Wza is one of 6 proteins that make up a complex of group 1 capsular proteins.

This complex functions to in both the synthesis and export of polysaccharides in gram-negative bacteria, in this case the E-Coli capsule serotype K30. Wza is the outer membrane auxiliary protein of this complex, and is completely contained in the cell wall. Wza's first domain rests in the peptidoglycan layer, the 2nd and third domains are in the periplasm, and the 4th domain is in the outer membrane.

This is the Octamer structure of Wza

The 8 monomers of Wza come together to form the 4 ring structure.

This view shows how the rings come together with ring 1 (made of D1) is colored blue, ring 2 is colored purple, ring 3 is colored orange, ring 4 is colored yellow, the N-terminal is colored green and the residues not listed as one of the domains are colored red.

This

shows the structure of one monomer of Wza, it is made up of 4 domains D1 is colored blue, D2 colored purple, D3 colored orange, D4 colored yellow, and the N terminus colored green. Notice how the N terminus is wrapped around ring 3 (colored orange) and is buried inside the protein. The C terminus (at the end of D4) is exposed to the cell surface.

Domain 1 contains both an anti-parallel beta-sandwich and an alpha-helix. The alpha-helix is on the surface of the protein. When the 8 copies of domain 1 come together they form ring 1 which creates a concaved surface at the base of Wza. In the center of the concaved region are 8 loops, one from each subunit; at the end of each loop is a Tyr 110.

Domain 2 is similar to domain 1 however there are a few differences. The 5 stranded beta-sheet is in the center and the alpha-helices are on the surface. When the 8 copies of domain 2 come together they form the ring known as ring 2.

Domain 3 is in fact a exact structural duplicate of domain 2, the 8 copies of this domain come together to form ring 3.

Domain 4 contains the C-terminus of the monomer it is completely helical. The helixes are not strait; they have a helical distortion on the third helix turn from the bottom. This domain is the only part of the protein that goes through the outer membrane.

The authors think this protein defines a new class of proteins. Until this protein was resolved it was thought that the all helical barrel transmembrane proteins that went through the outer membrane were beta-barrels. However resolving the structure of Wza suggests there might be many other proteins with outer transmembrane alpha-helical barrels.

The polysaccharide export sequence (PES)has traditional been thought of as conserved and includes residues between 82 and 195. It contains all of D1 and some of D2.

However because of the new structure the researchers suggest that PES domain, be defined as residues between 89 and 169, basically domain 1. this link shows you the space filled model of Wza.

if you rotate Wza so you are looking down the beta neck you can see the how the neck is large and there is only a very small hole in the bottom of Wza.

The internal cavity of Wza constantly changes width

The Polar residues (colored Purple)

are most concentrated on the outside of Wza below the 4th domain. The hydrophobic (colored Cyan) residues are most concentrated on the outside of the 4th domain and on the inside of the 1st, 2nd and 3rd domains. There are several exceptions such as several pockets of polar residues on the inside of the 3rd domain. There are few amphipathic residues (colored gray), these are most concentrated around the outside of the neck and the inside of domain 1.

The most interesting features of Wza’s polarity are the charged residues. At the bottom of Wza

there is a ring of negatively charged residues, this and the concaved surface is thought to be the site where Wza interacts with Wzc. These interactions might trigger a conformational change.

There is another interesting feature of Wza’s charged residues, there is a high concentration of positively charged amino acids around the top of the neck, and another high concentration of negatively charged amino acids on the inside of the neck right also near the top of the neck.

Finally it is proposed by the authors that Wza uses water to help export the polysaccharides from the cell.

It is thought that water is allowed into Wza when the polysaccharides are inside, the water molecules hydrogen bond to the polysaccharides and avoid the polysaccharides getting stuck in the protein. This also gives the protein an advantage since, allowing water to “plug the gaps” in the polysaccharides, allows the protein to be more flexible and bind to different types of polysaccharides. This theory is supported by the fact that both E-coli and Klebsiella pneumoniae have almost identical Wza proteins but export chemically distinct polysaccharides.

More interesting information includes how Wza protein is held together:

The N-terminal is held wrapped around the out side of Wza by salt bridges.

The Arg 33 (Green) is forming a salt bridge to Tyr 44 (Orange) and Asp 47 (Blue) is forming a salt bridge with Lys 34 (Magenta), both help to stabilize the Wza structure.

Also by hydrogen bonds that stabilize Wza, these include H-bonding between Tyr 341 (Cyan) and Gln 27 (Purple),

The first ring is stabilized by hydrogen bonds between Asp 99 (Red) and Tyr 130 (Light Green), Thr 116 (Yellow) and Arg 111 (Pink), Arg 111 and Arg 111 on an adjacent monomer.

The second ring is stabilized by Van der Waal’s interactions between Ile 190 (Brown) and Lys 172 (Blue), MSE 230 (Green) and Thr 176 (Purple), MSE 230 and Phe 248 (Yellow), and two hydrogen bonds (Asp 196 (Light Green) and Tyr 174 (Black), Asn 199 (Orange) and Thr 176 (Purple)).

The third ring is stabilized with salt-bridges (Asp 272 (Brown) and Lys 256 (Blue), Glu 280 (Green) and Lys 256 (Blue)), hydrogen bonds (Ile 288 (Yellow) and Gln 266 (Purple), Arg 273 (Light Green) and Asp 253(Black)), and a van der Waal’s contact between Ala 323 (Pink) and Tyr 341 (Orange).

The forth ring is stabilized with possible hydrogen bonds between Hist 365 (Green) and Glu 369 (Yellow) in adjacent transmembrane alpha-helices.