Structure of the large extracellular loop of FtsX and its interaction with the essential peptidoglycan hydrolase PcsB inStreptococcus pneumoniae

ABSTRACTStreptococcus pneumoniaeis a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically targeting essential proteins for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises an integral membrane protein (FtsX) and cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present NMR solution structural and crystallographic models of the large extracellular domain of FtsX, denoted ECL1. The structure of ECL1 reveals an upper extended β-hairpin and a lower α-helical lobe, each extending from a mixed α-β core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB, via the coiled-coil domain of PcsB (PcsB-CC). Characterization ofS. pneumoniaeD39 derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division ofS. pneumoniae.IMPORTANCEFtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as “mechanotransmitters,” proteins that relay signals from inside to the outside of the cell. Here we present a structural characterization of the large extracellular loop (ECL1) of FtsX from the human opportunistic pathogenStreptococcus pneumoniae. We show a direct interaction between the peptidoglycan hydrolase PcsB and FtsX, and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

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Structure of the Large Extracellular Loop of FtsX and Its Interaction with the Essential Peptidoglycan Hydrolase PcsB inStreptococcus pneumoniae

mBio ◽

10.1128/mbio.02622-18 ◽

2019 ◽

Vol 10 (1) ◽

pp. e02622-18 ◽

Cited By ~ 11

Author(s):

Britta E. Rued ◽

Martín Alcorlo ◽

Katherine A. Edmonds ◽

Siseth Martínez-Caballero ◽

Daniel Straume ◽

...

Keyword(s):

Cell Division ◽

Membrane Protein ◽

Cell Viability ◽

Integral Membrane Protein ◽

Peptidoglycan Hydrolase ◽

Physical Interaction ◽

Extracellular Loop ◽

Content Type ◽

Large Extracellular Loop

ABSTRACTStreptococcus pneumoniaeis a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically by targeting proteins that are essential for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises a cell division-regulating integral membrane protein (FtsX) and a cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present nuclear magnetic resonance (NMR) solution structural and crystallographic models of the large extracellular domain of FtsX, denoted extracellular loop 1 (ECL1). The structure of ECL1 reveals an upper extended β-hairpin and a lower α-helical lobe, each extending from a mixed α-β core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB via the coiled-coil domain of PcsB (PscBCC). Characterization ofS. pneumoniaestrain D39-derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division ofS. pneumoniae.IMPORTANCEFtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as “mechanotransmitters,” proteins that relay signals from the inside to the outside of the cell. Here, we present a structural characterization of the large extracellular loop, ECL1, of FtsX from the opportunistic human pathogenS.pneumoniae. We show the molecular nature of the direct interaction between the peptidoglycan hydrolase PcsB and FtsX and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

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Characterization of YmgF, a 72-Residue Inner Membrane Protein That Associates with the Escherichia coli Cell Division Machinery

Journal of Bacteriology ◽

10.1128/jb.00331-08 ◽

2008 ◽

Vol 191 (1) ◽

pp. 333-346 ◽

Cited By ~ 44

Author(s):

Gouzel Karimova ◽

Carine Robichon ◽

Daniel Ladant

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Membrane Protein ◽

Integral Membrane Protein ◽

Dependent Manner ◽

E Coli ◽

Division Site ◽

Inner Membrane Protein ◽

Two Hybrid

ABSTRACT Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Many of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. In the present study, we attempted to identify a novel putative component(s) of the E. coli cell division machinery by searching for proteins that could interact with known Fts proteins. To do that, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to perform a library screening in order to find putative partners of E. coli cell division protein FtsL. Here we report the characterization of YmgF, a 72-residue integral membrane protein of unknown function that was found to associate with many E. coli cell division proteins and to localize to the E. coli division septum in an FtsZ-, FtsA-, FtsQ-, and FtsN-dependent manner. Although YmgF was previously shown to be not essential for cell viability, we found that when overexpressed, YmgF was able to overcome the thermosensitive phenotype of the ftsQ1(Ts) mutation and restore its viability under low-osmolarity conditions. Our results suggest that YmgF might be a novel component of the E. coli cell division machinery.

