Journal of Molecular Biology
Volume 342, Issue 3, 17 September 2004, Pages 943-952
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Crystal Structure of Human ABAD/HSD10 with a Bound Inhibitor: Implications for Design of Alzheimer's Disease Therapeutics

https://doi.org/10.1016/j.jmb.2004.07.071Get rights and content

The enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10), also known as amyloid β-peptide-binding alcohol dehydrogenase (ABAD), has been implicated in the development of Alzheimer's disease. This protein, a member of the short-chain dehydrogenase/reductase family of enzymes, has been shown to bind β-amyloid and to participate in β-amyloid neurotoxicity. We have determined the crystal structure of human ABAD/HSD10 complexed with NAD+ and an inhibitory small molecule. The inhibitor occupies the substrate-binding site and forms a covalent adduct with the NAD+ cofactor. The crystal structure provides a basis for the design of potent, highly specific ABAD/HSD10 inhibitors with potential application in the treatment of Alzheimer's disease.

Introduction

A strong link has been established between the development of Alzheimer's disease (AD) and the accumulation of amyloid β-peptide (Aβ) in the brain.1, 2, 3, 4 Aβ is a proteolytic fragment of the integral membrane glycoprotein, amyloid-β precursor protein,5 and is the principal component of the extracellular plaques that are diagnostic of AD. Aggregated Aβ is toxic to neuronal cells in culture, although the mechanisms of Aβ neurotoxicity are not understood completely.

Aβ accumulates both inside and outside of nerve cells,6 and there is growing evidence that the interaction of Aβ with intracellular proteins could lead to cytotoxic events prior to the formation of extracellular plaques. The enzyme 17β-hydroxysteroid dehydrogenase type 10 (HSD10), also known as amyloid β-peptide-binding alcohol dehydrogenase (ABAD) is an intracellular protein found to bind Aβ in the yeast two-hybrid system.7 An identical protein was identified independently as a new l-3-hydroxyacyl-coenzyme A dehydrogenase, HADH2, with an apparent role in the mitochondrial fatty acid β-oxidation pathway.8 Subsequent studies have shown that the enzyme has significant steroid dehydrogenase activity, in addition to alcohol dehydrogenase and HADH2 activities,9 and the enzyme has been renamed HSD10. Sequence comparisons show that ABAD/HSD10 belongs to the short-chain dehydrogenase/reductase (SDR) family of enzymes.

A variety of evidence suggests that ABAD/HSD10 interacts with Aβ and can mediate its cytotoxic effects.7, 10 The cytotoxic effects of Aβ on neuroblastoma cells in culture were enhanced by overexpression of ABAD/HSD10 and blocked by anti-ABAD/HSD10 antibodies. Cells expressing catalytically inactive mutants of ABAD/HSD10, however, did not show enhanced sensitivity to Aβ, suggesting that enzymatic activity of ABAD/HSD10 is required for mediation of Aβ neurotoxicity. In addition, the brains of patients diagnosed with AD showed elevated levels of ABAD/HSD10. Synthetic Aβ fragments have been shown to bind and inhibit ABAD/HSD10 in vitro.11, 12

Although the precise role of ABAD/HSD10 in AD is not clear, mechanisms have been proposed in which Aβ neurotoxicity is mediated through the enzymatic activity of the enzyme. Although ABAD/HSD10 appears to be localized primarily in mitochondria, the protein translocates to the plasma membrane when cells are exposed to Aβ.7 This observation led to the proposal that ABAD/HSD10 might mediate the intraneuronal toxicity of Aβ by acting on abnormal substrates to generate cytotoxic aldehydes.10 It has been suggested that ABAD/HSD10 could contribute to AD pathogenesis by reducing neuroprotective estrogen levels in the brain, based on the finding that the enzyme can utilize 17β-estradiol as a substrate.13 Recently, attention has focused on the role of mitochondrial dysfunction in AD pathogenesis. Several lines of evidence indicate that Aβ causes mitochondrial dysfunction and neuronal cell death through the direct interaction of Aβ with catalytically active ABAD/HSD10 located in the mitochondria.14 Inhibition of ABAD/HSD10 enzymatic activity thus may provide a new approach to the treatment of AD. In addition to potential therapeutic applications, ABAD/HSD10 inhibitors could be valuable in delineating the role of the enzyme in both normal cellular function and in AD pathogenesis.

We have identified a potent inhibitor of human ABAD/HSD10 (IC50 92 nM), designated AG18051. As part of a structure-based drug design program, we have co-crystallized human ABAD/HSD10 with its NAD+ cofactor and AG18051. We describe here the three-dimensional structure of this complex at 2.0 Å resolution. The structure reveals several unique features of ABAD/HSD10 in comparison to other members of the SDR family, and shows the interactions responsible for the high-affinity binding of AG18051 to ABAD/HSD10, including the formation of a covalent linkage between the inhibitor and the NAD+ cofactor.

Section snippets

Results and Discussion

To facilitate crystallization, both the wild-type enzyme and several mutants were overexpressed in Escherichia coli. Crystals of ABAD/HSD10 with bound NAD+ and AG18051 were ultimately obtained using the C214R mutant of the enzyme. This mutant was engineered to avoid cysteine oxidation in the protein, with arginine selected as the replacement amino acid because of its occurrence at this position in other mammalian ABAD/HSD10 sequences.8 Enzymatic activity of the C214R mutant protein is

Cloning, overexpression, and purification

The coding sequence for full-length wild-type ABAD/HSD107, 8 was amplified by PCR23, 24 from a Marathon-Ready human lung cDNA library and the Advantage™ cDNA PCR kit, both from Clontech (Palo Alto, CA), using the manufacturer's instructions. The forward primer was:

  • 5′-GGGCACACCATGGCAGCAGCGTGTCGGAGCGTGAAGG-3′

and the reverse primer was:
  • 5′-AGCTTTCGGCCGTTAAGGCTGCATACGAATAGCCCCATCC-3′

The amplified DNA was digested with the restriction enzymes NcoI and EagI, ligated into the E. coli plasmid pMGH4,25, 26

Acknowledgements

We thank Dr Dave Matthews and Dr Karen Maegley for helpful discussions, and Sheila Wingfield for assistance in the preparation of this manuscript.

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