Domain Identification of Hormone-sensitive Lipase by Circular Dichroism and Fluorescence Spectroscopy, Limited Proteolysis, and Mass Spectrometry

Abstract

To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1-4 and 5-9, respectively, of which the latter contains an exposed regulatory module outside the catalytic ␣/␤-hydrolase fold core.

Acid and Chemical Induced Conformational Changes of Ervatamin B. Presence of Partially Structured Multiple Intermediates

“…In the case of cardiac troponin I (Morjana and Tal, 1998), the urea denaturation curves are biphasic, which indicates the presence of a stable folding intermediate, which might be related to the two-domain architecture of troponin I. Similar observations have suggested the presence of domains in the hormone-sensitive lipase (Osterlund et al, 1999) structure also.…”

Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores

“…Multiple potential transcription factor binding elements upstream of each transcriptional start site suggest the possibility of differential transcriptional regulation of HSL in different tissues and under various physiological conditions. According to the HSL domain structure model [the three-dimensional (3D) structure of the enzyme remains to be elucidated], the enzyme can be subdivided into three functional regions (41)(42)(43)(44). The N-terminal domain (amino acids 1-300) is believed to mediate enzyme dimerization (45) and interaction with FABP4, a fatty acid binding protein known to enhance HSL enzyme activity (46)(47)(48).…”

Interaction of Hormone-sensitive Lipase with Steroidogeneic Acute Regulatory Protein

“…The C-terminal portion of HSL displays secondary structural homology with that of acetylcholinesterase and several fungal lipases (5) and bacterial brefeldin A esterase (6), consisting of parallel ␤-sheets flanked by ␣-helical connections, which has allowed these proteins to be classified as ␣/␤-hydrolases (7). Using limited proteolysis, it has been suggested that HSL is composed of two major structural domains (8,9). Based on sequence alignment, structural homology with fungal lipases, and mutational analyses, the C-terminal domain has been shown to contain the catalytic triad and other residues important in hydrolytic activity, as well as a 150-amino acid insert that has been termed the regulatory module because several serines located within this region have been shown to be phosphorylated (1,10).…”

“…Based on sequence alignment, structural homology with fungal lipases, and mutational analyses, the C-terminal domain has been shown to contain the catalytic triad and other residues important in hydrolytic activity, as well as a 150-amino acid insert that has been termed the regulatory module because several serines located within this region have been shown to be phosphorylated (1,10). The N-terminal domain in rat HSL constitutes the first 323 amino acids, which are encoded by exons 1-4, and displays no sequence or structural similarity with any other known proteins (8,9).…”