Stalk Domain of the Dynamin-like MxA GTPase Protein Mediates Membrane Binding and Liposome Tubulation via the Unstructured L4 Loop

Alexander Von Der Malsburg,Inbal Abutbul-Ionita,Otto Haller,Georg Kochs,Dganit Danino

doi:10.1074/jbc.m111.249037

Alexander Von Der Malsburg, Inbal Abutbul-Ionita + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111.249037

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Abstract

The human MxA protein is an interferon-induced large GTPase with antiviral activity against a wide range of viruses, including influenza viruses. Recent structural data demonstrated that MxA oligomerizes into multimeric filamentous or ring-like structures by virtue of its stalk domain. Here, we show that negatively charged lipid membranes support MxA self-assembly. Like dynamin, MxA assembled around spherical liposomes inducing liposome tubulation. Cryo-transmission electron microscopy revealed that MxA oligomers around liposomes have a "T-bar" shape similar to dynamin. Moreover, biochemical assays indicated that the unstructured L4 loop of the MxA stalk serves as the lipid-binding moiety, and mutational analysis of L4 revealed that a stretch of four lysine residues is critical for binding. The orientation of the MxA molecule within the membrane-associated oligomer is in agreement with the proposed topology of MxA oligomers based on crystallographic data. Although oligomerization of wild-type MxA around liposomes led to the creation of helically decorated tubes similar to those formed by dynamin, this lipid interaction did not stimulate GTPase activity, in sharp contrast to the assembly-stimulated nucleotide hydrolysis observed with dynamin. Moreover, MxA readily self-assembles into rings at physiological conditions, as opposed to dynamin which self-assembles only at low salt conditions or onto lipids. Thus, the present results indicate that the oligomeric structures formed by MxA critically differ from those of dynamin.

Highlights

Previous sequence alignments and biochemical analysis showed that MxA has a multidomain structure similar to dynamin and other dynamin-like proteins (Fig. 1) (1, 8)
These are (i) the globular G domain, (ii) the bundle signaling element consisting of the C-terminal portion of the GTPase effector domain (GED) and two helices connected to the G domain (15) and (iii) an elongated stalk structure formed by the middle domain (MD) and the N-terminal part of the GED (8)
Incubation of MxA at 25 °C resulted in the formation of ring-like structures (Fig. 2e) whereas at 4 °C MxA oligomerized into long filamentous structures with a diameter of about 10 nm and a length of about 500 –1000 nm (Fig. 2d)

Summary

Introduction

Previous sequence alignments and biochemical analysis showed that MxA has a multidomain structure similar to dynamin and other dynamin-like proteins (Fig. 1) (1, 8). The MxA stalk contains a 40-amino acid long loop L4 (residues 533–572) which is located at equivalent position to the PH domain of dynamin (Fig. 1) (8). We used a salt concentration of 100 mM NaCl to analyze the MxA structures in more detail by cryo-TEM in the set of experiments.

Results

Conclusion