3D Structure of Sulfolobus solfataricus Carboxypeptidase Developed by Molecular Modeling is Confirmed by Site-Directed Mutagenesis and Small Angle X-Ray Scattering

Abstract

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.

Molecular analysis and solution structure from small-angle X-ray scattering of the human natural killer inhibitory receptor IRp60 (CD300a)

“…The processed experimental SAXS curve from IRp60 displayed in Fig. 4 yields the effective molecular mass (MM) of the protein compatible with the value predicted from the IRp60 sequence (12,500 Da). This points to the absence of the aggregation effects and suggests that the protein is monomeric in solution, as was also shown by gel filtration chromatography (Fig.…”

“…Several experimental techniques could be used for the validation of a given homology model. The structural information of macromolecules in solution under physiological conditions obtained by small-angle X-ray scattering (SAXS) [10] can be used to validate computermodeled protein structures [11][12][13].…”

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

“…These enzymes are classified into the M20 family of metallopeptidases, in which zinc is included as an essential metal for the catalytic activity [16,17]. The alignments clearly demonstrate that the sequences of these enzymes share highly conserved regions, especially in the vicinity of Cys104, His106, Glu139, Glu140, His164 and His361 (indicated by the positions in the Pho ACY sequence), which have been reported to be the amino acid residues related to metal binding from mutational analysis [18] and structural information from Bsb AH [Protein Data Bank (PDB) ID: 1YSJ]. With regard to functionally related enzymes with available structural information, the carboxypeptidase from Pseudomonas sp.…”

Binding of the Major Phasin, PhaP1, fromRalstonia eutrophaH16 to Poly(3-Hydroxybutyrate) Granules

“…In a SAXS experiment, a protein solution is exposed to a focused X-ray beam, and the scattered intensity is collected as a function of the scattering angle. The theoretical background, basic equations, and methods used for SAXS data analysis have been fully described previously (10,15,16,18,28,31,37,48,54).…”