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Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.

ABSTRACT: Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

SUBMITTER: Filippova EV

PROVIDER: S-EPMC3624434 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.

Filippova Ekaterina V EV Weston Leigh A LA Kuhn Misty L ML Geissler Brett B Gehring Alexandra M AM Armoush Nicola N Adkins Chinessa T CT Minasov George G Dubrovska Ievgeniia I Shuvalova Ludmilla L Winsor James R JR Lavis Luke D LD Satchell Karla J F KJ Becker Daniel P DP Anderson Wayne F WF Johnson R Jeremy RJ

The Journal of biological chemistry 20130219 15

Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large c ...[more]

PMID: 23430251

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Project description:UNLABELLED:The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ?kdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ?kdhAB mutant strain had a 50% lethal dose (LD(50)) 2 log(10) units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-?) and interleukin-1? (IL-1?) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ?kdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ?kdhAB mutant induced higher levels of TNF-? and IL-1? in a TLR2-dependent manner. In addition, TLR2(-/-) mice were more susceptible than wild-type mice to ?kdhAB bacterial infection. Finally, immunization of mice with ?kdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine. IMPORTANCE:The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification-Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal--has recently been reported in F. tularensis. We found that the kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.

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Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.

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Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.

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