The structure of truncated recombinant human bile salt-stimulated lipase reveals bile salt-independent conformational flexibility at the active-site loop and provides insights into heparin binding.
Human bile salt-stimulated lipase (BSSL), which is secreted from the pancreas into the digestive tract and from the lactating mammary gland into human milk, is important for the effective absorption of dietary lipids. The dependence of BSSL on bile acids for activity with water-insoluble substrates differentiates it from other lipases. We have determined the crystal structure of a truncated variant of human BSSL (residues 1-5.8) and refined it at 2.60 A resolution, to an R-factor of 0.238 and R(free) of 0.275. This variant lacks the C-terminal alpha-helix and tandem C-terminal repeat region of native BSSL, but retains full catalytic activity. A short loop (residues 115-126) capable of occluding the active-site (the active site loop) is highly mobile and exists in two conformations, the most predominant of which leaves the active-site open for interactions with substrate. The bile salt analogue 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid (CHAPS) was present in the crystallisation medium, but was not observed bound to the enzyme. However, the structure reveals a sulfonate group from the buffer piperizine ethane sulfonic acid (PIPES), making interactions with Arg63 and His115. His115 is part of the active-site loop, indicating that the loop could participate in the binding of a sulphate group from either the glycosaminoglycan heparin (known to bind BSSL) or a bile acid such as deoxycholate. Opening of the 115-126 active-site loop may be cooperatively linked to a sulphate anion binding at this site. The helix bundle domain of BSSL (residues 319-398) exhibits weak electron density and high temperature factors, indicating considerable structural mobility. This domain contains an unusual Asp:Glu pair buried in a hydrophobic pocket between helices alpha(H) and alpha(K) that may be functionally important. We have also solved the structure of full-length glycosylated human BSSL at 4.1 A resolution, using the refined coordinates of the truncated molecule as a search model. This structure reveals the position of the C-terminal helix, missing in the truncated variant, and also shows the active-site loop to be in a closed conformation.
Moore
S A
SA
Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand.
Kingston
R L
RL
Loomes
K M
KM
Hernell
O
O
Bläckberg
L
L
Baker
H M
HM
Baker
E N
EN
D000818:Animals; D001647:Bile Acids and Salts / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D003460:Crystallization; D018360:Crystallography, X-Ray; D003840:Deoxycholic Acid / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D018583:Pliability; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012997:Solvents / Q000378:metabolism; D002787:Sterol Esterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001647:Bile Acids and Salts; D011994:Recombinant Proteins; D012997:Solvents; D003840:Deoxycholic Acid; D006493:Heparin; C054784:bile salt-stimulated lipase; D002787:Sterol Esterase