Structural biology of integral membrane proteins - From methods to molecular mechanisms

Membrane proteins are vital components in the cell and crucial for the proliferation of all living organisms. Unfortunately our collective knowledge of structures of membrane proteins is very limited, as compared to the information available on soluble proteins. This is to a large extent due to the outstanding challenge of working with membrane proteins and the relatively high cost associated with determining a membrane protein structure.  Therefore, the establishment of efficient methods and means for the production and crystallization of membrane proteins is urgently needed. The two methods explored in this thesis  are aimed to achieve rapid optimization of expression and purification conditions of membrane proteins, thereby allowing for the rapid production of more suitable samples for crystallization trials. Despite the challenges in membrane protein structure determination two structures are presented in the thesis: The first structure determined is of the CorA magnesium transporter from Thermotoga maritima will be the focus of this thesis. The CorA revealed a pentameric protein in a closed state. The presence of two regulatory metal binding sites is suggested, as well as a putative magnesium ion bound in the ion conductive pathway. The second structure is of the human enzyme LTC4-synthase, which catalyzes the pivotal step in eicosanoid synthesis by the conjugation of glutathione to LTA4, a reactive epoxide-containing derivative from arachidonic acid. The products of this step, the so-called cysteinyl leukotrienes are potent inflammatory mediators making this enzyme a potential drug target. The structure reveals a charged binding pocket for a horseshoe-shaped glutathione, and a hydrophobic binding pocket for a lipophilic LTA4 molecule. Based on the structure a key residue for catalysis has been identified, Arg 104, which is proposed to play a critical role in activating the thiol group of glutathione for the nucleophilic attack on LTA4

Structure and inhibition of mouse leukotriene C4 synthase.

Leukotriene (LT) C4 synthase (LTC4S) is an integral membrane protein that catalyzes the conjugation reaction between the fatty acid LTA4 and GSH to form the pro-inflammatory LTC4, an important mediator of asthma. Mouse models of inflammatory disorders such as asthma are key to improve our understanding of pathogenesis and potential therapeutic targets. Here, we solved the crystal structure of mouse LTC4S in complex with GSH and a product analog, S-hexyl-GSH. Furthermore, we synthesized a nM inhibitor and compared its efficiency and binding mode against the purified mouse and human isoenzymes, along with the enzymes' steady-state kinetics. Although structural differences near the active site and along the C-terminal α-helix V suggest that the mouse and human LTC4S may function differently in vivo, our data indicate that mouse LTC4S will be a useful tool in future pharmacological research and drug development

Product formation controlled by substrate dynamics in leukotriene A4 hydrolase.

Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers

Steady state kinetic parameters of mLTC4S and hLTC4S against GSH and LTA₄.

<p>The enzyme activity was measured in 25mM Tris (pH 7.8), 0.1M NaCl, 0.05% DDM in the presence of either 30 µM LTA₄ and/or 5 mM GSH with 0.1 µg of enzyme.</p><p>**<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096763#pone.0096763-RinaldoMatthis2" target="_blank">[29]</a>.</p

Comparison of human and mouse LTC4S enzymes.

<p>A. Amino acid sequence alignment of human and mouse LTC4S generated with the program ClustalW. Species differences are highlighted in white. B. Mapping the amino acid differences (in red) between mouse and human trimeric LTC4S structures. The active site in one monomer is depicted with a bound GSH (green). In blue is the Phe50Tyr exchange positioned close to the active site.</p

Positional shift of Arg51 and loss of salt bridge at the active site of mLTC4S.

<p>A. Close up of the mLTC4S complex with SO₄²⁻, showing a shift in the position of Arg51 due to Phe50Tyr exchange. Human LTC4S is colored in green and mLTC4S is colored in gray. GSH is shown as green “lines”. *indicates that it is positioned on the neighboring subunit. B. Trimer of mLTC4S showing the amino acid exchange at position 50 where Phe in mLTC4S fails to make a salt bridge with Arg51. In hLTC4S, the Tyr50-Arg51 couple will likely contribute to trimer stability.</p

Binding of S-hexyl GSH to the active site of mLTC4S.

<p>A. The trimeric form of mLTC4S with three bound S-hexyl GSH. B. Electron density 2fo-fc map, contoured at 1.0 σ around S-hexyl GSH. C. The hydrophobic cavity with S-hexyl GSH bound (yellow stick carbons) in the hydrophobic cleft. Amino acids facing the cavity are from monomers A (yellow) and B (green).</p

Binding of GSH at the active site of mLTC4S.

<p>A. Electron density 2fo-fc map contoured at 1.0 σ around GSH with Arg104 coordinating the sulfur in GSH. B. GSH bound at the active site, coordinated by several amino acids where the Arg51 - Tyr50 (indicated with a line) interaction in the human enzyme, is lost in the mLTC4S, which has a Phe in position 50.</p

The LTC₄ synthase reaction.

<p>A. Schematic drawing of the catalytic reaction of LTC4S where the allylic epoxide LTA₄ is conjugated with GSH at C6, to form LTC₄. B. Structure of the product analog S-hexyl GSH.</p

TK04 is a nanomolar competitive inhibitor of LTC4S.

<p>A. Chemical structure of TK04, the inhibitor used in this study. Dose-response curves for inhibition of mouse and human LTC4S by TK04. 100% activity corresponds to the enzyme activity without inhibitor, which was 44.0 µmol min⁻¹ mg⁻¹ for the mouse enzyme (red line) and 69.7 µmol min⁻¹ mg⁻¹ for the human enzyme (black line). The concentrations of substrates GSH and LTA₄ used in the assay were 5 mM and 20 µM, respectively. The IC₅₀ for mLTC4S was 135±30 nM and for the hLTC4S it was 134±16 nM.</p