John G. Primm

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.

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.

Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins.

The use of the Protemist XE, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported. The soluble Galdieria sulphuraria protein DCN1 was obtained in greater than 2mg total synthesis yield per mL of reaction mixture from the Protemist XE, and the structure was subsequently solved by X-ray crystallography using material from one 10 mL synthesis (PDB ID: 3KEV). The Protemist XE was also capable of membrane protein translation. Thus human sigma-1 receptor was translated in the presence of unilamellar liposomes and bacteriorhodopsin was translated directly into detergent micelles in the presence of all-trans-retinal. The versatility, ease of use, and compact size of the Protemist XE robot demonstrate its suitability for large-scale synthesis of many classes of proteins.

Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence

Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling.

Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or 1H–15N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.

X‐ray structure of Arabidopsis At1g77680, 12‐oxophytodienoate reductase isoform 1

Brian G. Fox,* Thomas E. Malone, Kenneth A. Johnson, Stacey E. Madson, David Aceti, Craig A. Bingman, Paul G. Blommel, Blake Buchan, Brendan Burns, John Cao, Claudia Cornilescu, Jurgen Doreleijers, Jason Ellefson, Ronnie Frederick, Holokere Geetha, David Hruby, Won Bae Jeon, Todd Kimball, John Kunert, John L. Markley, Craig Newman, Andrew Olson, Francis C. Peterson, George N. Phillips Jr., John Primm, Bryan Ramirez, Nathan S. Rosenberg, Mike Runnels, Kory Seder, Jeff Shaw, David W. Smith, Hassan Sreenath, Jikui Song, Michael R. Sussman, Sandy Thao, Donna Troestler, Ejan Tyler, Robert Tyler, Eldon Ulrich, Dimitriy Vinarov, Frank Vojtik, Brian F. Volkman, Gary Wesenberg, Russell L. Wrobel, Jie Zhang, Qin Zhao, and Zolt Zolnai University of Wisconsin Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, Madison, Wisconsin Molecular and Environmental Toxicology Program, University of Wisconsin–Madison, Madison, Wisconsin Biophysics Doctoral Program, University of Wisconsin–Madison, Madison, Wisconsin

The structure at 2.4 A resolution of the protein from gene locus At3g21360, a putative Fe(II)/2-oxoglutarate-dependent enzyme from Arabidopsis thaliana.

The crystal structure of the gene product of At3g21360 from Arabidopsis thaliana was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 19.3% (Rfree = 24.1%) at 2.4 A resolution. The crystal structure includes two monomers in the asymmetric unit that differ in the conformation of a flexible domain that spans residues 178-230. The crystal structure confirmed that At3g21360 encodes a protein belonging to the clavaminate synthase-like superfamily of iron(II) and 2-oxoglutarate-dependent enzymes. The metal-binding site was defined and is similar to the iron(II) binding sites found in other members of the superfamily.

Technology Development Highlights Generated from the Center for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) aims to be the leading center for developing and disseminating tested technologies to efficiently solve structures of eukaryotic proteins. We create, evaluate, and optimize innovative protocols for producing eukaryotic proteins in active forms. We seek to improve the efficiency of all stages from target selection-design to three-dimensional structure determination by X-ray crystallography or NMR spectroscopy, including development of bioinformatic techniques and LIMS tools. Using our protein production platform, we refine methods for improving the yield of structures from high-value targets, in particular proteins from humans and other vertebrates. CESG has a substantial outreach component; more than 400 targets from outside requestors have been accepted for study with a structure success rate of 5%, which compares favorably with the eukaryotic success rates for the total PSI effort. These eukaryotic targets frequently present unique challenges. All CESG protein production protocols and Technology Dissemination Reports are accessible through the PSI Knowledgebase: http://kb-psi-structuralgenomics.org/KB/ and CESGs website. Selected technology developments are presented here. These include advances in expression vector design, enhanced methodology for cell based and wheat germ cell-free expression systems, new software to improve the quality and reduce time for structure determination by X-ray crystallography and NMR, and optimized techniques for the production of TEV protease for use in our protein production platform. We actively share our advances with the biotechnology, pharmaceutical, and academic communities through collaborations, oral presentations, peer-reviewed articles, submissions to public databases and material distribution channels, including PepcDB, PDB, BMRB, PSI Materials Repository, and technology transfer workshops.Supported by NIH/NIGMS Protein Structure Initiative grants GM074901 and GM064598. We acknowledge ALL members of the CESG team for their dedicated work.