Structural characterization of the tetrameric form of the major cat allergen Fel d 1.

Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin.

DMSO-related effects in protein characterization.

DMSO is the standard solvent for preparing stock solutions of compounds for drug discovery. The assay concentration of DMSO is normally 0.1% to 5% (v/v) or 14 to 715 mM. Thus, DMSO is often one of the principal additives in assay buffers. This standardization of stock solutions does not eliminate possible pitfalls associated with the effects of the DMSO-containing solutions on individual proteins. In this article, the authors want to emphasize the importance of detailed studies of these effects in the early stages of drug discovery. Two protein systems, the extracellular soluble domain of the human growth hormone receptor (hGHbp) and the phosphatase domain of PFKFB1 (BPase), were used for the study on effects of DMSO on protein stability, protein aggregation, and binding of drug compounds. The study revealed significant differences in the proteins' behavior in the presence and absence of low amounts of DMSO. The addition of DMSO resulted in destabilization of the proteins investigated and also changed the apparent binding property of 1 protein. The authors have also shown that low DMSO concentrations influence the ionization process in electrospray ionization mass spectrometry (ESI-MS).

Determination of dissociation constants for protein-ligand complexes by electrospray ionization mass spectrometry.

A fully automated biophysical assay based on electrospray ionization mass spectrometry (ESI-MS) for the determination of the dissociation constants (KD) between soluble proteins and low molecular mass ligands is presented. The method can be applied to systems where the relative MS response of the protein and the protein-ligand complexes do not reflect relative concentrations. Thus, the employed approach enables the use of both electrostatically and nonpolar bound complexes. The dynamic range is wider than for most biological assays, which facilitates the process of establishing a structure-activity relationship. This fully automated ESI-MS assay is now routinely used for ligand screening. The entire procedure is described in detail using hGHbp, a 25-kDa extracellular soluble domain of the human growth hormone receptor, as a model protein.

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.

The development of a continuous isothermal titration calorimetric method for equilibrium studies.

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12-20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60-180 min to generate 20-30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba(2+) to 18-crown-6 and the binding of cytidine 2'-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.

Folding into a beta-hairpin can prevent amyloid fibril formation.

The tetrapeptide KFFE is one of the shortest amyloid fibril-forming peptides described. Herein, we have investigated how the structural environment of this motif affects polymerization. Using a turn motif (YNGK) or a less rigid sequence (AAAK) to fuse two KFFE tetrapeptides, we show by several biophysical methods that the amyloidogenic properties are strongly dependent on the structural environment. The dodecapeptide KFFEAAAKKFFE forms abundant thick fibril bundles. Freshly dissolved KFFEAAAKKFFE is monomeric and shows mainly disordered secondary structure, as evidenced by circular dichroism, NMR spectroscopy, hydrogen/deuterium exchange measurements, and molecular modeling studies. In sharp contrast, the dodecapeptide KFFEYNGKKFFE does not form fibrils but folds into a stable beta-hairpin. This structure can oligomerize into a stable 12-mer and multiples thereof, as shown by size exclusion chromatography, sedimentation analysis, and electrospray mass spectrometry. These data indicate that the structural context in which a potential fibril forming sequence is present can prevent fibril formation by favoring self-limiting oligomerization over polymerization.

Mechanism of action of pyridazine analogues on protein tyrosine phosphatase 1B (PTP1B).

The inhibitory effect on PTP1B caused by the addition of pyridazine analogues has been investigated. Biophysical techniques, that is, mass spectrometry (MS), nuclear magnetic resonance (NMR), and isothermal titration calorimetry (ITC) were used for the characterization. Pyridazine analogues cause catalytic oxidation of the reducing agent, generating hydrogen peroxide that oxidizes the active site cysteine on the enzyme, leading to enzyme inactivation. Two additional compound classes show the same effect. We found one common structural feature in these molecules that allows the reaction with triplet molecular oxygen to be less endothermic. A proposed mechanism for the catalytic redox cycle is described.

Crystal structure of the heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation.

The nuclear receptor heterodimers of liver X receptor (LXR) and retinoid X receptor (RXR) are key transcriptional regulators of genes involved in lipid homeostasis and inflammation. We report the crystal structure of the ligand-binding domains (LBDs) of LXRalpha and RXRbeta complexed to the synthetic LXR agonist T-0901317 and the RXR agonist methoprene acid (Protein Data Base entry 1UHL). Both LBDs are in agonist conformation with GRIP-1 peptides bound at the coactivator binding sites. T-0901317 occupies the center of the LXR ligand-binding pocket and its hydroxyl head group interacts with H421 and W443, residues identified by mutational analysis as critical for ligand-induced transcriptional activation by T-0901317 and various endogenous oxysterols. The topography of the pocket suggests a common anchoring of these oxysterols via their 22-, 24- or 27-hydroxyl group to H421 and W443. Polyunsaturated fatty acids act as LXR antagonists and an E267A mutation was found to enhance their transcriptional inhibition. The present structure provides a powerful tool for the design of novel modulators that can be used to characterize further the physiological functions of the LXR-RXR heterodimer.

Activation of erythropoietin receptor through a novel extracellular binding site.

Activation of erythropoietin (EPO) receptor (EPOR) by a small peptide (ERP) was reported previously. ERP binds to a different receptor site than EPO, and binding of ERP does not change the dissociation constant and maximal binding for EPO binding to the receptor. The extracellular binding site for ERP is now characterized. The site is located in the membrane proximal, extracellular part of the receptor. ERP binds to a region on the EPOR that contains the same sequence as ERP. It is speculated that ERP binds to its identical sequence on EPOR, as ERP self-interacts. ERP is specific for EPOR and associates noncovalently with EPOR in a ratio 1:1. Peptide binding to the receptor results in receptor-mediated cellular proliferation, intracellular signaling, and erythroid colony-forming unit formation in bone marrow cells. The activity is comparable to that of EPO. Recognition of such receptor sites represents a new and important concept in receptor function.