Russell L. Wrobel

Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination.

AbstractThe Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsisopen reading frames into Escherichia coli expression vectors are presented along with an analysis of results from ~2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold–function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel GatewayTM protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the GatewayTM system.

Comparison of Cell-Based and Cell-Free Protocols for Producing Target Proteins from the Arabidopsis thaliana Genome for Structural Studies

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell‐based and cell‐free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N‐labeled proteins to be analyzed by 1H‐15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell‐based and two involved wheat germ cell‐free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H‐15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC± (protein partially disordered or not in a single stable conformational state), HSQC− (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X− for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell‐free platform offers the advantage of greater genome coverage for NMR‐based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X‐ray structure determination. Proteins 2005.

Mitochondrial COQ9 is a lipid-binding protein that associates with COQ7 to enable coenzyme Q biosynthesis

Significance Coenzyme Q (CoQ) is a requisite component of the mitochondrial oxidative phosphorylation machinery that produces more than 90% of cellular ATP. Despite the discovery of CoQ more than 50 years ago, many aspects of its biosynthesis remain obscure. These include the functions of uncharacterized CoQ-related proteins whose disruption can cause human diseases. Our work reveals that one such protein, COQ9, is a lipid-binding protein that enables CoQ biosynthesis through its physical and functional interaction with COQ7, and via its stabilization of the entire CoQ biosynthetic complex. Unexpectedly, COQ9 achieves these functions by repurposing an ancient bacterial fold typically used for transcriptional regulation. Collectively, our work adds new insight into a core component of the CoQ biosynthesis process. Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1–9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis.

The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands

Background: Sigma-1 receptor (S1R) is an integral membrane ligand-binding receptor. Results: Gel filtration chromatography revealed oligomeric states that are stabilized by ligand binding and destabilized by mutations in the GXXXG integral membrane dimerization domain. Conclusion: Purified S1R binds small molecule ligands as an oligomer but not as a monomer. Significance: The results provide new insight into the function of S1R with ligands and proteins partners. Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[3H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed.

Mitochondrial ADCK3 Employs an Atypical Protein Kinase-like Fold to Enable Coenzyme Q Biosynthesis

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

Cell-free translation of integral membrane proteins into unilamelar liposomes.

Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies.

Flexi Vector Cloning

A protocol for ligation-dependent cloning using the Flexi Vector method in a 96-well format is described. The complete protocol includes PCR amplification of the desired gene to append Flexi Vector cloning sequences, restriction digestion of the PCR products, ligation of the digested PCR products into a similarly digested acceptor vector, transformation and growth of host cells, analysis of the transformed clones, and storage of a sequence-verified clone. The protocol also includes transfer of the sequence-verified clones into another Flexi Vector plasmid backbone. Smaller numbers of cloning reactions can be undertaken by appropriate scaling of the indicated reaction volumes.

The Center for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) is a “specialized” or “technology development” center supported by the Protein Structure Initiative (PSI). CESG’s mission is to develop improved methods for the high-throughput solution of structures from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes. Notably, 30% of all structures of human proteins solved by the PSI Centers were determined at CESG. Whereas eukaryotic proteins generally are considered to be much more challenging targets than prokaryotic proteins, the technology now in place at CESG yields success rates that are comparable to those of the large production centers that work primarily on prokaryotic proteins. We describe here the technological innovations that underlie CESG’s platforms for bioinformatics and laboratory information management, target selection, protein production, and structure determination by X-ray crystallography or NMR spectroscopy.

X-ray structure of Arabidopsis At2g06050, 12-oxophytodienoate reductase isoform 3

Thomas E. Malone, Stacey E. Madson, Russell L. Wrobel, Won Bae Jeon, Nathan S. Rosenberg, Kenneth A. Johnson, Craig A. Bingman, David W. Smith, George N. Phillips, Jr., John L. Markley, and Brian G. Fox* Molecular and Environmental Toxicology Program, University of Wisconsin–Madison, Madison, Wisconsin Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, Madison, Wisconsin Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin

Crystal structure of At2g03760, a putative steroid sulfotransferase from Arabidopsis thaliana

David W. Smith, Kenneth A. Johnson, Craig A. Bingman, David J. Aceti, Paul G. Blommel, Russell L. Wrobel, Ronnie O. Frederick, Qin Zhao, Hassan Sreenath, Brian G. Fox, Brian F. Volkman, Won Bae Jeon, Craig S. Newman, Eldon L. Ulrich, Adrian D. Hegeman, Todd Kimball, Sandy Thao, Michael R. Sussman, John L. Markley, and George N. Phillips, Jr.* Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin Center for Eukaryotic Structural Genomics, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin