Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis

Filippova, E.V.; Weston, Leigh A.; Kuhn, Misty L.; Geissler, Brett; Gehring, Alexandra M.; Armoush, Nicola; Adkins, Chinessa T.; Minasov, G.; Dubrovska, I.; Shuvalova, L.; Winsor, J.; Lavis, Luke D.; Satchell, K.J.F.; Becker, Daniel P.; Anderson, W.F.; Johnson, R. Jeremy

doi:10.1074/jbc.m112.446625

Cited by 31 publications

(51 citation statements)

References 63 publications

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“…This hydrophobic channel shows varying degrees of openness between APT1, APT1·ML348 and APT2·ML349 (Figures 2c–d and S1). In the distant bacterial homologue FTT258 (PDB: 4F21), the analogous loop domain demonstrates dynamic conformational changes proposed to define the active and inactive states, suggesting the loop closes upon substrate binding to initiate hydrolysis 19 . However, this substrate driven conformational change could differ among APTs, especially because the shorter G1 helix and the non-canonical β4–β5 sheet replace this loop in all vertebrate APTs (Figures S2a–b).…”

Section: Resultsmentioning

confidence: 99%

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

et al. 2016

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Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively re-distribute S-palmitoylated proteins towards different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The de-palmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.

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Section: Resultsmentioning

confidence: 99%

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

et al. 2016

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“…41;42;51 Additionally, the fluorogenic library encompasses acyl substituents covering the major classes of serine hydrolase substrates and provides single bond resolution of substrate specificity (Figure 2B). 38;39;42;52 Measuring the Michaelis-Menten kinetics of Mm LipW against the full range of fluorogenic substrates provided a detailed description of the substrate specificity profile for LipW (Figure 2C–2E and Supplemental Table 1). …”

Section: Resultsmentioning

confidence: 99%

Structural Basis for the Strict Substrate Selectivity of the Mycobacterial Hydrolase LipW

et al. 2016

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The complex life cycle of Mycobacterium tuberculosis requires diverse energy mobilization and utilization strategies facilitated by a battery of lipid metabolism enzymes. Among lipid metabolism enzymes, the Lip family of mycobacterial serine hydrolases is essential to lipid scavenging, metabolic cycles, and reactivation from dormancy. Based on the homologous rescue strategy for mycobacterial drug targets, we have characterized the three-dimensional structure of full length LipW from Mycobacterium marinum, the first structure of a catalytically active Lip family member. LipW contains a deep, expansive substrate-binding pocket with only a narrow, restrictive active site, suggesting tight substrate selectivity for short, unbranched esters. Structural alignment reinforced this strict substrate selectivity of LipW, as the binding pocket of LipW aligned most closely with the bacterial acyl esterase superfamily. Detailed kinetic analysis of two different LipW homologues confirmed this strict substrate selectivity, as each homologue selected for unbranched propionyl ester substrates, irrespective of the alcohol portion of the ester. Using comprehensive substitutional analysis across the binding pocket, the strict substrate selectivity of LipW for propionyl esters was assigned to a narrow funnel in the acyl-binding pocket capped by a key hydrophobic valine residue. The polar, negatively charged alcohol-binding pocket also contributed to substrate orientation and stabilization of rotameric states in the catalytic serine. Together the structural, enzymatic, and substitutional analysis of LipW provide a connection between the structure and metabolic properties of a Lip family hydrolase that refines its biological function in active and dormant tuberculosis infection.

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“…Introduction of a serine hydrolase to these protected fluorogenic substrates catalyzes their activation, with the relative efficiency of enzyme-catalyzed hydrolysis dependent upon the specific recognition of the acyl-chain structure of these fluorogenic esters by the serine hydrolase (Lavis et al ., 2011; Hedge et al ., 2012; Tian et al ., 2012). Thus, a series of bis-acyloxymethyl ether derivatives of fluorescein can serve as a tool for characterizing the substrate specificity of mycobacterial serine hydrolases (Hedge et al ., 2012; Ellis et al ., 2013; Filippova et al ., 2013; Johnson et al ., 2014a; Lukowski et al ., 2014) and for developing novel neural-signaling sensors (Tian et al ., 2012). …”

Section: Methodsmentioning

confidence: 99%

Implementation of a Collaborative Series of Classroom-Based Undergraduate Research Experiences Spanning Chemical Biology, Biochemistry, and Neurobiology

Kowalski

Johnson

2016

LSE

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Large Scale Structural Rearrangement of a Serine Hydrolase from Francisella tularensis Facilitates Catalysis

Cited by 31 publications

References 63 publications

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2)

Structural Basis for the Strict Substrate Selectivity of the Mycobacterial Hydrolase LipW

Implementation of a Collaborative Series of Classroom-Based Undergraduate Research Experiences Spanning Chemical Biology, Biochemistry, and Neurobiology

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