Amino acids of the bacterial toxin SopE involved in G nucleotide exchange on Cdc42.
RhoGTPases are central switches in all eukaryotic cells. There are at least two known families of guanine nucleotide exchange factors that can activate RhoGTPases: the Dbl-like eukaryotic G nucleotide exchange factors and the SopE-like toxins of pathogenic bacteria, which are injected into host cells to manipulate signaling. Both families have strikingly different sequences, structures, and catalytic core elements. This suggests that they have emerged by convergent evolution. Nevertheless, both families of G nucleotide exchange factors also share some similarities: (a) both rearrange the G nucleotide binding site of RhoGTPases into virtually identical conformations, and (b) two SopE residues (Gln-109SopE and Asp-124SopE) engage Cdc42 in a similar way as equivalent residues of Dbl-like G nucleotide exchange factors (i.e. Asn-810Dbs and Glu-639Dbs). The functional importance of these observations has remained unclear. Here, we have analyzed the effect of amino acid substitutions at selected SopE residues implicated in catalysis (Asp-124SopE, Gln-109SopE, Asp-103SopE, Lys-198SopE, and Gly-168SopE) on in vitro catalysis of G nucleotide release from Cdc42 and on in vivo activity. Substitutions at Asp-124SopE, Gln-109SopE, and Gly-168SopE severely reduced the SopE activity. Slight defects were observed with Asp-103SopE variants, whereas Lys-198SopE was not found to be required in vitro or in vivo. Our results demonstrate that G nucleotide exchange by SopE involves both catalytic elements unique to the SopE family (i.e. 166GAGA169 loop, Asp-103SopE) and amino acid contacts resembling those of key residues of Dbl-like guanine nucleotide exchange factors. Therefore, besides all of the differences, the catalytic mechanisms of the SopE and the Dbl families share some key functional aspects.
Institute of Microbiology, ETH Zürich, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland.
Markus C
MC
Schlumberger
Andrea
A
Friebel
Gretel
G
Buchwald
Klaus
K
Scheffzek
Alfred
A
Wittinghofer
Wolf-Dietrich
WD
Hardt
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D004269:DNA, Bacterial / Q000235:genetics; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; C103987:SopE protein, Salmonella; D020764:cdc42 GTP-Binding Protein