Geoffrey A. Mueller

A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.

The structure of the dust mite allergen Der p 7 reveals similarities to innate immune proteins.

BACKGROUND Sensitization to house dust mite allergens is strongly correlated with asthma. Der p 7 elicits strong IgE antibody and T-cell responses in patients with mite allergy. However, the structure and biological function of this important allergen are unknown. Allergen function might contribute to allergenicity, as shown for the protease activity of group 1 mite allergens and the interaction with the innate immune system by group 2 mite allergens. OBJECTIVE We sought to determine the crystal structure of Der p 7 and to investigate its biological function. METHODS X-ray crystallography was used to determine the Der p 7 structure. Nuclear magnetic resonance analysis and biochemical assays were used to examine the binding of Der p 7 to predicted ligands. RESULTS Der p 7 has an elongated structure, with two 4-stranded antiparallel beta-sheets that wrap around a long C-terminal helix. The fold of Der p 7 is similar to that of LPS-binding protein (LBP), which interacts with Toll-like receptors after binding LPS and other bacterially derived lipid ligands. Nuclear magnetic resonance and biochemical assays indicate that Der p 7 does not bind LPS but binds with weak affinity to the bacterial lipopeptide polymyxin B in the predicted binding site of Der p 7. CONCLUSIONS Der p 7 binds a bacterially derived lipid product, a common feature of some allergens. The finding that the group 7, as well as the group 2, mite allergens are structurally similar to different proteins in the Toll-like receptor pathway further strengthens the connections between dust mites, innate immunity, and allergy.

Dependence of Amino Acid Side Chain 13C Shifts on Dihedral Angle: Application to Conformational Analysis

Chemical shift data from the BiomagResDataBank and conformational data derived from the protein data bank have been correlated in order to explore the conformational dependence of side chain (13)C resonance shifts. Consistent with predictions based on steric compression, upfield shifts for Cgamma resonances of Thr, Val, Ile, Leu, Met, Arg, Lys, Glu, and Gln residues correlate with both the number of heavy atom (nonproton) gamma-substituents and with gauche conformational orientations of gamma-substituents. The (13)C shift/conformation correlations are most apparent for Cgamma carbons but also can be observed at positions further from the backbone. Intraresidue steric conflict leads to a correlation between upfield-shifted side chain (13)C resonances and statistically lower probabilities in surveys of protein side chain conformation. Illustrative applications to the DNA pol lambda lyase domain and to dihydrofolate reductase are discussed. In the latter case, (13)C shift analysis indicates that the conformation of the remote residue V119 on the betaF-betaG loop is correlated with the redox state of the bound pyridine nucleotide cofactor, providing one basis for discrimination between substrate and product. It is anticipated that (13)C shift data for protein sidechains can provide a useful basis for the analysis of conformational changes even in large, deuterated proteins. Additionally, the large dependence of the leucine methyl shift difference, deltaCdelta1-deltaCdelta2, on both chi1 and chi2 is sufficient to allow this parameter to be used as a restraint in structure calculations if stereospecific assignment data are available.

Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity

To cite this article: Mueller GA, Gosavi RA, Pomés A, Wünschmann S, Moon AF, London RE, Pedersen LC. Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity. Allergy 2011; 66: 878–885.

Crystal Structure of Calmodulin Binding Domain of Orai1 in Complex with Ca2+/Calmodulin Displays a Unique Binding Mode

