The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis

Bellizzi, John J.; Widom, Joanne; Kemp, Christopher W.; Lu, Jui Yun; Das, Amit Kumar; Hofmann, Sandra L.; Clardy, Jon

doi:10.1073/pnas.080508097

Cited by 143 publications

(131 citation statements)

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“…4. The position of the palmitate residue corresponds to the position observed in the crystal structure of PPT1 (for review, see "Results and Discussion") (10). b, fatty acid specificity of PPT1 and -2.…”

Section: Resultsmentioning

confidence: 99%

“…By 10 months of age, most PPT1 knockout mice are dead, whereas mortality in the PPT2 knockout mice is only 20%; however, most mice show spasticity by that age. The crystal structure of PPT1 alone and in complex with palmitate solved in our laboratory (10) showed that PPT1 has an ␣-␤ hydrolase fold with an additional lipid binding domain. This information provided the structural basis for understanding the severity of phenotypes associated with different PPT1 mutations.…”

Section: Neuronal Ceroid Lipofuscinosis (Ncl)mentioning

confidence: 99%

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The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Calero

Gupta

Nonato

et al. 2003

Journal of Biological Chemistry

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Mutations in palmitoyl protein thioesterase-1 (PPT1) have been found to cause the infantile form of neuronal ceroid lipofuscinosis, which is a lysosomal storage disorder characterized by impaired degradation of fatty acid-modified proteins with accumulation of amorphous granular deposits in cortical neurons, leading to mental retardation and death. Palmitoyl protein thioesterase-2 (PPT2) is a second lysosomal hydrolase that shares a 26% identity with PPT1. A previous study had suggested that palmitoyl-CoA was the preferred substrate of PPT2. Furthermore, PPT2 did not hydrolyze palmitate from the several S-palmitoylated protein substrates. Interestingly, PPT2 deficiency in a recent transgenic mouse model is associated with a form of neuronal ceroid lipofuscinosis, suggesting that PPT1 and -2 perform nonredundant roles in lysosomal thioester catabolism. In the current paper, we present the crystal structure of PPT2 at a resolution of 2.7 Å. Comparisons of the structures of PPT1 and -2 show very similar architectural features; however, conformational differences in helix ␣4 lead to a solvent-exposed lipid-binding groove in PPT1. The limited space between two parallel loops (␤3-␣A and ␤8-␣F) located immediately above the lipidbinding groove in PPT2 restricts the binding of fatty acids with bulky head groups, and this binding groove is significantly larger in PPT1. This structural difference accounts for the ability of PPT2 to hydrolyze an unbranched structure such as palmitoyl-CoA but not palmitoylcysteine or palmitoylated proteins. Furthermore, differences in fatty acid chain length specificity of PPT1 and -2, also reported here, are explained by the structure and may provide a biochemical basis for their non-redundant roles.

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Section: Resultsmentioning

confidence: 99%

Section: Neuronal Ceroid Lipofuscinosis (Ncl)mentioning

confidence: 99%

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Calero

Gupta

Nonato

et al. 2003

Journal of Biological Chemistry

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“…The reported finding that inhibition of protein synthesis by DB in MCF-7 human breast carcinoma cells occurs at concentrations that are about 1 order of magnitude lower than those required for induction of apoptosis via caspase activation has been taken as evidence for a second mechanism of action and/or a second target protein. 44 PPT-1 has apparently failed to fulfill the role of a secondary pharmacological target, 8 and no obvious structural similarity is apparent between this enzyme 45 and eEF1A beyond the common R/ hydrolase fold. For this reason, it might be worth taking into account that, apart from being an essential partner in the mechanism of translational elongation, eEF1 is considered to be an important multifunctional (moonlighting) protein 46 whose levels are positively correlated with the proliferative state of cells.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for the Binding of Didemnins to Human Elongation Factor eEF1A and Rationale for the Potent Antitumor Activity of These Marine Natural Products

