined protein sample and incubated at room temperature for 30min. Tris-HCl buffer was added to quench the reaction and the quenched reaction was stored at-20°C.
SDS-PAGE and Western Blot Analysis Pull-down and crosslinking samples were analyzed by SDS-PAGE Gel Electrophoresis.Samples were run on two 4-20% polyacrylamide gels. The gel containing pull-down samples wasstained using GelCode stain while the gel containing the crosslinking samples waselectroblotted to a membrane. The membrane was subjected to staining with Anti-His HRPConjugate antibodies.
Results:Bioinformatics Analysis of Orf811 In order to glance at Orf811’s possible structure and function, a bioinformatics approachwas used to find homologs. The fragment of sequenced genome was translated using ExPASyand run through BLASTP which showed a high relation to Glycine Oxidase from Bacillus subtilis(Max Score: 447, E-value: 2e-156, top hit). This homolog is ThiO, an FAD dependent oxidase,and is implicated in thiamine biosynthesis by its close proximity to ThiS, a sulfur carrier, andThiF, which is involved in thiamine/molybdopterin sytnthesis. Structural data shows it exists asa homotetramer (3). Based on its sequence similarity, it is likely that Orf811 will be similar infunction to ThiO.Preparation and Purification of Bacillus muhlenbergius Cell Lysate Cultures of B. muhlenbergius were harvested at OD600 = 0.6 and lysed via BeadBeating toobtain CFCE. Cellular protein was purified to 12.46 mg/mL and stored with glycerol at -20°C.Protein samples were aliquoted in 800uL amounts for future use. Although the presence ofproteins involved in thiamine biosynthesis was unable to be determined, dark bands close tothe molecular weights of involved proteins (ThiS: 7.89 kDa, ThiG, 26.9 kDa) suggest an increasein expression of these enzymes.Pull Down Assay A pull-down assay involving His-tagged Orf811 was performed to locate stableprotein:protein interactions within the thiamine biosynthetic pathway. SDS-PAGE analysis of allsamples, including flow through and positive controls was performed to view any boundproteins. Control samples consisted of Bait Control (BC) with no prey protein solution added tothe column and Resin Control (RC) with no Orf811 bound to the column. Flow thru after theaddition of both bait and prey proteins were collected for analysis. Gel bands for experimentalsamples were identical to that of bait control (fig. 1), indicating that no stable interactions werepresent. The double band shown for Orf811 is consistent with an addition of a small protein;however, with no difference between sample and control lanes, no additional proteins seem to
have been purified. Due to lack of binding, gel bands were not excised for peptide massfingerprinting.
Cross-Linking Studies Cross-linking studies involving a protein mix of Orf811 and CFCEwere performed using BS3 and EDC. In order to perform these studies, protein solutions werebuffer exchanged into KPO3 monobasic buffer to avoid cross-linker interactions with solvent.The cross-linked solutions were analyzed via western blotting using His-tag specific antibodiesto observe any changes in molecular weight of Orf811. No bands were observed for EDC cross-linking, but two additional bands were present for BS3 (fig. 2). Both bands are higher inmolecular weight than Orf811 but do not correspond to an addition of 7.2 kDa (ThiS) or 26.9kDa (ThiG) (Settembre et al 2004). Molecular weights are consistent with dimer and trimerformation of GO due to the even spacing of gel bands and their apparent increase in molecularweight of 47 kDa.
