3D structure of Sulfolobus solfataricus carboxypeptidase developed by molecular modeling is confirmed by site-directed mutagenesis and small angle X-ray scattering.
Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a M(r) of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His(108), Asp(109), and His(168). We also identified Glu(142) as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data.
Occhipinti
Emanuela
E
Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, I-20126 Milano, Italy.
Martelli
Pier Luigi
PL
Spinozzi
Francesco
F
Corsi
Federica
F
Formantici
Cristina
C
Molteni
Laura
L
Amenitsch
Heintz
H
Mariani
Paolo
P
Tortora
Paolo
P
Casadio
Rita
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D003198:Computer Simulation; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein / Q000379:methods*; D013329:Structure-Activity Relationship; D016944:Sulfolobus / Q000737:chemistry* / Q000201:enzymology; D014961:X-Ray Diffraction / Q000379:methods*; D015032:Zinc / Q000737:chemistry*
D002268:Carboxypeptidases; D015032:Zinc