gijs van der schot simone wanningen
DESCRIPTION
Gijs van der Schot Simone Wanningen. Bacteriophages. Bacteriophages. Bacteriophages. Host cell lysis. Large double stranded DNA phages: Employ an invariable holin Make use of endolysin Single stranded nucleic acid bacteriophages: Expression of single gene No muralytic enzyme needed - PowerPoint PPT PresentationTRANSCRIPT
Gijs van der Schot
Simone Wanningen
Bacteriophages
Bacteriophages
Bacteriophages
Host cell lysis
• Large double stranded DNA phages:– Employ an invariable holin– Make use of endolysin
• Single stranded nucleic acid bacteriophages:– Expression of single gene– No muralytic enzyme needed
– Example: Gene E from Microviridae ΦX174
Gene E from ΦX174
• Encodes a membrane protein of 91 residues• α-helical shape• Causes lysis of several Gram-negative hosts
• Protein E causes lysis by inhibiting MraY
MraY
out
inA AA E KMUDP
A AA E KM
Lipid I
MraY
UDPG
Lipid IIMurG
G
A AA E KM
AA AEK M
G
Lipid II
A AA E KM
G
A AA E KM
G
MraY and E
out
inA AA E KMUDP
A AA E KM
Lipid I
MraY
UDPG
Lipid IIMurG
G
A AA E KM
AA AEK M
G
Lipid II
A AA E KM
G
A AA E KM
G
MraY and E
out
inA AA E KMUDP
A AA E KM
Lipid I
MraY
UDPG
Lipid IIMurG
G
A AA E KM
AA AEK M
G
Lipid II
A AA E KM
G
A AA E KM
G
MraY catalyzes formation of Lipid I
Phytol Phosphate
Mechanism Inhibition MraY (I)
• Mutations in MraY lead to E-resistance
• MraY from Bacillus suptilis is resistant (BSMraY)
Mechanism Inhibition MraY (II)
• Two models explaining Inhibition:– E affects functioning MraY directly– E affects functioning MraY indirectly
(i.e. assembly heteromultimeric complex)
• Epep fragment contains 37 N-terminal residues:– Lysis of membrane containing overexpressed MraY– No lysis in detergent-solubilized membranes
In this article/study:
• First purifiction of full-length E-protein• Characterization of the ability of E-protein to inhibit MraY
Overproduction of E6his
• Induction E allele lethal
Overproduction of E6his
• Induction E allele and BsMraY overcomes lethality
Purification of E6his
• Yield of extracted protein: 54uM, 84% pure
Quantification of E6his in vivo
• Previous indirect in vivo approaches:
– ~100-300 molecules/cell
– ~1000 molecules/cell
• This study used purified E6his
– ~500 molecules/cell
• We think:– ~750
molecules/cell
Fluorescent analysis of MraY
Substrates used:– UDP-MurNAc-pentapeptide-DNS
– Phytol-P
• Fluorescent labeled product:– Phytol-P-P-MurNAc-pentapeptide-DNS
Michaelis-Menten kinetics
V0 = Initial reaction rate
VMax = Maximum rate
KM = Michaels constant[S] = substrate concentration
Determination of Km values
Al-Dabbagh et al. (ref 27):C55-P – 0.2 mM
UM5 – 0,94 mM
E resistance is not due to an altered substrate affinity
E-mediated inhibition of MraY (I)
• E inhibits MraY specifically when both are present in same membrane
Reversible Inhibitors
E-mediated inhibition of MraY (II)
Km parameters for both substrates unchanged in presence of E
Vmax in both substrates decreased in presence of E
E is a non-competitive inhibitor of MraY with respect to both lipid and sugar-nucleotide substrates
– Ki averages of 0,53 +/- 0,12 uM
Sensitivity of MraY mutant alleles
• Ability of E to inhibit the MraY proteins form the 5 mutant alleles
• 5 mutants in 3 classes:– MraYG186S and MraYV291M
– MraYp170L and MraY∆L172
– MraYF288L
• Matches classes of apparent affinities
Conclusions
• Overproduction of protein E achieved– Possible to do structural and biophysical characterization of E
• E acts as a non-competitive inhibitor with respect to both lipid and sugar-nucleotide substrates of MraY
New model: Inhibition by direct binding
• Interaction of one TMD of E and TMD 5 and 9 of MraY• Non-competitive binding results in conformational change