structure and function of the first full-length murein ... · in this cell wall recycling enzyme....
TRANSCRIPT
Paper Presentation PLoS ONE 2011
Structure and Function of the First Full-Length MureinPeptide Ligase (Mpl) Cell Wall Recycling ProteinDebanu Das, Mireille Herve, Julie Feuerhelm, etc. and Dominique Mengin-Lecreulx*, lan A. Wilson*
Hai Liang
11/20/2013
Paper Presentation PLoS ONE 2011
Structure and Function of the First Full-Length MureinPeptide Ligase (Mpl) Cell Wall Recycling ProteinDebanu Das, Mireille Herve, Julie Feuerhelm, etc. and Dominique Mengin-Lecreulx*, lan A. Wilson*
Hai Liang
11/20/2013
In the United States, about 30% of total waste is recycled in 2009. (data from Wikipedia)
Background
During each generation, ~30-60% of bacterial cell wall is recycled in Gram-negative bacteria.
We should do better!!!
What is peptidoglycan?
• Important bacterial cell wall component.
• Mesh-like layer outside the plasma membrane of bacteria.
• Structural strength, protection, osmotic pressure counteracting……
From Thomas M. Terry (2000).
Bacterial cell wall
Background
Simplified model of G+ bacterial cell wall
From en.wikipedia
Background
Background
Biosynthesis of peptidoglycan
From Catherine Grimes
Background
What’s the advantage of bacterial cell wall recycling?
From Jean van Heijenoort
Biosynthesis of peptidoglycan
Some of these compounds are uneasy to make.
If the bacteria decide to buy all the compounds they need from Sigma…
UDP-GlcNAc: $938.0/g
L-Ala: $11.7/g
D-Glu: $16.1/g
A2pm: $2472.0/g
D-Ala-D-Ala: $369.5/g
……
Background
From Jean van Heijenoort
Biosynthesis of peptidoglycan
What’s the advantage of bacterial cell wall recycling?
Cell wall recycling would be beneficial in bacterial growth and give bacteria the advantage in competition with others.
Background
Peptidoglycan recycling pathway
Mpl: Murein Peptide Ligase• Important enzyme in cell wall recycling.
• Link the peptidoglycan breakdown component to UDP-MurNAc.
BackgroundExperimental flowchart:
1. Mpl gene from Psychrobacter arcticus genome was cloned, and PaMpl protein was produced and crystallized.
2. MAD data were collected and protein structure was solved and refined.
3. Biochemical character and enzymatic activity of PaMpl were studied.
Experiment methods:Polymerase Incomplete Primer Extension (PIPE) cloning method:In this method, all major cloning operations are achieved by transforming competent cells with PCR products immediately following amplification. Special PCR primers with overlapping sequences are used in this method.
Results:Part 1. Crystal parameters, data collection and refinement statistics
Results:Part 2. Sequence comparison of Mpl to MurC-F enzymes
Results:Part 2. Sequence comparison of Mpl to MurC-F enzymes
Sequence identity between Mpl and MurC is ~21–25%, between Mpland MurD-F is ~15–17%.
Overexpression of E. coli mpl gene was shown to complement an E. coli murC mutant.
Results:Part 3. Overall structure analysis
Secondary structure of PaMpl, based on domains and primary sequence.
PaMpl can be divided into 3 distinct domains
1. N-terminal UDP-MurNAc-binding domain (ND, residues 1-102).
2. Middle ATP-binding domain (MD, residues 103-357).
3. C-terminal tripeptide-binding domain (CD, residues 358-505).
Results:Part 3. Overall structure analysis
• PaMpl is present as a monomer in crystal asymmetric unit.
• Ramachandran plot shows 98.1% of the residues are in the favored regions with none in disallowed regions.
• All three domains are linked to form a triangular-shaped molecule with dimensions of ~56X60X47 Å3.
ND, residues 1-102MD, residues 103-357CD, residues 358-505
Results:Part 4. Structure comparisons
Comparison of crystal structures of full-length PaMpl (Blue) and truncated NmMpl (grey, ND and MD only, PDB 3eag).
Sequence identity is 57%, R.m.s.d. is 2.2 Åover 311 Cα atoms.
