RESUMO
Glucosyltransferases (GtfB/C/D) in Streptococcus mutans are responsible for synthesizing water-insoluble and water-soluble glucans from sucrose and play very crucial roles in the formation of dental plaque. A monoclonal antibody against a 19-mer peptide fragment named Gtf-P1 was found in GtfC to reduce the enzyme activity to 50%. However, a similar experiment suggested almost unchanged activity in GtfD, despite of the very high sequence homology between the two enzymes. No further details are yet available to elucidate the biochemical mechanism responsible for such discrimination. For a better understanding of the catalytic behavior of these glucosyltransferases, structural and functional analyses were performed. First, the exact epitope was identified to specify the residue(s) required for monoclonal antibody recognition. The results suggest that the discrimination is determined solely by single residue substitution. Second, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, a three-dimensional homology model for GtfC was built. Structural analysis for the region communicating between Gtf-P1 and the catalytic triad revealed the possibility for an "en bloc" movement of hydrophobic residues, which may transduce the functional influence on enzyme activity from the surface of molecule into the proximity of the active site. Figure Side chain interactions between Gtf-P1 and catalytic Asp-477 in GtfC. Calpha-tracing of GtfC with the two crucial peptides (Gtf-P1, orange; Gtf-P2, blue) and the catalytic triad residues ( red) highlighted to show their relative spatial organization. Side chains for the residues are also depicted according to their atom types. The structure is viewed with the barrel opening facing down
Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Streptococcus mutans/enzimologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Catálise , Domínio Catalítico/genética , Domínio Catalítico/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Streptococcus mutans/genéticaRESUMO
The carboxyl terminus of the S3 segment (S3C) in voltage-gated potassium channels was suggested to be the binding site of gating modifier toxins like hanatoxin. It has also been proposed to have a helical secondary structural arrangement. The currently available structures in high resolution for such channel molecules are restricted to regions illustrating the pore function. Therefore no further direct experimental data to elucidate the detailed mechanism for such toxin binding can be derived. In order to examine the putative three-dimensional structure of S3C and to analyze the residues required for hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of hanatoxin and the structural information from mutational scanning data for the S3C fragment in Kv2.1. Our results indicate that hydrophobic and electrostatic interactions are both utilized to stabilize the toxin binding. Precise docking residues and the appropriate orientation for binding regarding amphipathic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of the S3 segment in voltage-gated potassium channels can therefore be further emphasized and analyzed. The possible location/orientation for toxin binding with respect to membrane distribution around the S3C segment is also discussed in this paper.