et al. 2004

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Didemnins and tamandarins are closely related marine natural products with potent inhibitory effects on protein synthesis and cell viability. On the basis of available biochemical and structural evidence and results from molecular dynamics simulations, a model is proposed that accounts for the strong and selective binding of these compounds to human elongation factor eEF1A in the presence of GTP. We suggest that the p-methoxyphenyl ring of these cyclic depsipeptides is inserted into the same pocket in eEF1A that normally lodges either the 3′ terminal adenine of aminoacylated tRNA, as inferred from two prokaryotic EF-Tu‚GTP‚tRNA complexes, or the aromatic side chain of Phe/Tyr-163 from the nucleotide exchange factor eEF1BR, as observed in several X-ray crystal structures of a yeast eEF1A:eEF1BR complex. This pocket, which has a strong hydrophobic character, is formed by two protruding loops on the surface of eEF1A domain 2. Further stabilization of the bound depsipeptide is brought about by additional crucial interactions involving eEF1A domain 1 in such a way that the molecule fits snugly at the interface between these two domains. In the GDP-bound form of eEF1A, this binding site exists only as two separate halves, which accounts for the much greater affinity of didemnins for the GTP-bound form of this elongation factor. This binding mode is entirely different from those seen in the complexes of the homologous prokaryotic EF-Tu with kirromycin-type antibiotics or the cyclic thiazolyl peptide antibiotic GE2270A. Interestingly, the set of interactions used by didemnins to bind to eEF1A is also distinct from that used by eEF1BR or eEF1B , thus establishing a competition for binding to a common site that goes beyond simple molecular mimicry. The model presented here is consistent with both available biochemical evidence and known structure-activity relationships for these two classes of natural compounds and synthetic analogues and provides fertile ground for future research.

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“…We wondered whether there might be structural and/or mechanistic differences between PPT1 and these other lipolytic enzymes that would account for this observation. We have recently determined the three-dimensional crystal structure of PPT1 in the presence and absence of palmitoyl-CoA and shown that palmitate modifies serine 115 of the enzyme (5), confirming that a serine residue is the catalytic nucleophile.…”

mentioning

confidence: 99%

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

Das¹,

Bellizzi²,

Tandel³

et al. 2000

Journal of Biological Chemistry

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Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K i of the inhibition was 125 M (in the presence of 1.5 mM Triton X-100), and the catalytic rate constant for sulfonylation (k 2 ) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 Å resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.Palmitoyl-protein thioesterase-1 (PPT1) 1 is a newly described lysosomal hydrolase that removes long chain fatty acids from lipid-modified cysteine residues in fatty acylated proteins (reviewed in Ref. 1). Deficiency of the enzyme leads to a lysosomal storage disease, infantile neuronal ceroid lipofuscinosis, which causes blindness, seizures, and cortical atrophy of the brain (2). Lysosomal inclusion bodies, termed granular osmiophilic deposits, accumulate in all tissues, and resemble lipofuscin deposits that occur during normal aging. In keeping with this important lipid-metabolizing role, PPT1 is one of the most abundant lysosomal enzymes in the brain (3). In contrast to most hydrolytic enzymes (such as proteases, lipases, esterases, and thioesterases), PPT1 is insensitive to the serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) (4). The insensitivity of PPT1 to these reagents was an important feature of PPT1 because it allowed for the identification of PPT activity in the presence of contaminating lipase and protease activities. We wondered whether there might be structural and/or mechanistic differences between PPT1 and these other lipolytic enzymes that would account for this observation. We have recently determined the three-dimensional crystal structure of PPT1 in the presence and absence of palmitoyl-CoA and shown that palmitate modifies serine 115 of the enzyme (5), confirming that a serine residue is the catalytic nucleophile.In the current study, we show that although PPT1 is insensitive to inactivation by the ser...

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The crystal structure of palmitoyl protein thioesterase 1 and the molecular basis of infantile neuronal ceroid lipofuscinosis

Cited by 143 publications

References 41 publications

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

The Crystal Structure of Palmitoyl Protein Thioesterase-2 (PPT2) Reveals the Basis for Divergent Substrate Specificities of the Two Lysosomal Thioesterases, PPT1 and PPT2

Structural Basis for the Binding of Didemnins to Human Elongation Factor eEF1A and Rationale for the Potent Antitumor Activity of These Marine Natural Products

Structural Basis for the Insensitivity of a Serine Enzyme (Palmitoyl-Protein Thioesterase) to Phenylmethylsulfonyl Fluoride

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