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.

FIGURE 1

Structural superimposition of CPTvu with the catalytic domain of CPG2. CPTvu is in green and the catalytic domain of CPG2 (residues 1–192 and 304–393) is in blue. Green spheres highlight the Cα atoms of the residues coordinating to the zinc ion in CPTvu; blue spheres represent the Cα atoms of residues coordinating to the first zinc ion in CPG2.

FIGURE 2

Models for CPSso. The model based on the structure of CPTvu is depicted in A, while the model based on the structure of CPG2 is depicted in B. α-helices, β-strands, and turns are in magenta, yellow, and blue respectively. The zinc ions and the water molecules are represented with green and cyan spheres, respectively. The residues coordinating to the zinc ion and the water molecules are represented by sticks.

FIGURE 3

SAXS data. Experimental SAXS data have been normalized for the I(0) value obtained by applying the Guinier law (see Eq. 3) and are shown in different forms. (a) Guinier plot (ln I(Q)/I(0) vs. Q²). The solid line corresponds to the best fit obtained by using the Guinier law (see Eq. 3). (b) semilogarithmic plot (ln I(Q)/I(0) vs. Q). The dotted line corresponds to the fit obtained using the multipole expansion method. The corresponding best fitting parameters are reported in Table 2. The position of the Shannon channels (Ns = 7) is shown by open circles. (c) Kratky plot (Q²I(Q)/I(0) vs. Q). The best fit curves calculated by using the Monte Carlo method from the two computer-designed models are given. Model A (CPTvu-based) and B (CPG2-based), both in monomeric and in oligomeric form, are represented by the dashed and the dotted lines, respectively. Model B, tetrameric assembly, σ = 1.5 ± 0.6 Å; Model A, hexameric assembly, σ = 0.5 ± 0.3 Å. The solid line was obtained by the multipole expansion method.

FIGURE 4

Distance distribution function. Comparison between the p(r) functions obtained by fitting the SAXS experimental data with the Monte Carlo method from the two computer-designed models (dashed line, CPG2, tetrameric assembly, σ = 1.5 ± 0.6 Å; dotted line, CPTvu, hexameric assembly, σ = 0.5 ±0.3 Å) and with the multipole expansion method (solid line).

FIGURE 5

Reconstructed CPSso shape function. Scaled representation of the shape of the scattering particle, as obtained by multiple expansion analysis of the SAXS experimental data. Three different orientations are reported. To illustrate the agreement with the computer-designed model, the CPG2 tetrameric structure is superimposed to the reconstructed shape. The black and the white spheres represent the C- and the N-terminal ends, respectively, of the four polypeptide chains.