Irena Ekiel

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Structure and function of the C-terminal PABC domain of human poly(A)-binding protein

We have determined the solution structure of the C-terminal quarter of human poly(A)-binding protein (hPABP). The protein fragment contains a protein domain, PABC [for poly(A)-binding protein C-terminal domain], which is also found associated with the HECT family of ubiquitin ligases. By using peptides derived from PABP interacting protein (Paip) 1, Paip2, and eRF3, we show that PABC functions as a peptide binding domain. We use chemical shift perturbation analysis to identify the peptide binding site in PABC and the major elements involved in peptide recognition. From comparative sequence analysis of PABC-binding peptides, we formulate a preliminary PABC consensus sequence and identify human ataxin-2, the protein responsible for type 2 spinocerebellar ataxia (SCA2), as a potential PABC ligand.

Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two‐hybrid system

Calnexin and ERp57 act cooperatively to ensure a proper folding of proteins in the endoplasmic reticulum (ER). Calnexin contains two domains: a lectin domain and an extended arm termed the P‐domain. ERp57 is a protein disulfide isomerase composed of four thioredoxin‐like repeats and a short basic C‐terminal tail. Here we show direct interactions between the tip of the calnexin P‐domain and the ERp57 basic C‐terminus by using NMR and a novel membrane yeast two‐hybrid system (MYTHS) for mapping protein interactions of ER proteins. Our results prove that a small peptide derived from the P‐domain is active in binding ERp57, and we determine the structure of the bound conformation of the P‐domain peptide. The experimental strategy of using the MYTHS two‐hybrid system to map interaction sites between ER proteins, together with NMR, provides a powerful new strategy for establishing the function of ER complexes.

Folding-related Dimerization of Human Cystatin C

With the aim to improve our understanding of the structural basis for protein self-association and aggregation, in particular in relationship to protein refolding and amyloid formation, folding-related processes for human cystatin C have been studied. Using NMR spectroscopy together with chromatographic and electrophoretic methods, a self-association process resulting in dimer formation for protein samples treated with denaturing agents as well as for samples subjected to low pH or high temperature conditions could be studied with amino acid resolution. In all three cases, the dimerization involves properly folded molecules and proceeds via the reactive site of the inhibitor, which leads to complete loss of its biological activity. This dimerization process has potential relevance for amyloid formation by the brain hemorrhage-causing Leu-Gln variant of cystatin C. The results also indicate that cystatin C dimerization and inactivation may occur in acidified compartments in vivo, which could be relevant for the physiological regulation of cysteine proteinase activity.

Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C‐terminal domain of poly(A)‐binding protein (PABC) is a peptide‐binding domain found in poly(A)‐binding proteins (PABPs) and a HECT (homologous to E6‐AP C‐terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP‐interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP‐interacting protein‐1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of β‐turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein–peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull‐down assays. The results provide insight into the specificity of PABC in mediating PABP–protein interactions.

Solution Structure of the PDZ2 Domain from Cytosolic Human Phosphatase hPTP1E Complexed with a Peptide Reveals Contribution of the β2–β3 Loop to PDZ Domain–Ligand Interactions

The solution structure of the second PDZ domain from human phosphatase hPTP1E in complex with a C-terminal peptide from the guanine nucleotide exchange factor RA-GEF-2 has been determined using 2D and 3D heteronuclear NMR experiments. Compared to previously solved structures, the hPTP1E complex shows an enlarged interaction surface with the C terminus of the bound peptide. Novel contacts were found between the long structured beta2/beta3 loop of the PDZ domain and the sixth amino acid residue from the C terminus of the peptide. This work underlines the importance of the beta2/beta3 loop for ligand selection by PDZ domains.

A novel core lipid isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Abstract The membrane lipids of Methanothrix concilii are based on two types of core lipid structures, namely, the ubiquitous 2,3-di- O -phytanyl- sn -glycerol and a second previously unidentified diether core lipid, each accounting for 70 and 30% (wt. %), respectively, of the total polar lipids. Preparation of the novel diether core lipid from either purified glycolipid-1 (GL-1) or total polar lipid in 2.5% methanolic HCl resulted in the formation of several lipid artifacts, thereby necessitating the utilization of new hydrolytic conditions. We found that mild acid hydrolysis in 0.18% methanolic HCl produced only one core lipid product from GL-1 or two core lipids from the total polar lipid fraction. This new core lipid material differed from the authentic C 20,20 diether in that it contained a hydroxyl group on C-3 of the phytanyl chain at the sn -3 position of glycerol. The full chemical designation for this novel diether is: 2- O -[3,7,11,15-tetramethyl]hexadecy l -3- O -[3′ -hydroxy-3′,7′,11′,15′-tetramethyl]hexadecyl- sn -glycerol. 1 H-NMR of the total polar lipid extract could detect the presence of this new core lipid by exploiting the appearance of the methyl (C-17′) singlet at 1.24 ppm. The presence of this methyl singlet in the 1 H-NMR spectrum would subsequently determine whether a strong or a mild acid hydrolysis should be used for the preparation of core lipids in methanogens.

Synthesis, Conformation and Hydrolytic Stability of p1,p3−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate Analogs

P1,P3−Dinucleoside triphosphates, N(5′)G(5′)G, have been prepared in which N is 7-Me-, 7-Et-, 7-Bn, N2, 7-diMe- or N2,N2, 7-triMe-guanosine. Conformations of the nucleoside moieties have been deter...

The state of the lipids in the purple membrane of Halobacterium cutirubrum as seen by 31P NMR.

Summary The purple membrane from Halobacterium cutirubrum and aqueous dispersions of its principal phospholipid, phosphatidyl-glycerophosphate, were studied by 31P NMR at 121.5 MHz. The spectra manifest separate powder patterns for the mono- and diesterified phosphate groups, the latter having the larger chemical shift anisotropy. Spectral simulation was used to derive accurate values of the chemical shift anisotropy. Contrary to some earlier reports, there is no evidence for a lipid phase transition in the membrane or the isolated lipids over the range 5 to 60°C; at all temperatures the lipid is in a bilayer arrangement. Combination with protein and neutral lipids in the purple membrane leads to reduced amplitude and rate of motion of the phosphate moieties of the major phospholipid.

Structure of the major polar lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Abstract About 10% of the cell dry weight of the aceticlastic methanogen, Methanothrix concilii , was found to be lipid consisting of 93% (wt.%) polar and 7% (wt.%) neutral lipids, respectively. Several minor phospholipids and glycolipids were detected; however, the major lipid components, a phospholipid and two glycolipids, accounted for approx. 84% (wt.%) of the total polar fraction. The three major polar lipids were identified as: (1) phospholipid; 2, 3- di -O- phytanyl -sn- glycero -1- phosphoryl -1′-myo- l - inositol ; (2) glycolipid-1; 2- O - phytanyl-3- O -[3′-hydroxy-3′, 7′, 11′, 15′- tetramethyl]hexadecyl -1-O-[β- d - galactopyranosyl -(1→ 6) -β- d - galactopyranosyl ]-sn- glycerol and (3) glycolipid-2; 2, 3- di -O- phytanyl -1-O-[α- d - mannopyranosyl - (1→ 3)-β- d - galactopyranosyl ]-sn- glycerol .