Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein.
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.
Schwemmle
M
M
Abteilung Virologie, Universität Freiburg, Germany.
Richter
M F
MF
Herrmann
C
C
Nassar
N
N
Staeheli
P
P
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins; D007372:Interferons / Q000502:physiology*; D008969:Molecular Sequence Data; D064547:Myxovirus Resistance Proteins; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000998:Antiviral Agents; D064547:Myxovirus Resistance Proteins; D011506:Proteins; D011994:Recombinant Proteins; D007372:Interferons; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins