Abstract
Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3',5'-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3',5'-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence.
Highlights
Experimental ProceduresProtein Expression, and Purification—The gene lpg1496 from L. pneumophila strain Philadelphia was cloned into pLR652 as a N-terminal GST-tagged fusion protein and expressed in a BL21 Star Escherichia coli strain
Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease
Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and hijack eukaryotic biochemical processes for the benefit of the pathogen
Summary
Protein Expression, and Purification—The gene lpg1496 from L. pneumophila strain Philadelphia was cloned into pLR652 as a N-terminal GST-tagged fusion protein and expressed in a BL21 Star Escherichia coli strain. The expression and purification conditions were the same as for the N-terminal domain, except size-exclusion chromatography was performed using buffer C (10 mM HEPES, pH 7.5, 100 mM NaCl). The His tag in the pET15b construct was cleaved with thrombin before injecting the protein into a sizeexclusion column. Crystals of the middle domain of lpg1496 were obtained in approximately 1 week from a 1:1 mixture of the protein solution (16.6 mg/ml) and the reservoir solution (0.1 M HEPES, pH 7.5, 25% (w/v) PEG 3350). Free phosphate was determined by measuring absorbance at 610 nm
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