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Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein
M Schwemmle1, M F Richter, C Herrmann
1Abteilung Virologie, Universität Freiburg, Germany.
Abstract:
MxA is an interferon-induced 76-kDa GTPase that inhibits the multiplication of several RNA viruses. Deleting seven amino acids from the COOH terminus reduced the GTPase activity of purified MxA to 1.4%. MxA mutants with COOH-terminal deletions of 63 or more amino acids lost all ability to hydrolyze GTP and failed to bind guanine nucleotides. By contrast, an MxA deletion mutant consisting of 301 amino acids from the NH2 terminus and 87 amino acids from the COOH terminus retained about 9% of wild-type GTPase activity, underscoring the pivotal role of COOH-terminal sequences. Limited proteolysis of wild-type MxA with proteinase K resulted in two resistant polypeptides of 60 and 10 kDa, respectively, which copurified as a stable complex. The p60-p10 complex exhibited high GTPase activity, suggesting that it included all MxA domains required for this biochemical activity. Sequencing revealed that the NH2 terminus of the 60-kDa polypeptide mapped to leucine 41 and the NH2 terminus of the 10-kDa polypeptide to glutamine 564 of the MxA sequence. Based on these results we propose a model that suggests that the GTP-binding consensus element located in the NH2-terminal half of MxA is held in an active conformation by strong physical interactions with amino acids from the COOH-terminal region.
Insights
MxA protein
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- MxA is an interferon-induced GTPase crucial for inhibiting RNA virus replication.
- Its GTPase activity is essential for antiviral functions.
Purpose of the Study:
- To investigate the role of MxA's C-terminal region in its GTPase activity and guanine nucleotide binding.
- To identify the domains responsible for MxA's biochemical function.
Main Methods:
- Site-directed mutagenesis was used to create MxA deletion mutants.
- GTPase activity assays and guanine nucleotide binding experiments were performed.
- Limited proteolysis with proteinase K and subsequent sequencing were employed.
Main Results:
- COOH-terminal deletions significantly reduced or abolished GTPase activity and nucleotide binding.
- A specific deletion mutant retained partial GTPase activity, highlighting the C-terminus's importance.
- Limited proteolysis yielded a stable p60-p10 complex with high GTPase activity.
Conclusions:
- The C-terminal region of MxA plays a critical role in maintaining GTPase activity and guanine nucleotide binding.
- A model is proposed where the N-terminal GTP-binding domain is stabilized by interactions with the C-terminal region.