Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography.
BACKGROUND
The lysosomal 66.3 kDa protein from mouse is a soluble, mannose 6-phosphate containing protein of so far unknown function. It is synthesized as a glycosylated 75 kDa precursor that undergoes limited proteolysis leading to a 28 kDa N- and a 40 kDa C-terminal fragment.


RESULTS
In order to gain insight into the function and the post-translational maturation process of the glycosylated 66.3 kDa protein, three crystal structures were determined that represent different maturation states. These structures demonstrate that the 28 kDa and 40 kDa fragment which have been derived by a proteolytic cleavage remain associated. Mass spectrometric analysis confirmed the subsequent trimming of the C-terminus of the 28 kDa fragment making a large pocket accessible, at the bottom of which the putative active site is located. The crystal structures reveal a significant similarity of the 66.3 kDa protein to several bacterial hydrolases. The core alphabetabetaalpha sandwich fold and a cysteine residue at the N-terminus of the 40 kDa fragment (C249) classify the 66.3 kDa protein as a member of the structurally defined N-terminal nucleophile (Ntn) hydrolase superfamily.


CONCLUSIONS
Due to the close resemblance of the 66.3 kDa protein to members of the Ntn hydrolase superfamily a hydrolytic activity on substrates containing a non-peptide amide bond seems reasonable. The structural homology which comprises both the overall fold and essential active site residues also implies an autocatalytic maturation process of the lysosomal 66.3 kDa protein. Upon the proteolytic cleavage between S248 and C249, a deep pocket becomes solvent accessible, which harbors the putative active site of the 66.3 kDa protein.
Lakomek
Kristina
K
Department of Molecular Structural Biology, Institute of Microbiology and Genetics, GZMB, Georg-August University Goettingen, Justus-von-Liebig-Weg 11, D-37077 Goettingen, Germany. klakome@gwdg.de
Dickmanns
Achim
A
Kettwig
Matthias
M
Urlaub
Henning
H
Ficner
Ralf
R
Lübke
Torben
T
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D010399:Penicillin Amidase / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D040681:Structural Homology, Protein
D001426:Bacterial Proteins; D006023:Glycoproteins; D011506:Proteins; C018589:lysosomal proteins; D010399:Penicillin Amidase
J Proteome Res. 2008 Jul;7(7):3010-21
18507433
Curr Opin Genet Dev. 2002 Apr;12(2):142-8
11893486
J Biol Chem. 1966 Mar 25;241(6):1308-13
5935346
Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
15299374
Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9371-6
11470906
Nat Protoc. 2007;2(11):2728-33
18007608
Acta Crystallogr D Biol Crystallogr. 2009 Mar;65(Pt 3):220-8
19237744
Prostaglandins Other Lipid Mediat. 2002 Aug;68-69:521-34
12432941
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2288-94
15572783
Chem Phys Lipids. 2000 Nov;108(1-2):135-50
11106787
Nucleic Acids Res. 1997 Mar 15;25(6):1087-93
9092614
Science. 2001 Aug 10;293(5532):1074-80
11498575
Infect Immun. 2004 Jun;72(6):3171-8
15155618
Mol Cell Proteomics. 2006 Apr;5(4):686-701
16399764
Biochem Biophys Res Commun. 1990 Nov 30;173(1):317-22
2256921
Nature. 1981 Jun 11;291(5815):489-91
7015152
Nature. 1995 Nov 23;378(6555):416-9
7477383
Prog Lipid Res. 1990;29(1):1-43
2087478
Nature. 1995 Jan 19;373(6511):264-8
