Involvement of FtsE ATPase and FtsX Extracellular Loops 1 and 2 in FtsEX-PcsB Complex Function in Cell Division of Streptococcus pneumoniae D39

Abstract

The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1FtsX) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsBCC(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature.

Structural basis of PcsB-mediated cell separation in Streptococcus pneumoniae

“…The energy required for the coordinated movement of a-helices in the CC PcsB domain is in all likelihood produced by hydrolysis of ATP by FtsE. In agreement with this, it has been shown that the conserved amino acids in the ATP binding site of FtsE are essential for pneumococcal growth, supporting the idea that ATPase activity by FtsE is required for FtsEX-PcsB signalling and coordination of cell separation 14 . Once released, the catalytic domain would not require further opening of the active-site loops.…”

“…To release the CHAP domain, the 'molecular tweezers', which consists of a1-a3 on one side and a4-a5 on the other, must be opened on both sides. Previous studies involving Ts and suppressor mutants of PcsB and FtsX, respectively, revealed mutations in the large (ECL1) and small (ECL2) extracellular loops of FtsX that indicate that the FtsXPcsB interaction occurs between these loops and the CC domain of PcsB 13,14 . The Ts mutations in CC PcsB are located just in the middle of a1 and a3 helices and at the base of a4 helix ( Supplementary Fig.…”

“…The Ts mutations in CC PcsB are located just in the middle of a1 and a3 helices and at the base of a4 helix ( Supplementary Fig. 15), while the connecting regions in FtsX involve the distal portion of the ECL1 and ECL2 loops 14 . These results point to a specific interaction between the FtsX loops and both sides of the 'molecular tweezers'.…”

“…FtsEX is essential or conditionally essential in a great variety of bacterial species 14,34 . FtsEX structurally resembles an ABC transporter 35 , but recent studies indicate that FtsEX acts as a signal transduction system rather than as a transporter of an unknown substrate 34 .…”

“…Recently, it was discovered that PcsB interacts with the membrane-embedded protein FtsX through its N-terminal CC domain. FtsX has two extracellular loops, ECL1 and ECL2, which are involved in this interaction 13,14 . At the cytoplasmic side of the membrane, FtsX, which has four transmembrane segments, interacts with FtsE.…”