Structural insight into the binding mode between the targeting domain of ALE-1 (92AA) and pentaglycine of peptidoglycan.

ALE-1 is a glycylglycine endopeptidase that selectively targets and lyses Staphylococcus aureus, and is expected to be a next generation antibacterial agent because of its substrate specificity to pathogenic bacteria. It has a central catalytic domain and a targeting domain called 92AA. 92AA has been shown to recognize pentaglycine, but the molecular mechanism by which it recognizes and interacts with pentaglycine has not been elucidated. To predict the binding modes of pentaglycine is important for estimating the catalytic reaction mechanism of ALE-1. In the present study, we characterized the binding cleft of 92AA by a computational method and modeled the complexes formed between 92AA and the pentaglycine of peptidoglycan by a binding simulation. In addition, we performed precise simulations of the molecular dynamics by which the complexes identify the amino acid residues interacting with the pentaglycine. We also experimentally constructed mutants in which the amino acid residues present in the binding cleft were changed by site-directed mutagenesis and assessed their ability to bind to peptidoglycan by ELISA. Based on the results of these analyses, we proposed a mode of binding between 92AA and the pentaglycine of peptidoglycan, and modeled the energetically stable complexes between 92AA and the pentaglycine.

Heterologous expression of Na+/H+ antiporter gene (CvNHA1) from salt-tolerant yeast Candida versatilis in Saccharomyces cerevisiae Na+-transporter deficient mutants.

A Na(+)/H(+) antiporter gene (CvNHA1) was cloned from the salt-tolerant yeast Candida versatilis. CvNHA1 encodes an antiporter with a typical yeast plasma membrane Na(+)/H(+) antiporter structure. Transcription of CvNHA1 in C. versatilis cells was dependent on the salinity of the culture. When CvNHA1 was expressed in salt-sensitive Saccharomyces cerevisiae cells, increased salt-tolerance was observed, indicating that Cvnha1p possesses an Na(+)/H(+) antiporter function, because the increased salt-tolerance was dependent on the extracellular pH. It appears that Cvnha1p mediates only the transport of Na(+). In an S. cerevisiae transformant harboring a CvNHA1-EGFP fusion plasmid in which the greater part of the C-terminal hydrophilic region of Cvnha1p was deleted by fusion with enhanced green fluorescent protein (EGFP), the Cvnha1-EGFP fusion protein was localized mainly in the plasma membrane, and the NaCl-tolerance of this transformant was greater than that of a strain harboring the entire CvNHA1 gene.