Said Eshaghi

Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase.

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 Å resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane–enzyme interface. In addition, the structure provides a model for how the ω-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular ‘ruler’ to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins.

The recombinant expression of integral membrane proteins is considered a major challenge, and together with the crystallization step, the major hurdle toward routine structure determination of membrane proteins. Basic methodologies for high‐throughput (HTP) expression optimization of soluble proteins have recently emerged, providing statistically significant success rates for producing such proteins. Experimental procedures for handling integral membrane proteins are generally more challenging, and there have been no previous comprehensive reports of HTP technology for membrane protein production.

Increased Hydrophobicity at the N Terminus/Membrane Interface Impairs Gating of the Severe Combined Immunodeficiency-related ORAI1 Mutant

Patients with severe combined immune deficiency (SCID) suffer from defective T-cell Ca2+ signaling. A loss of Ca2+ entry has been linked at the molecular level to single missense mutation R91W in the store-operated Ca2+ channel ORAI1. However, the mechanistic impact of this mutation on ORAI1 function remains unclear. Confocal Förster resonance energy transfer microscopy revealed that dynamic store-operated coupling of STIM1 to ORAI1 R91W was largely sustained similar to wild-type ORAI1. Characterization of various point mutants at position 91 by whole cell patch clamp recordings displayed that neutral or even negatively charged amino acids did not abolish ORAI1 function. However, substitution by hydrophobic leucine, valine, or phenylalanine resulted in non-functional ORAI1 channels, despite preserved STIM1 coupling. Besides conformational constraints at the N terminus/membrane interface predicted for the hydrophobic mutants, additional key factor(s) were suggested to determine ORAI1 functionality. Calculation of the probability for the 1st transmembrane domain and its hydrophobicity revealed a substantial increase for all hydrophobic substitutions that lead to non-functional ORAI1 R91X mutants in contrast to those with hydrophilic residues. Hence, increased hydrophobicity might lead to disrupted permeation/gating, as an ORAI1 channel with increased pore size and R91W mutation failed to recover activity. In conclusion, the increase in hydrophobicity at the N terminus/membrane interface represents the major cause for yielding non-functional ORAI1 channels.

The CorA family: Structure and function revisited

Abstract.The CorA family is a group of ion transporters that mediate transport of divalent metal ions across biological membranes. Metal ions are essential elements in most cellular processes and hence the concentrations of ions in cells and organelles must be kept at appropriate levels. Impairment of these systems is implied in a number of pathological conditions. CorA proteins are abundant among the prokaryotic organisms but homologues are present in both human and yeast. The activity of CorA proteins has generally been associated with the transport of magnesium ions but the members of the CorA family can also transport other ions such as cobalt and nickel. The structure of the CorA from Thermotoga maritima, which also was the first structure of a divalent cation transporter determined, has opened the possibilities for understanding the mechanisms behind the ion transport and also corrected a number of assumptions that have been made in the past.

Catalysis within the lipid bilayer-structure and mechanism of the MAPEG family of integral membrane proteins.

Integral membrane enzymes of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) family catalyze glutathione-dependent transformations of lipophilic substrates harvested from the lipid bilayer. Recent studies of members of this family have yielded extensive insights into the structural basis for their substrate binding and catalytic activity. Most informative are the structural studies of leukotriene C4 synthase, revealing a narrow hydrophobic substrate binding pocket allowing extensive recognition of the aliphatic chain of the LTA(4) substrate. A key feature of the pocket is a tryptophan residue that pins down the omega-end of the aliphatic chain into the active site. Since MAPEG members cannot utilize a hydrophobic effect for substrate binding, this novel mode of substrate recognition appears well suited for harvesting lipophilic substrates from the membrane. The binding mode also allows for the specific alignment of the substrate in the active site, positioning the C6 of the substrate for conjugation with glutathione. The glutathione is in turn bound in a polar pocket submerged into the protein core. Structure-based sequence alignments of human MAPEG members support the notion that the glutathione binding site is highly conserved among MAPEG enzymes and that they use a similar mechanism for glutathione activation.

The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20

Fractionation of proteins from the thylakoid lumen of spinach chloroplasts combined with peptidyl‐prolyl cis/trans isomerase (PPIase) measurements revealed a major isomerase activity that was ascribed to a novel enzyme TLP20 ( hylakoid umen PIase of kDa). TLP20 was inhibited by cyclosporin A and mass spectrometric sequencing of tryptic peptides confirmed its classification as a cyclophilin. Genes encoding similar putative thylakoid cyclophilins with a unique insert of three amino acids NPV in their N‐termini were found in chromosome 5 of both Arabidopsis and rice. TLP20 is suggested to be the major PPIase and protein folding catalyst in the thylakoid lumen of plant chloroplasts.

Engineering membrane protein overproduction in Escherichia coli

Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent‐solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent‐solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent‐solubilized membrane protein was increased 40‐fold.

Exploring the activity of tobacco etch virus protease in detergent solutions

Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.

Co2+ Selectivity of Thermotoga maritima CorA and Its Inability to Regulate Mg2+ Homeostasis Present a New Class of CorA Proteins

CorA is a family of divalent cation transporters ubiquitously present in bacteria and archaea. Although CorA can transport both Mg2+ and Co2+ almost equally well, its main role has been suggested to be that of primary Mg2+ transporter of prokaryotes and hence the regulator of Mg2+ homeostasis. The reason is that the affinity of CorA for Co2+ is relatively low and thus considered non-physiological. Here, we show that Thermotoga maritima CorA (TmCorA) is incapable of regulating the Mg2+ homeostasis and therefore cannot be the primary Mg2+ transporter of T. maritima. Further, our in vivo experiments confirm that TmCorA is a highly selective Co2+ transporter, as it selects Co2+ over Mg2+ at >100 times lower concentrations. In addition, we present data that show TmCorA to be extremely thermostable in the presence of Co2+. Mg2+ could not stabilize the protein to the same extent, even at high concentrations. We also show that addition of Co2+, but not Mg2+, specifically induces structural changes to the protein. Altogether, these data show that TmCorA has the role of being the transporter of Co2+ but not Mg2+. The physiological relevance and requirements of Co2+ in T. maritima is discussed and highlighted. We suggest that CorA may have different roles in different organisms. Such functional diversity is presumably a reflection of minor, but important structural differences within the CorA family that regulate the gating, substrate selection, and transport.

High-Throughput Expression and Detergent Screening of Integral Membrane Proteins

Integral membrane proteins are a major challenge within structural genomics. These proteins are not only difficult to produce in quantities sufficient for analysis by X-ray diffraction or NMR, but also require extraction from their lipid environment, which leads to a new dimension of difficulties in purification and subsequent structural analysis. To overcome these problems requires new strategies enabling screening larger number of parameters dealing with expression and purification. For this reason, we have developed high-throughput methods for screening extracting and purifying detergents as well as other purification parameters, e.g. salt and pH. The method requires standard laboratory equipments, but can also be automated.