NmMpl lacks the segments 141-152(green), 262-285(orange) and 444-450(cyan).
NmMpl residues 152-163 are positioned differently compared to the PaMpl residues 161-171(yellow).
PaMpl residues 210-215(red) are disordered.
Results:Part 4. Structure comparisons
Comparison of crystal structures of PaMpl (Blue), EcMurC (PDB 2f00, pale green), HiMurC (PDB 1p31, orange), and TmMurC (PDB 1j6u, grey).
Results shows a common structural core of 355 Cα residues with an r.m.s.d. of 2.7 Å.
Results:Part 4. Structure comparisons
ND, residues 1-102MD, residues 103-357CD, residues 358-505
A. apo-PaMpl.
B. EcMurC with bound Mg2+.
C. HiMurC bound to UDP-MurNAc-L-Ala (UMA) and AMPPNP(ANP), and metal.
D. Apo-EcMurF.
Surface representation of different domain dispositions in Mur family structure.
These structures illustrate the conformational variability of these Mur enzymes. All molecules are in the same orientation.
CD of the apo-PaMpl is rotated 30°when compared to MurCstructure.
Results:Part 5. Oligomerization of PaMpl protein
Static light scattering measurements indicated that a dimer form of PaMpl protein is prominent in solution.
Refractive index signal against elution time
EcMpl is also dimeric in solution. EcMurD (PDB 1uag), EcMurE (PDB 1e8c) and EcMurF(PDB 1gg4), have been reported to be monomers in the crystal and in solution.
EcMurC (PDB 2f00) is also a dimer in the crystal structure but in solution, displays a dynamic equilibrium between monomeric and dimeric forms. Both forms were active.
Some residues in EcMurCwhich involved in dimerizationare not found in PaMpl, which means the mode of dimerizationin PaMpl may be different.
Results:Part 6. PaMpl residues involved in substrates interactions (ND and MD).
Interactions between HiMurC and ligandsUMA and ANP.
Majority of these residues in HiMurC(redlabels) ND and MD are conserved in PaMpl(blue labels).
Mpl have similar interaction with ATP and UDP-MurNAc.
Results:Part 6. PaMpl residues involved in substrates interactions.
Mpl-specific residues in ND, MD and CD that may be involved in substrate interactions.
Tyr35, Asn69, Arg73 and Gly74 are conserved residues in the vicinity of UDP-MurNAc-binding pocket.
Tyr182 is unique to Mpl and located prior to the start of Mpl-specific loop, it could be involved in substrate recognition.
Tyr182, Gln223 and Phe224 are in the ATP-binding pocket.
Glu181 will aid the stability of Mg2 binding.
Results:Part 6. PaMpl residues involved in substrates interactions.
CD of Mur families are functionally unique, they have the most variation in this domain.
Arg357, Arg358, Phe374, Ala375, His376, His377, Glu402, Pro403, Arg404, Ser405, Asn406, Thr407, Ser483, Asn484 and Gly485 may be important in binding substrates.
Based on their location, the CD should undergo a conformational change in the interaction with peptide.
Results:Part 7. Enzymatic studies of PaMpl
Kinetic parameters of PaMpl
Results:Part 7. Enzymatic studies of PaMpl
Substrate specificity of PaMpl→
Preference for A2pm at the third position.
Results:Part 7. Enzymatic studies of PaMpl
→EcMpl
Optimal pH: 8.4
Optimal Mg concentration: 5mM
Optimal temperature: 30 ºC
At low temperature(~15 ºC), activity of PaMpl was almost twice that of EcMpl. And at 42 ºC, activity of PaMpl was almost completely abolished.
Psychrobacter species are permafrost bacteria.
Conclusion & Discussion
1. High-resolution crystal structure of the full-length PaMpl provides insights into the structure and relative orientations of the three domains in this cell wall recycling enzyme.
2. Various conserved Mpl residues which are potentially involved in substrate interaction provide a guide for site-directed mutagenesis to test their functional roles.
3. Temperature dependent manner of PaMpl is also a topic in future study.
4. Deletion of Mpl gene and other genes involved in peptidoglycanrecycling could increase antibiotic susceptibility of some bacterial strains. In future the design of Mpl inhibitor turns to be possible based on the Mpl active sites.