. Ng and . Romr, Required for Motility Response Regulator;YFP, yellow fluorescent protein

, A phylogenetic tree based on MglB sequences with a Ct-helix extension (longer than 15 residues beyond the RBl/LC7 domain) and a corresponding positively charged ? 5 helix. The bacterial phyla demonstrate that these belong to varied classes and are not restricted to Myxococcus. MxMglB (UniProt ID: Q1DB03) and TtMglB (UniProt ID: Q5SJ82) are highlighted in shaded boxes, whereas the sequences for which the positively charged residues are not present in the corresponding coupled MglA sequences are highlighted by a star, S5 Fig. Conservation of D/E rich Ct-helix in MglA-like proteins

D. ,

. Mgla, Mutual gliding motility A; MglB, Mutual gliding motility B

M. Mxmglb and . Mglb,

/. Rbl, . Lc7, /. Roadblock, T. Lc7;-ttmglb, and . Mglb,

, MxMglAB complex superposed on Yptp1 GTPase and TRAPP1 complex (PDB ID: 3CUE). The GTPase domains are shown in shades of green (dark green for eukaryotic GTPases, and pale green for MxMglA), the Rbl/LC7 domains in shades of magenta (dark shades for eukaryotic proteins and light pink for MxMglB), and insertions to the Rbl/LC7 fold and other associated proteins in gray. The insertions to the Rbl/LC7 fold or associated protein loops that contribute to the GEF activity are highlighted in dark orange and boxed, whereas the Ct-helix of MglB is in light orange, S6 Fig. Eukaryotic GTPases associated with GEFs of Roadblock/LC7 domain. (A) MxMglAB complex superposed on Rab GTPase and its GEF Mon1-Ccz1 complex (PDB ID: 5LDD). (B)

. Mglb, Mutual gliding motility B

M. Mxmglb and . Mglb,

M. Mglb,

P. Id, Protein Data Bank identification

/. Rbl and . Lc7,

, Comparison of MxMglB Ct-helix with RomR Glu-rich sequence. (A) Sequence alignment of RomR Glu-rich C-terminal sequence with MxMglB Ct-helix sequence. Negatively charged residues are shown in blue, hydrophobic residues in red, and positively charged residues in pink. The conservation of amino acids is marked according to ClustalO format, S7 Fig

&. , denote identity, similarity, and conservation of polar residues. (B) Secondary structure prediction of C-terminal end of RomR highlighting that the C-terminal region has helical features (highlighted by pink cylinder). Only the helical region

, GTPase activities of wild-type MxMglA only (green) and in the presence of MxMglB (dark purple; MglAB), MxMglB ?Ct (magenta; MglAB ?Ct ), and MxMglB Rhelix (orange

M. Rhelix, MxMglA and MxMglB variants were used in a ratio of 1:2 considering monomeric molecular weight of MxMglB. The release of GDP was estimated using NADH-based enzyme-coupled assay. The lines corresponding to MxMglAB and MxMglB Rhelix are shown with shaded zones depicting the error represent the average of at least 3 repeats, whereas MxMglB ?Ct and MxMglA are shown without highlighting error because the data in this figure panel represent only 2 repeats. The numerical data for the figure panel have been provided in the respective sheets in S1 Data

G. Glu,

M. ?ct, MglA and MglB with Ct-helix truncated; MglAB Rhelix , MglA and MglB with RomR helix instead of Ct-helix

M. Mxmgla and . Mgla,

M. Mxmglb and . Mglb,

M. ?ct, MglB with Ct-helix truncated

M. Rhelix, MglB with RomR helix instead of Ct-helix

. Romr, Required for Motility Response Regulator

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