The SRP signal sequence of KdpD.

KdpD is a four-spanning membrane protein that has two large cytoplasmic domains at the amino- and at the carboxyterminus, respectively. During its biogenesis KdpD binds to the signal recognition particle (SRP) of Escherichia coli that consists of a 48-kDa protein Ffh and a 4.5S RNA. The protein is targeted to the inner membrane surface and is released after contacting the SRP receptor protein FtsY. The information within the KdpD protein that confers SRP interaction was found in the amino-terminal cytoplasmic domain of KdpD, particularly at residues 22-48. Within this sequence a Walker A motif is present at residues 30-38. To determine the actual sequence specificity to SRP, a collection of mutants was constructed. When the KdpD peptides (residues 22-48) were fused to sfGFP the targeting to the membrane was observed by fluorescence microscopy. Further, nascent chains of KdpD bound to ribosomes were purified and their binding to SRP was analysed by microscale thermophoresis. We found that the amino acid residues R22, K24 and K26 are important for SRP interaction, whereas the residues G30, G34 and G36, essential for a functional Walker A motif, can be replaced with alanines without affecting the affinity to SRP-FtsY and membrane targeting.