Homologous modeling of the lysosomal protective protein/carboxypeptidase L: structural and functional implications of mutations identified in galactosialidosis patients.
The deficiency of the lysosomal protective protein/carboxypeptidase L (CARB L) causes the lysosomal storage disorder, galactosialidosis, characterized by neuraminidase and beta-galactosidase deficiencies in patients' cells. The three enzymes form a complex inside the lysosome, and the neuraminidase and beta-galactosidase deficiencies are secondary to CARB L deficiency. Sequence similarity and common enzymological properties suggest that the protomeric tertiary structure of CARB L is conserved within a family of serine carboxypeptidases which includes the yeast carboxypeptidase Y, killer expression I gene product and several plant carboxypeptidases. We used this homology to build a model of the CARB L structure based on the recently published X-ray atomic coordinates of the wheat carboxypeptidase II (CPDW-II) which shares 32% primary structure identity with CARB L. Small insertions and deletions were accommodated into the model structure by energy minimization using the DREIDING II force field. The C alpha atomic coordinates of the final CARB L model have a RMS shift of 1.01 A compared to the corresponding conserved residues in the CPDW-II template structure. The correct orientation of the homologous catalytic triad residues Ser150, His429 and Asp392, the potential energy calculations and the distribution of hydrophobic and hydrophillic residues in the structure all support the validity of the CARB L model. Most missense mutations identified in galactosialidosis patients were located in secondary structural elements except for the Tyr211-->Asn mutation which is in a loop. The other mutant residues have their side chains deeply buried in the central beta-sheet of the model structure except for the Phe412-->Val mutation which is located in the dimer interface. The predicted effects of specific mutations on CARB L structural stability correlates well with recently published transient expression studies of mutant CARB L (Shimmoto, M. et al., J. Clin. Invest., 91:2393-2399, 1993).
Elsliger
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MA
Hôpital Sainte-Justine, Département de Pédiatrie, Université de Montréal, Québec, Canada.
Potier
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics; D003198:Computer Simulation; D004789:Enzyme Activation; D006801:Humans; D016464:Lysosomal Storage Diseases / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D002268:Carboxypeptidases; C055587:carboxypeptidase L