Peter S. Horanyi

Structure of the Integral Membrane Protein CAAX Protease Ste24p

Lamin Loppers The nuclear lamina provides mechanical stability to the nuclear envelope and is involved in regulation of cellular processes such as DNA replication. Defects in the nuclear lamina lead to diseases such as progeria and metabolic disorders. One of the components of the nuclear lamina, lamin A, undergoes a complex maturation process. A key player is an inner nuclear membrane zinc metalloprotease (ZMP) that is responsible for two proteolysis steps (see the Perspective by Michaelis and Hrycyna). Quigley et al. (p. 1604) report the crystal structure of human ZMPSTE24 and Pryor et al. (p. 1600) that of the yeast homolog Ste24p. The structures provide insight into the mechanism of catalysis and into why mutations in ZMPSTE24 lead to laminopathies. Structures of two transmembrane zinc proteases reveal a barrel of seven helices surrounding a large cavity. [Also see Perspective by Michaelis and Hrycyna] Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.

Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin labels intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.

Investigation of the role of 3-hydroxyanthranilic acid in the degradation of lignin by white-rot fungus Pycnoporus cinnabarinus.

An aminophenol, 3-hydroxyanthranilic acid (3-HAA), has been proposed to play important roles in lignin degradation. Production of 3-HAA in Pycnoporus cinnabarinus was completely inhibited by a combination of tryptophan and S-(2-aminophenyl)-L-cysteine S,S-dioxide (APCD) while the fungus grew well and produced high amounts of laccase. The biosynthesis of 3-HAA is mainly through the metabolism of tryptophan in the kynurenine pathway. A minor pathway for 3-HAA synthesis is through the hydroxylation of anthranilic acid during the biosynthesis of tryptophan in the shikimic acid pathway. Through UV irradiation of wild-type P. cinnabarinus (WT-Pc) spores, a 3-HAA-less mutant was produced. Both WT-Pc, under the inhibitory culture condition, and the 3-HAA-less mutant were found to degrade lignin in unbleached kraft pulp as efficiently as the WT-Pc, which unambiguously demonstrated that 3-HAA does not play an important role in the fungal degradation of lignin.

Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B(12). Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.

Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited

The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes.

The high-throughput protein-to-structure pipeline at SECSG.

Using a high degree of automation, the crystallography core at the Southeast Collaboratory for Structural Genomics (SECSG) has developed a high-throughput protein-to-structure pipeline. Various robots and automation procedures have been adopted and integrated into a pipeline that is capable of screening 40 proteins for crystallization and solving four protein structures per week. This pipeline is composed of three major units: crystallization, structure determination/validation and crystallomics. Coupled with the protein-production cores at SECSG, the protein-to-structure pipeline provides a two-tiered approach for protein production at SECSG. In tier 1, all protein samples supplied by the protein-production cores pass through the pipeline using standard crystallization screening and optimization procedures. The protein targets that failed to yield diffraction-quality crystals (resolution better than 3.0 A) become tier 2 or salvaging targets. The goal of tier 2 target salvaging, carried out by the crystallomics core, is to produce the target proteins with increased purity and homogeneity, which would render them more likely to yield well diffracting crystals. This is performed by alternative purification procedures and/or the introduction of chemical modifications to the proteins (such as tag removal, methylation, surface mutagenesis, selenomethionine labelling etc.). Details of the various procedures in the pipeline for protein crystallization, target salvaging, data collection/processing and high-throughput structure determination/validation, as well as some examples, are described.

Molecular Origin of Electron Paramagnetic Resonance Line Shapes on β-Barrel Membrane Proteins: The Local Solvation Environment Modulates Spin-Label Configuration

