Protecting us… even in our tears…

Lysozyme Artwork PDB ID 1lmn
01 February 2021

We are still making discoveries about lysozyme and its many roles in the arms-race between our body’s defences and bacterial invaders. 

The artwork this month highlights lysozyme’s protective role in our bodies.   One can almost imagine a cartoon-like “kaa-pow” sound effect as lysozyme is breaking-through…   breaking-down bacterial walls and destroying bacterial invaders.   The blue around lysozyme appears to be breaking apart in almost an explosive manner, but the blue also seems fluid-like.   This fluidity may bring-to-mind the many bodily fluids that contain lysozyme, such as our tears, saliva, mucus and more.

In tears and in the fluid on our eyeballs, 40% of the protein content is lysozyme.   Thus, lysozyme is considered by researchers as part of the product development for contact lenses.   Certain brands promote that, for their contact lens, lysozyme maintains its 3D shape even after being absorbed onto the lens.   Lysozyme does not end-up as aggregated and denatured protein build-up, even after being absorbed into these contact lenses, thus enabling this protein to be on “standby” ready to wage war against bacterial invaders.

 

Lysozyme is a deceptively simple protein. It even has a simple name.

Alexander Fleming gave lysozyme its name in 1922.  Fleming was systematically testing a wide variety of biological mixtures to see if they could kill bacteria.  Once he found a mixture that killed bacteria, he would follow-up by trying to isolate the “active ingredient” in the mixture.  After discovering nasal secretions could kill bacteria, Fleming isolated lysozyme as the “active ingredient” in mucus.

On a remarkable bacteriolytic element found in tissues and secretions

Link to full article: On a remarkable bacteriolytic element found in tissues and secretions

 

The “zyme” part of the name lysozyme is given because this protein catalyses change.   In the case of lysozyme, it catalyses a “lytic” change, that is the breakdown of a carbohydrate-peptide layer found in bacteria.   This carbohydrate-peptide layer helps bacteria maintain their shape and osmotic balance.   The action of lysozyme leads to bacterial envelope rupture and bacterial death due to the degradation of the carbohydrate-peptide layer - by the cleavage of multiple carbohydrate bonds.

How lysozyme fights bacteria in our saliva

Lysozyme-C (Black; PDB ID 1LZ1) catalyses the breaking of a carbohydrate bond found in the carbohydrate-peptide layer in bacteria.   This layer, also called the peptidoglycan, is the outer layer in gram positive bacteria, such as the streptococcus bacterial species that can cause strep throat.   This layer in bacteria commonly involves alternating units of carbohydrates referred to as NAM and NAG (for N-acetylmuramic acid and N-acetyl-D-glucosamine, respectively) and has peptides linking the carbohydrate chains to make a strong polymer layer to protect and enable bacteria to maintain their shape.

Image created with PyMOL, Biorender.com and Adobe Photoshop.

 

Lysozyme: a pioneer protein structure.

Lysozyme is among the first 30 protein structures ever deposited in the PDB, but its structure actually pre-dates the establishment of the PDB.

 

The structure of lysozyme was discovered in the 1960s when it was still a rarity for scientists to use X-ray crystallography to study proteins because it was primarily being used to study minerals and chemicals.   In the 1960s various methods were being trailed with lysozyme crystals and it was considered a “pioneer structure”.   Even today, lysozyme is a favourite “pioneer” protein – often being used as a test case to advance new methods to study proteins and other biological macromolecules structures.   Lysozyme under certain conditions will form nice protein crystals and is readily available from egg white, where its biological role is to protect unborn chicks from bacteria.   Partially because lysozyme is a favourite “pioneer” protein, there are now over 904 structures of chicken (Gallus gallus) lysozyme (also known as HEWL) in the PDB and you can see an overview of all the available structures at the PDBe-KB page.



Lysozyme’s mechanism of attack.

Structures of lysozyme deposited in the PDB show that its amino acids come together and form a 3D shape with a groove that recognizes and binds a chain of carbohydrate molecules.   The groove is relatively open and this enables lysozyme to act on the carbohydrate chain when it is part of the larger polymer found in peptidoglycan.

Due to lysozyme’s excellent capacity to cleave carbohydrates so far not even a single structure has been determined with a peptidoglycan-like substrate bound to it.   In the example structures shown, the carbohydrate chains either show cleavage to have already occurred (PDB 9LYZ; NAM-NAG-NAM) or those that cannot be cleaved by lysozyme (PDB ID; NAG-NAG-NAG-NAG-NAG).

