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Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily
Jaeseok Bang1, Jaehui Park1, Sung-Hee Lee1
1College of Pharmacy, Chungbuk National University, Cheongju 28160, Republic of Korea.
Abstract:
Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.
Insights
Researchers identified a novel protein, Sav2152, in Methicillin-resistant Staphylococcus aureus (MRSA). This haloacid dehalogenase-like protein
Area of Science:
- Biochemistry and Molecular Biology
- Structural Biology
- Antimicrobial Resistance Research
Background:
- Methicillin-resistant Staphylococcus aureus (MRSA) is a significant public health threat due to its rapid spread.
- Genomic analysis of MRSA identified Sav2152, a protein homologous to haloacid dehalogenase (HAD)-like hydrolases, as a potential drug target.
Purpose of the Study:
- To determine the crystal structure of the MRSA protein Sav2152.
- To investigate the role of magnesium ions (Mg2+) and specific residues in Sav2152's phosphatase activity and structural stability.
Main Methods:
- X-ray crystallography was used to determine the three-dimensional structure of Sav2152.
- Site-directed mutagenesis was employed to create mutant variants of Sav2152.
- Biochemical assays were performed to assess phosphatase activity and Mg2+ binding.
Main Results:
- The crystal structure revealed Sav2152 comprises a C2-type cap domain and a core domain containing motifs for Mg2+-dependent phosphatase activity.
- Residues D10, D12, and D233 are critical for Mg2+ binding and phosphatase activity.
- Mutations D10A and D12A differentially affected Mg2+ binding, structural stability, and phosphatase activity, with D237 showing potential to substitute for D12.
Conclusions:
- Sav2152 possesses Mg2+-dependent phosphatase activity, with specific residues playing crucial roles in metal ion binding and catalysis.
- Magnesium ions paradoxically destabilize local regions of Sav2152, highlighting a novel aspect of HAD-like enzyme regulation.
- Understanding Sav2152's structure-function relationship provides insights into novel antimicrobial drug development targeting MRSA.
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