7816145
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
Chem Biol. 2001 Dec;8(12):1253-64
11755403
EMBO J. 1991 Jan;10(1):51-8
1703489
Structure. 1996 Jul 15;4(7):801-10
8805567
Electrophoresis. 2000 Oct;21(16):3411-9
11079561
Cell. 1999 Sep 3;98(5):651-61
10490104
J Comput Chem. 2002 Jan 15;23(1):128-37
11913378
Science. 2002 Nov 29;298(5599):1793-6
12459591
FEBS Lett. 2006 Oct 16;580(24):5747-52
17007843
Biochim Biophys Acta. 2007 Mar;1774(3):368-72
17258946
Nat Struct Biol. 1999 May;6(5):414-6
10331865
Mol Cell Proteomics. 2006 Oct;5(10):1942-56
16709564
Br J Pharmacol. 2001 Nov;134(5):945-50
11682441
Biochemistry. 2007 May 1;46(17):5050-62
17407260
Chem Phys Lipids. 2000 Nov;108(1-2):107-21
11106785
Br J Pharmacol. 2001 Oct;134(3):563-70
11588110
Prostaglandins Leukot Essent Fatty Acids. 2002 Feb-Mar;66(2-3):173-92
12052034
J Alzheimers Dis. 2006;9(3 Suppl):277-89
16914867
Eur J Pharmacol. 1997 Jul 9;330(2-3):231-40
9253958
J Neurochem. 1996 Mar;66(3):1157-66
8769879
Chem Biodivers. 2007 Aug;4(8):1914-25
17712833
Biochemistry. 2003 Apr 15;42(14):4084-93
12680762
Proteomics. 2002 Aug;2(8):1026-40
12203898
Biochemistry. 2005 Apr 19;44(15):5739-48
15823032
J Mol Biol. 2007 Sep 21;372(3):774-97
17681537
Biochem J. 2007 Mar 15;402(3):449-58
17105447
J Mol Biol. 2006 Jun 30;360(1):105-16
16725155
J Mol Biol. 2001 Nov 9;313(5):1093-102
11700065
Curr Pharm Des. 2002;8(7):533-47
11945157
Biochim Biophys Acta. 2009 Apr;1793(4):625-35
18977398
J Biol Chem. 1985 Nov 15;260(26):14145-9
4055775
Experientia. 1992 Feb 15;48(2):130-51
1740186
J Biol Chem. 2001 Dec 21;276(51):48376-81
11604409
Proteomics. 2005 Oct;5(15):3966-78
16145712
Acta Crystallogr D Biol Crystallogr. 2002 May;58(Pt 5):872-4
11976508
Anal Biochem. 1989 Aug 1;180(2):195-204
2510544
Methods Enzymol. 1996;266:653-62
8743712
Biochemistry. 1997 Sep 2;36(35):10718-26
9271502
Biochem J. 2004 Sep 1;382(Pt 2):441-9
15193148
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Mol Microbiol. 2008 May;68(4):933-46
18430083
Eur J Biochem. 1999 Jun;262(3):713-9
10411632
Protein Sci. 2000 Dec;9(12):2329-37
11206054
Genes Dev. 2001 Sep 15;15(18):2343-60
11562345
Nature. 2005 Feb 24;433(7028):865-8
15729342
Anal Chem. 1996 Mar 1;68(5):850-8
8779443
Biochem J. 2004 Mar 1;378(Pt 2):363-71
14616088
J Mol Biol. 1993 Jun 20;231(4):1049-67
8515464
Structure. 2000 Oct 15;8(10):1059-68
11080627
Eur J Pharmacol. 1996 Apr 11;300(3):227-36
8739213
Nature. 2000 Jan 6;403(6765):41-5
10638745
Arch Biochem Biophys. 1998 Jun 15;354(2):303-10
9637740
Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3376-80
7724569
J Biol Chem. 2005 Mar 25;280(12):11082-92
15655246
Mech Dev. 1996 Sep;59(1):11-27
8892229
Life Sci. 1998;63(8):PL125-9
9718090
Prostaglandins Other Lipid Mediat. 2000 Apr;61(1-2):19-28
10785539
J Cell Sci. 2002 Aug 15;115(Pt 16):3253-63
12140257
Nature. 1997 Apr 3;386(6624):463-71
9087403
J Lipid Res. 2006 Jul;47(7):1352-7
16627894
Nat Struct Biol. 1995 Dec;2(12):1102-8
8846222
Cancer Res. 2005 Apr 15;65(8):2993-5
15833821
Biochem Biophys Res Commun. 1979 Sep 27;90(2):628-33
508325
J Biol Chem. 2002 Jan 25;277(4):2823-9
11706000
Proc Natl Acad Sci U S A. 2006 Feb 7;103(6):1732-7
16446446
Chem Biol. 2008 May;15(5):501-12
18482702
Prostaglandins Leukot Essent Fatty Acids. 2002 Feb-Mar;66(2-3):363-76
12052050
J Mol Biol. 2001 Oct 12;313(1):139-50
11601852