Lysozyme's carbohydrate binding groove

Bacterial peptidoglycan typically involves alternating units of NAG and NAM.   Structures of lysozyme (e.g. PDB ID 1SFB and PDB ID 9LYZ) revealed multiple binding sites for carbohydrate rings (A, B, C, D, and E sites).   The groove orientates the carbohydrate chain, with a β-(1,4) linkage positioned between sites.   Lysozyme only catalyses cleavage of the β-(1,4) linkage between sites A and B, and cleavage only occurs when NAG is bound in site A and NAM in site B.   Although NAG can bind in site B (PDB ID 1SFB), NAG is orientated in a very different way than NAM (PDB ID 9LYZ).

Image created with PyMOL, Biorender.com, ChemDraw and Adobe Photoshop.

A structure of lysozyme (PDB ID 1H6M) was determined in 2001 with catalysis “stalled”. It had been theorized that lysozyme’s capacity to break bacterial wall components involved formation of a covalent bond with a key amino acid residue, an asparagine (Asn52). A mutation of another key amino acid in the groove, in combination with a chemical analogue of carbohydrate (2-deoxy-2-fluoro-alpha-D-glucopyranose; enabled the capture of a “stalled” catalysis. In this lysozyme structure, a key glutamic acid residue (Glu35), was replaced by a glutamine (Gln35).

Lysozyme's mechanism for cleaving carbohydrates

The mechanism shown here is the sort of insight that 3D structures can help us to gain.   A structure of lysozyme-C (Grey; PDB ID 1H6M) was determined with a designed change to an active site residue (Glu35 to Gln35).   This mutation prevents lysozyme from performing the final steps of its mechanism.   This lysozyme is less active due to the mutation, however the fluorinated carbohydrate analogue is more reactive than lysozyme’s native carbohydrate substrate and thus the less active lysozyme can act if it has the more reactive substrate.   This combination enabled the capture of an intermediate state that occurs as part of the process performed by lysozyme-C on bacterial peptidoglycan.

Image created with PyMOL, ChemDraw and Adobe Photoshop.

 

Lysozyme: still things to be discovered

A group of scientists published in January 2021 new insights into the role of lysozyme.   Their publication provides experimental evidence that lysozyme is the key component in blood serum that works in combination with our immune system’s membrane attack complex (MAC) to destroy bacterial invaders.   How MAC kills gram negative bacteria has been an ongoing question, but recent insight with cryo-electron microscopy structures (e.g. PDB ID 6DLW) has indicated it generates a pore only in the outermost layers of the bacterial envelope.   The combination of lysozyme and MAC is a powerful approach for attacking gram negative bacteria because MAC gives lysozyme access to the carbohydrate-peptide layer, and when this layer is degraded, bacteria are unable to maintain their shape, causing them to rupture and die.   This newly published experimental evidence shows that lysozyme is protecting us in ways we are still uncovering.

 How lysozyme fights bacteria in our blood

Lysozyme-C (Black; PDB ID 1LZ1) and MAC (Rainbow; PDB ID 6DLW) are part of the immune response.   From 2016 onwards, detailed structures of the MAC pore were being determined by cryo-electron microscopy and deposited in the PDB, bringing new insights into how these complexes function.   The MAC structure shown here is a simplified version of the pore with 22 identical subunits of the most abundant component of human MAC.

Image created with PyMOL, Biorender.com and Adobe Photoshop 

 

Genevieve Evans

The artwork

This artwork for the month of February was created by Katie Wardley from the Leys, in Cambridge, UK.   Katie etched the protein and printed it in black ink on top of a blue Lino print which represents the bodily fluids, such as tears.

 

 

THINGS TO EXPLORE:

 

FURTHER STRUCTURES 

Lysozyme bound with bacterial proteins that bind & inhibit it:

(a) IVY: A NEW FAMILY OF PROTEIN 

PDB ID 1UUZ

(b) Structure of ivy complexed with its target, HEWL

PDB ID 1GPQ

(c) Crystal structure of Escherichia coli PliG in complex with Atlantic salmon g-type lysozyme

PDB ID 4G9S

More structures of the membrane attack complex:

(d) The poly-C9 component of the Complement Membrane Attack Complex 

PDB ID 5FMW

(e) OPEN CONFORMATION OF THE MEMBRANE ATTACK COMPLEX

PDB ID 6H03

(f) Closed conformation of the Membrane Attack Complex

PDB ID 6H04

 

FURTHER READING:

1. On contact lenses & lysozyme:

Biological and Clinical Implications of Lysozyme Deposition... : Optometry and Vision Science

2. On the discovery of the first lysozyme structures:

(International Tables for Crystallography) How the structure of lysozyme was actually determined

3. On lysozyme’s catalytic mechanism

The lysozyme mechanism sorted — after 50 years

Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

4. On lysozyme & the membrane attack complex (MAC) - January 2021 article

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

5. On the membrane attack complex (MAC)

The mystery behind membrane insertion: a review of the complement membrane attack complex