Crystal Structure of Cockroach Allergen Bla g 2, an Unusual Zinc Binding Aspartic Protease with a Novel Mode of Self-inhibition

This paper is dedicated to the memory of Dr Venugopal Dhanaraj, who modeled the structure of Bla g 2
https://doi.org/10.1016/j.jmb.2005.02.062Get rights and content

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity of the catalytic aspartate residues, increasing the distance between them to ∼4 Å and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.

Introduction

Cockroaches excrete a potent allergen, Bla g 2, that is associated with IgE production and asthma.1 Sensitization and exposure to cockroach allergens is a major risk factor for asthma mortality and morbidity among children living in inner-city areas of the United States, and is strongly associated with asthma in other parts of the world.2, 3, 4, 5, 6, 7, 8 Bla g 2 appears to be an especially potent allergen, which elicits IgE responses at exposure levels that are 10–100-fold lower than other common indoor allergens, such as dust mite and cat.9 Several mite allergens function as proteolytic enzymes, including Der p 1 (cysteine protease), and Der p 3, Der p 6 and Der p 9 (serine proteases).10, 11 A body of evidence suggests that these enzyme allergens play an important role in asthma by potentiating IgE responses, inducing pro-inflammatory cytokine release, and causing direct damage to airway epithelium.12, 13, 14, 15 However, unlike dust mite, none of the cockroach allergens that have been cloned, except Bla g 2, shows homology to proteolytic enzymes.

Bla g 2 is an important allergen that causes IgE responses in ∼60% of cockroach allergic patients and has unusual structural features. Bla g 2 is a 36 kDa glycoprotein that shares homology with the family of aspartic proteases.1 Aspartic proteases catalyze the hydrolysis of a peptide bond utilizing an acid–base mechanism via nucleophilic attack by the water molecule which is polarized by two catalytic aspartate residues. They share similar overall fold of a bilobal molecule with a well-defined substrate-binding cleft. Each domain contains a conserved DTG triad with a catalytic aspartate. However, molecular modeling and functional studies using standard enzyme assays showed that Bla g 2 was an inactive aspartic protease, with critical amino acid substitutions around the catalytic residues.16, 17 In Bla g 2, the two glycine residues in DTG triads are substituted by threonine and serine, and the conserved tyrosine 75 in the “flap” region of the enzyme is substituted by phenylalanine. Surprisingly, Bla g 2 is related in its primary structure to a group of inactive mammalian aspartic proteases, known as pregnancy-associated glycoproteins (PAG), which are expressed in the chorion of pregnant females from ungulates, including pig, horse, cow, and sheep.18, 19 A common feature of Bla g 2 and most PAGs is the lack of enzymatic activity in standard aspartic protease assays, for which neither the structural basis nor a molecular mechanism have been established.

Here, we report a high-resolution crystal structure of partially deglycosylated, recombinant Bla g 2 that was expressed in the yeast Pichia pastoris. The structure reveals a novel mode of self-inhibition that explains why the catalytic activity of this protein is impaired. Unlike any aspartic protease of known structure, Bla g 2 is a zinc-binding protein.

Section snippets

Crystal structure of Bla g 2

Native Bla g 2 is a glycoprotein that contains three potential N-glycosylation sites involving residues Asn93, Asn268, and Asn317. Since the attempts to crystallize the recombinant native protein were not successful, we tried to reduce the glycosylation state of the molecule by mutating these asparagine residues to glutamine. Several mutants were made, and one of them, the N93Q mutant, was expressed reasonably well and produced the crystals used in this study.

Initial attempts to solve the

Discussion

The structure of Bla g 2 that is reported here was obtained using partially deglycosylated, recombinant enzyme expressed in yeast Pichia pastoris and was solved at near-atomic resolution of 1.3 Å, assuring high quality of the resulting model. As expected, the overall structure of Bla g 2 has the bilobal fold of a non-viral aspartic protease with a large cleft located between two structurally homologous domains. However, important interactions in the active site area are significantly modified

Expression and purification of a recombinant Bla g 2 mutant

A partially deglycosylated recombinant Bla g 2 mutant (rBla g 2 N93Q) was expressed in P. pastoris after substitution of an asparagine residue by glutamine in the putative N-glycosylation motif NLT at position 93. The template used was Bla g 2 cDNA without the signal peptide, inserted into the P. pastoris expression vector pGAPZαC (Invitrogen) for constitutive expression of the allergen. Site-directed mutagenesis was performed using a QuikChange™ Kit (Stratagene) and custom-designed primers.

Acknowledgements

We thank Dr Zbyszek Dauter for the measurements of fluorescence data from a crystal of Bla g 2 and Jerry Alexandratos for assistance in preparation of the Figures. This work was supported in part by Philip Morris USA Inc, and in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-24000. Diffraction data used in the final refinement were collected at the Southeast Regional Collaborative Access Team (SER-CAT) beamline 22-ID, located

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