Background: Calmodulin (CaM) binds the calmodulin binding domain (CMBD) of Orai1 causing Ca2+·CaM-dependent inhibition (CDI). Results: Orai1-CMBD binds the C-lobe of CaM in crystal, whereas in solution Orai1-CMBD interacts with both N- and C-lobes of CaM. Conclusion: One CaM, in an extended conformation, binds to two Orai1-CMBDs. Significance: This expands our knowledge of the interaction of CaM with its targets, and provides important information for understanding CDI. Orai1 is a plasma membrane protein that in its tetrameric form is responsible for calcium influx from the extracellular environment into the cytosol in response to interaction with the Ca2+-depletion sensor STIM1. This is followed by a fast Ca2+·calmodulin (CaM)-dependent inhibition, resulting from CaM binding to an Orai1 region called the calmodulin binding domain (CMBD). The interaction between Orai1 and CaM at the atomic level remains unknown. Here, we report the crystal structure of a CaM·Orai1-CMBD complex showing one CMBD bound to the C-terminal lobe of CaM, differing from other CaM-target protein complexes, in which both N- and C-terminal lobes of CaM (CaM-N and CaM-C) are involved in target binding. Orai1-CMBD binds CaM-C mainly through hydrophobic interactions, primarily involving residue Trp76 of Orai1-CMBD, which interacts with the hydrophobic pocket of CaM-C. However, NMR data, isothermal titration calorimetry data, and pulldown assays indicated that CaM-N and CaM-C both can bind Orai1-CMBD, with CaM-N having ∼4 times weaker affinity than CaM-C. Pulldown assays of a Orai1-CMBD(W76E) mutant, gel filtration chromatography data, and NOE signals indicated that CaM-N and CaM-C can each bind one Orai1-CMBD. Thus our studies support an unusual, extended 1:2 binding mode of CaM to Orai1-CMBDs, and quantify the affinity of Orai1 for CaM. We propose a two-step mechanism for CaM-dependent Orai1 inactivation initiated by binding of the C-lobe of CaM to the CMBD of one Orai1 followed by the binding of the N-lobe of CaM to the CMBD of a neighboring Orai1.

Revised structure of the AbrB N-terminal domain unifies a diverse superfamily of putative DNA-binding proteins

New relationships found in the process of updating the structural classification of proteins (SCOP) database resulted in the revision of the structure of the N‐terminal, DNA‐binding domain of the transition state regulator AbrB. The dimeric AbrB domain shares a common fold with the addiction antidote MazE and the subunit of uncharacterized protein MraZ implicated in cell division and cell envelope formation. It has a detectable sequence similarity to both MazE and MraZ thus providing an evolutionary link between the two proteins. The putative DNA‐binding site of AbrB is found on the same face as the DNA‐binding site of MazE and appears similar, both in structure and sequence, to the exposed conserved region of MraZ. This strongly suggests that MraZ also binds DNA and allows for a consensus model of DNA recognition by the members of this novel protein superfamily.

Future directions for allergen immunotherapy

Over the last 30 years several approaches to modify immunotherapy have been tested, including allergoids, alum precipitation, and most recently peptides. However, none of these have replaced the traditional regimens. Over the same period our scientific understanding of allergic disease has been transformed. Today it is possible to identify and monitor changes occurring during treatment and to target many different aspects of the immune system. Recombinant technology provides a powerful technique both for sequencing proteins and producing allergens in commercial quantities. The recombinant proteins can be modified by site-directed mutagenesis so as to decrease their reactivity with IgE antibodies while maintaining reactivity with T cells. Knowledge of the tertiary structure of allergens will make it simpler to identify and change surface epitopes. A completely different approach is to use plasmids to introduce the genes for an allergen. The strength of this technique is that the plasmid can be designed to control expression and also to influence the cytokine profile of the response or the isotype of antibodies produced. Finally, different adjuvants can be used with proteins to alter the response. These include IL-12, immunostimulatory sequences of DNA, and bacterial proteins such as those used in HibVax. It is now possible to identify the cells that control the immune response to allergens and to design treatments that will either downregulate or change the response of T cells. The challenge is to transform this information into an effective treatment for allergic disease.

The novel structure of the cockroach allergen Bla g 1 has implications for allergenicity and exposure assessment.

BACKGROUND Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of approximately 100 amino acids, but the fold of the protein and its biological function are unknown. OBJECTIVE We sought to determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays. METHODS nBla g 1 and recombinant constructs were compared by using ELISA with specific murine IgG and human IgE. The structure of Bla g 1 was determined by x-ray crystallography. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to examine the ligand-binding properties of the allergen. RESULTS The structure of an rBla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by x-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic, and stearic acids were associated with nBla g 1 from cockroach frass. One unit of Bla g 1 was equivalent to 104 ng of allergen. CONCLUSIONS Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with nonspecific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure.

Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.

Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)‐modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy.

Der p 5 crystal structure provides insight into the group 5 dust mite allergens.

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of ∼3000 Å3 that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.