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