The role of polyproline motifs in the histidine kinase EnvZ

Abstract

Although distinct amino acid motifs containing consecutive prolines (polyP) cause ribosome stalling, which necessitates recruitment of the translation elongation factor P (EF-P), they occur strikingly often in bacterial proteomes. For example, polyP motifs are found in more than half of all histidine kinases in Escherichia coli K-12, which raises the question of their role(s) in receptor function. Here we have investigated the roles of two polyP motifs in the osmosensor and histidine kinase EnvZ. We show that the IPPPL motif in the HAMP domain is required for dimerization of EnvZ. Moreover, replacement of the prolines in this motif by alanines disables the receptor’s sensor function. The second motif, VVPPA, which is located in the periplasmic domain, was found to be required for interaction with the modulator protein MzrA. Our study also reveals that polyP-dependent stalling has little effect on EnvZ levels. Hence, both polyP motifs in EnvZ are primarily involved in protein-protein interaction. Furthermore, while the first motif occurs in almost all EnvZ homologues, the second motif is only found in species that have MzrA, indicating co-evolution of the two proteins.

Elongation factor P is required for EIIGlc translation in Corynebacterium glutamicum due to an essential polyproline motif

“…A CadC variant in which the triproline motif is replaced by a pair of alanines is characterized by threefold higher copy number and a less sensitive stress response. On the contrary, the polyproline motif in EnvZ did not affect receptor copy number, but was found to be essential for dimerization and interaction with the modulator MzrA (Motz and Jung, 2018). Here, we propose that the polyproline motif in C. glutamicum EII Glc has a dual function: it is essential for permease activity, but also fine-tunes the amount of the transporter that is produced.…”

“…In EII Glc , the EF-P-dependent polyproline motif is predicted to be located in a transmembrane domain of the EIIC subunit of EII Glc , which is involved in dimer formation. Avoiding a bottleneck in EII Glc production by replacing one or the two prolines of this motif the role of polyproline motifs in membrane-integrated proteins have focused on the E. coli acid stress receptor CadC (Ude et al, 2013) and the osmosensor EnvZ (Motz and Jung, 2018). CadC has a strong polyproline motif that fine-tunes its copy number (Ude et al, 2013).…”

“…In C. glutamicum, EF-P is required for the synthesis of many polyproline-containing enzymes of primary and secondary metabolism, as well as regulatory proteins (Pinheiro et al, 2020). Various studies have shown that polyproline motifs can play important functional roles in the catalytic center of enzymes, in the downregulation of the copy numbers of receptors, and in protein-protein interactions (Motz and Jung, 2018;Starosta et al, 2014b;Ude et al, 2013). These motifs might also be important in protein folding and membrane insertion (Qi et al, 2018).…”

“…The requirement of EF-P for the translation of EII Glc raises the question on the function of the polyproline motif in this protein, because XPPX motifs are required for copy number regulation or dimerization or catalytic activity (Hummels and Kearns, 2019;Motz and Jung, 2018;Starosta et al, 2014b;Ude et al, 2013). To answer this question, we substituted alanines for the prolines at positions 235 and 236, and the corresponding construct mNG-ptsG-PP235/236AA…”

Employing scan-SCAM to identify residues that compose transmembrane helix 1 (TM1) of EnvZ

“…MzrA and resultant modulation of EnvZ signal output [18,19,42]. A related structure of the periplasmic domain of Salmonella enteritica serovar Typhimurium PhoQ (PhoQ-PD) possesses a similar hairpin-like β-strand composed of β1 and β2 [43].…”