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CD45, an integral membrane protein tyrosine phosphatase. Characterization of enzyme activity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)86999-x ◽

1990 ◽

Vol 265 (18) ◽

pp. 10674-10680 ◽

Cited By ~ 8

Author(s):

N K Tonks ◽

C D Diltz ◽

E H Fischer

Keyword(s):

Enzyme Activity ◽

Membrane Protein ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Integral Membrane Protein ◽

Protein Tyrosine

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Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)37635-5 ◽

1988 ◽

Vol 263 (30) ◽

pp. 15634-15642 ◽

Cited By ~ 1

Author(s):

B M Denker ◽

B L Smith ◽

F P Kuhajda ◽

P Agre

Keyword(s):

Membrane Protein ◽

Integral Membrane Protein ◽

Partial Characterization ◽

Renal Tubules

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An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.564-570.1988 ◽

1988 ◽

Vol 8 (2) ◽

pp. 564-570

Author(s):

P A Maher ◽

S J Singer

Keyword(s):

Molecular Weight ◽

Membrane Protein ◽

Protein Complex ◽

Apparent Molecular Weight ◽

Integral Membrane Protein ◽

Beta Chain ◽

Attachment Sites ◽

Wide Range ◽

Membrane Attachment

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.

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Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.256.6.g1070 ◽

1989 ◽

Vol 256 (6) ◽

pp. G1070-G1081 ◽

Cited By ~ 13

Author(s):

T. Urushidani ◽

D. K. Hanzel ◽

J. G. Forte

Keyword(s):

Membrane Protein ◽

Parietal Cell ◽

Apical Membrane ◽

Peptide Mapping ◽

Integral Membrane Protein ◽

Gastric Glands ◽

Low Speed ◽

Calcium Dependent ◽

Apical Membranes

When isolated rabbit gastric glands were stimulated with histamine plus isobutylmethylxanthine, a redistribution of H+-K+-ATPase, from microsomes to a low-speed pellet, occurred in association with the phosphorylation of an 80-kDa protein (80K) in the apical membrane-rich fraction purified from the low-speed pellet. Histamine alone or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), but not carbachol, also stimulated both the redistribution of H+-K+-ATPase and phosphorylation of 80K. Under stimulated conditions, 80K copurified in the apical membrane fraction along with H+-K+-ATPase and actin; whereas purified microsomes from resting stomach were highly enriched in H+-K+-ATPase but contained neither 80K nor actin. Treatment of the apical membranes with detergents, salts, sonication, and so on, led us to conclude that 80K is a membrane protein, unlike actin; however, the mode of association of 80K with membrane differed from H+-K+-ATPase, an integral membrane protein. Isoelectric focusing and peptide mapping revealed that 80K consists of six isomers of slightly differing pI, with 32P occurring only in the three most acidic isomers and exclusively on serine residues. Moreover, stimulation elicited a shift in the amount of 80K isomers, from basic to acidic, as well as phosphorylation. We conclude that 80K is an apical membrane protein in the parietal cell and an important substrate for cAMP-dependent, but not calcium-dependent, pathway of acid secretion.

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Essential PcsB putative peptidoglycan hydrolase interacts with the essential FtsXSpn cell division protein in Streptococcus pneumoniae D39

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1108323108 ◽

2011 ◽

Vol 108 (45) ◽

pp. E1061-E1069 ◽

Cited By ~ 92

Author(s):

L.-T. Sham ◽

S. M. Barendt ◽

K. E. Kopecky ◽

M. E. Winkler

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Peptidoglycan Hydrolase ◽

Cell Division Protein

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Cloning and characterization of the SmIMP25 integral membrane protein of the parasitic helminth Schistosoma mansoni

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(94)90178-3 ◽

1994 ◽

Vol 1218 (3) ◽

pp. 273-282 ◽

Cited By ~ 6

Author(s):

Alexander Markovics ◽

Daniela Ram ◽

Grossman Zehava ◽

Etty Ziv ◽

Frida Lantner ◽

...

Keyword(s):

Membrane Protein ◽

Schistosoma Mansoni ◽

Integral Membrane Protein ◽

Parasitic Helminth

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

mBio ◽

10.1128/mbio.00431-13 ◽

2013 ◽

Vol 4 (4) ◽

Cited By ~ 28

Author(s):

Lok-To Sham ◽

Katelyn R. Jensen ◽

Kevin E. Bruce ◽

Malcolm E. Winkler

Keyword(s):

Signal Transduction ◽

Amino Acids ◽

Cell Division ◽

Streptococcus Pneumoniae ◽

Amino Acid ◽

Stationary Phase ◽

Coiled Coil ◽

Extracellular Loop ◽

Coiled Coil Domain ◽

Loop Domain

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 pcsB CC(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. IMPORTANCE Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsB CC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

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