CONFORMATIONAL PLASTICITY OF VARIANTS L-ASPARAGINASE TYPE 2 FROM Escherichia coli

INTRODUCTION: l-asparaginase type 2 (EcA2) from Escherichia coli is known as a tetrameric enzyme which converts L-Asp and L-Gln to L-Asp and L-Glu respectively, and ammonia. EcA2 is used in the treatment of Acute Lymphoblastic Leukemia (ALL), A main EcA2 biopharmaceutical therapeutically known as Elspar® has long been used, with sequence equivalent to the BL21(DE3) E coli strain, and other biosimilar differing in sequence are available in the market. AIMS: To characterize the crystal structure (PX) and the conformational distribution in solution for two EcA2. MATERIAL AND METHODS: The recombinant EcA2 variants from K12 and BL21(DE3) were crystallized in complex with L-Asp, diffracted in the Manaca beamline, and solved by Molecular Replacement. Conformational distribution in solution was investigated by Ion mobility spectrometry – mass spectroscopy (IMS). Normal modes dynamic was evaluated by WEBnm@ v3.0 RESULTS:  The EcA2 from both E coli strains showed similar conformation in crystal phase, with overall RMSD lower than 0.5 Å. Solution measurements by IMS-MS revealed up to three conformers for the monomeric EcA2 at 5 µM, while no other oligomeric form was identified by this method. Normal modes analysis revealed a complex open-closed transition for the two strains, differing between them. CONCLUSION: Despite similar overall crystal structure and conformational distribution in solution, the sequence difference between EcA2 from K12 and BL21(DE3) apparently are sufficient to impose dissimilar conformational transition pathways between major populated states, which requires further investigation concerning therapeutic safety and efficacy.

This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional pra o Desenvolvimento Científico e Tecnológico (CNPq), by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and by CNPEM.