Probing the Dimeric Structure of Porcine Aminoacylase 1 by Mass Spectrometric and Modeling Procedures
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- 作者：Chiara D'Ambrosio ; Fabio Talamo ; Rosa Maria Vitale ; Pietro Amodeo ; Gianluca Tell ; Lino Ferrara ; and Andrea Scaloni
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：April 22, 2003
- 年：2003
- 卷：42
- 期：15
- 页码：4430 - 4443
- 全文大小：470K
- 年卷期：v.42,no.15(April 22, 2003)
- ISSN：1520-4995
文摘
Aminoacylase 1 is a zinc-binding metalloprotease catalyzing the hydrolysis of N
-acylatedL-amino acids; it presents altered expression levels in different renal and small cell lung carcinomas. Adescription of its redox and oligomerization state was achieved by combined biochemical and massspectrometric procedures. A topological analysis of the enzyme structural architecture was derived fromlimited proteolysis and selective chemical modification experiments, using a broad range of proteasesand chemical reagents. The analysis of the reaction products by different mass spectrometric techniquesidentified 26 amino acids as being accessible on the molecular surface, defining polypeptide regions exposedin the structure of the dimeric protein. The nature of the intermolecular contact zone between monomerswas investigated by cross-linking reaction and mass mapping experiments. The cross-linked dimer wasisolated, and the intermolecular cross-linked peptides were characterized, thus demonstrating the spatialproximity of Lys220 and Lys231 at the dimerization interface. Standard modeling procedures based onautomatic alignment on the structure of members of the M20 peptidase family failed to produce a dimericmodel consistent with experimental data. Discrepancies were observed mainly at the dimer interface andat loop regions. Therefore, a refined model for this dimeric protease was calculated by selecting the oneable to generate a structure fully compatible with experimental findings, among all possible suboptimalsequence alignments. According to this model, each aminoacylase monomer consists of two domains: aglobular catalytic subunit (residues 1-188 and 311-399) consisting of a 
-sheet sandwiched between-helices and a second -sheet located on the surface, and the dimerization domain (residues 189-310)folding into a -sheet flanked on one side by two -helices. These results indicate that reliable approachessuch as limited proteolysis, selective chemical modification, and cross-linking coupled to mass spectrometrycan be used to test and optimize molecular models of multimeric proteins and highlight problems inautomatic model building.
-acylatedL-amino acids; it presents altered expression levels in different renal and small cell lung carcinomas. Adescription of its redox and oligomerization state was achieved by combined biochemical and massspectrometric procedures. A topological analysis of the enzyme structural architecture was derived fromlimited proteolysis and selective chemical modification experiments, using a broad range of proteasesand chemical reagents. The analysis of the reaction products by different mass spectrometric techniquesidentified 26 amino acids as being accessible on the molecular surface, defining polypeptide regions exposedin the structure of the dimeric protein. The nature of the intermolecular contact zone between monomerswas investigated by cross-linking reaction and mass mapping experiments. The cross-linked dimer wasisolated, and the intermolecular cross-linked peptides were characterized, thus demonstrating the spatialproximity of Lys220 and Lys231 at the dimerization interface. Standard modeling procedures based onautomatic alignment on the structure of members of the M20 peptidase family failed to produce a dimericmodel consistent with experimental data. Discrepancies were observed mainly at the dimer interface andat loop regions. Therefore, a refined model for this dimeric protease was calculated by selecting the oneable to generate a structure fully compatible with experimental findings, among all possible suboptimalsequence alignments. According to this model, each aminoacylase monomer consists of two domains: aglobular catalytic subunit (residues 1-188 and 311-399) consisting of a -sheet sandwiched between-helices and a second -sheet located on the surface, and the dimerization domain (residues 189-310)folding into a -sheet flanked on one side by two -helices. These results indicate that reliable approachessuch as limited proteolysis, selective chemical modification, and cross-linking coupled to mass spectrometrycan be used to test and optimize molecular models of multimeric proteins and highlight problems inautomatic model building.