In this work, electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography were used to examine the origins of EPR line shapes from spin-labels at the protein-lipid interface on the β-barrel membrane protein BtuB. Two atomic-resolution structures were obtained for the methanethiosulfonate spin-label derivatized to cysteines on the membrane-facing surface of BtuB. At one of these sites, position 156, the label side chain resides in a pocket formed by neighboring residues; however, it extends from the protein surface and yields a single-component EPR spectrum in the crystal that results primarily from fast rotation about the fourth and fifth bonds linking the spin-label to the protein backbone. In lipid bilayers, site 156 yields a multicomponent spectrum resulting from different rotameric states of the labeled side chain. Moreover, changes in the lipid environment, such as variations in bilayer thickness, modulate the EPR spectrum by modulating label rotamer populations. At a second site, position 371, the labeled side chain interacts with a pocket on the protein surface, leading to a highly immobilized single-component EPR spectrum that is not sensitive to hydrocarbon thickness. This spectrum is similar to that seen at other sites that are deep in the hydrocarbon, such as position 170. This work indicates that the rotameric states of spin-labels on exposed hydrocarbon sites are sensitive to the environment at the protein-hydrocarbon interface, and that this environment may modulate weak interactions between the labeled side chain and the protein surface. In the case of BtuB, lipid acyl chain packing is not symmetric around the β-barrel, and EPR spectra from labeled hydrocarbon-facing sites in BtuB may reflect this asymmetry. In addition to facilitating the interpretation of EPR spectra of membrane proteins, these results have important implications for the use of long-range distance restraints in protein structure refinement that are obtained from spin-labels.

How hydrophobic molecules traverse the outer membranes of Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria provides an effective barrier to their often-harsh extracellular milieu. In particular, the outer leaflet of the OM is not a canonical monolayer of phospholipids. Rather, it is composed of lipopolysaccharide (LPS), a molecule generally consisting of a core of Lipid A decorated with inner and outer core oligosaccharides. The oligosaccharides extend ∼30 A above the plane of the lipid headgroups of the outer leaflet. As such, it is an effective permeability barrier against potentially harmful compounds (1). However, obviously, permeability is required for bacterial survival; no bacterium is an island, as it were. For example, uptake of nutrients is essential, and OM transport proteins are required to conduct this function. The recent paper of Lepore et al. (2) has significantly extended our understanding of how hydrophobic molecules are transported across the OM. With rare exception (e.g., ref. 3), virtually all OM proteins are β-barrels, consisting of an even number of eight to twenty-four of β-strands forming a pore-like structure. Many of these OM pore-like β-barrels are classified as porins, and most nutrient uptake is accomplished by them. The effective aperture of the porin is dictated by the number of β-strands, and the aperture size then dictates the size (and shape) of the solutes that can diffuse through them. Porins function passively, permitting the energy-independent diffusion of solute molecules with a molecular mass of 600 Da or less downhill across a concentration gradient, through the porins β-barrel, and into the periplasm. Another class of energy-independent OM transporters uses low-affinity binding sites that effectively serve to amplify small concentration gradients at the site of the transporter (4).

Natural Conformational Sampling of Human TNFα Visualized by Double Electron-Electron Resonance

Double electron-electron resonance in conjunction with site-directed spin labeling has been used to probe natural conformational sampling of the human tumor necrosis factor α trimer. We suggest a previously unreported, predeoligomerization conformation of the trimer that has been shown to be sampled at low frequency. A model of this trimeric state has been constructed based on crystal structures using the double-electron-electron-resonance distances. The model shows one of the protomers to be rotated and tilted outward at the tip end, leading to a breaking of the trimerous symmetry and distortion at a receptor-binding interface. The new structure offers opportunities to modulate the biological activity of tumor necrosis factor α through stabilization of the distorted trimer with small molecules.

Three‐dimesional structure of GlcNAcα1‐4Gal releasing Endo‐β‐Galactosidase from Clostridium perfringens

Introduction. Clostridium perfringens is a ubiquitous anaerobic bacterium that is commonly found not only in soil but also in the gastrointestinal tract of higher animals. Its pathogenicity has been reviewed extensively. As a major cause of gastrointestinal infections in humans and animals, C. perfringens is suspected to employ hydrolytic enzymes in the degradation of gastrointestinal mucous glycoproteins. A unique endo-galactosidase, Endo-GalGnGa, capable of releasing GlcNAc 1-4Gal from porcine gastric mucin was discovered serendipitously as a contaminant of a commercially available C. perfringens sialidase preparation. Further characterization revealed small but significant sequence similarities with known glycosidases, including several enzymes from Bacillus sp. Endo-GalGnGa has been cloned and overexpressed in Escherichia coli. Single-crystal X-ray diffraction studies of Endo-GalGnGa were undertaken in order to explain the distinctive behavior of these enzymes based on a structural model in atomic detail.