Discussion: We present here an attempt to identify stable and transient protein:protein interactionsbetween the homotetrameric protein, ThiO and other enzymes in the thiamin biosynthesispathway. Although ThiO is known to catalyze the FAD dependent oxidation of glycine to imino-glycine in this pathway (Settembre et al 2003), several components of the progression remainunsolved. One unsolved component of this pathway includes the highly reactive imine productof ThiO; although this product is readily hydrolyzed, the next reaction in the pathway requiresthe non-hydrolyzed imine to occur. This reaction, which includes the binding of bothiminoglycine and ThiS to the active site of ThiG, poses the question as to how ThiS andiminoglycine reach ThiG (Mörtl et al 2004). Since the active site of ThiO is known to be solventexposed (Mörtl et al 2004), it is possible that the imine product is transferred to the active siteof ThiG through intermediate shuffling to prevent hydrolysis of iminoglycine (Settembre et al2004). Analysis of the protein:protein interactions between ThiO and other enzymes in thethiamin biosynthesis pathway, such as ThiS, may aid in elucidating this potential intermediateshuffling and the further mapping of the pathway mechanisms. This work includes a pull-downassay and cross-linking studies to study both stable and transient protein:protein interactions,respectively. The newly discovered species of bacteria, Bacillus muhlenbergius, was used in this study.Prior characterization of this species has revealed a novel enzyme, ORF811, which was used inall experiments. By utilizing the Basic Local Alignment Search Tool, we found that closelyrelated homologs of ORF811 were annotated as glycine oxidases, indicating that ORF811 mayalso function as a glycine oxidase. Using the BRENDA enzyme database, it was found that aglycine oxidation reaction catalyzed by an ORF811 homolog consists of glycine, water, andmolecular oxygen reacting to form glycoxylate, ammonia, and hydrogen peroxide; this providessome insight as to what type of reaction ORF811 may catalyze. Additionally, a genomic contextanalysis of closely related homologs of ORF811 revealed that the homologous gene ThiO, as
well as its surrounding genes, appears to function in thiamine biosynthesis; this indicates thatORF811 may play similar roles in metabolism. Thus, the apparent close homology betweenORF811 and ThiO allowed us to use recombinant His-tagged ORF811 to facilitate the study ofprotein:protein interactions between ThiO and other enzymes in the thiamin biosynthesispathway. The study was initiated by growing B. muhlenbergius in nutrient deficient media. Thisforced the bacteria to produce large amounts of their own thiamin, as well as the proteinsimplicated in thiamin biosynthesis, ideally to improve binding of ORF811 to its potentialpartners in the pathway. Growth and harvesting of B. muhlenbergius was successful, and 9.5 mlof cell lysate was recovered from 2 L of culture; a Bradford assay revealed the concentration ofthis CFCE to be 12.46 mg/ml. To study any stable protein:protein interactions, a pull-down assay was conducted,followed by an SDS-polyacrylamide gel to analyze results. By immobilizing the recombinant His-tagged ORF811 bait to a column with a cobalt chelate resin, we were able to run a sample of B.muhlenbergius cell-free crude extract through the column to determine if any proteins in theextract bound to the immobilized ORF811. However, an SDS-PAGE gel of the eluant revealed nodifference between the bait control, which contained only ORF811 bound to the resin, and theexperimental sample, which contained both the ORF811 bait and the CFCE prey (Fig. 1). Thus,no stable protein-protein interactions between this close homolog of ThiO and other enzymesin the thiamin biosynthesis pathway were observed. Cross-linking studies allowed any transient protein:protein interactions with ORF811 tobe assessed. Following the cross-linking reaction, a Western blot was performed to comparethe size of the band to a control sample of just ORF811; an increase in size would indicate thatbinding may have occurred. A solution of ORF811 and B. muhlenbergius CFCE were cross-linkedwith both BS3 and EDC. When EDC was used as the cross-linker, no signal was observed on theWestern blot. Prior to the cross-linking reaction, the solution of CFCE and ORF811 was bufferexchanged three times, which resulted in a significant decrease in protein concentration. Thus,it is possible that the concentration of protein was too low for any cross-linking to be visualized
on the blot. Another EDC cross-linking reaction with a higher concentration of protein is neededto confirm this conclusion. Although bands were observed when BS 3 was used as the cross-linker, it is not likely that these bands correspond to any relevant protein:protein interactionswith ThiO. Previous studies have indicated that ThiO is a 47 kDa protein (Job et. al. 2001), andthe three bands on the Western blot (Fig. 2) appear to be equally spaced apart by about 47kDa; the bands are about 47 kDa, 94 kDa, and 141 kDa. This 47 kDa spacing between bandssuggests that ORF811 formed a homodimer, and is thus not indicative of any relevant transientprotein:protein interactions. However, this conclusion needs to be confirmed by proteinsequencing of the bands. Although no stable or transient protein:protein interactions with B. muhlenbergiusORF811 and other enzymes involved in thiamin biosynthesis were observed, further research isneeded to investigate these possible interactions. Addition of a glycine or sulfite substrate mayfacilitate the close relation of the possible binding partners, such as ThiS, and thus aid inassessing these interactions. Further elucidation of the method in which iminoglycine and ThiSare passed to the active site of ThiG may provide a potential antibiotic target for pathogens thatare susceptible to antibiotics that target thiamin synthesis, such as P. falciparum, the infectionsagent responsible for Malaria (Muller et al 2010).References: 1. Job V, Marcone GL, Pilone MS, Pollegioni, L. 2001. . Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein. Biological Chemistry [Internet]. [Cited 2011 Oct 28] 277(9):6985-93. Available from: http://www.jbc.org/content/277/9/6985.long#cited-by 2. Krishnamurthy M, Dugan A, Nowkoye A, Fung YH, Lancia JK, Majmudar CY and Mapp AK. Caught in the act: covalent crosslinking captures activator-coactivator interactions in vivo (2011). ACS Chem Biol. [Epub ahead of print]. 3. McMurry, John and Begley, Tadhg. The Organic Chemistry of Biological Pathways. Englewood, Colorado: Roberts and Company Publishers. 2005.
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