1. 5. Beta Domains By Betul Akcesme
STRUCTURAL BIOLOGY
5. Beta Domains
By Betul Akcesme

2. Functionally, this group is the most diverse;
Beta Structures
Antiparallel β-structures comprise second large group of protein domain structures.
Functionally, this group is the most diverse;
enzymes,
transport proteins,
antibodies,
cell surface proteins,
virus coat proteins.
4 beta strands (with β-loop-β structure), there are 24 ways.
more beta strands, the more theoretical arrangements.
BUT!
There are only a few observed structures.

3. Common properties Built up from 4 to over 10 beta strands
b strands are arranged in predominantly antiparallel fashion
The β strands usually form 2 twisted β sheets that are joined together and packed against each other, resembling barrel or distorted barrel (=double b sandwich)
4 different β Domains:
Up-and-down barrel
Greek Keys
Jelly roll barrels
Beta helix domains ( quite new fold)
Surface formed by residues from the loops and from the beta strands
a core of hydrophobic side chains

4. 1) Up-and-down barrels have a simple topology
The simplest topology
successive β strand is added adjacent to
the previous strand until the last strand is
joined by hydrogen bonds to the first
strand and the barrel is closed.
is similar to that in the α/β-barrel structures,
EXCEPT
the strands are antiparallel and all the connections are hairpins ( not alpha helix)
adjacent beta strands in the amino
acid sequence are also adjacent in the 3D structure

5. 1st example of up and down barrel: The retinol-binding protein (RBP)
Closed end
Function:
transporting the lipid alcohol vitamin A (retinol) from its storage site in the liver to vitamin-A dependent tissues
A 4-turn α
RBP is degraded after transporting one retinol
In plasma, RBP-retinol complex
is bound and stabilized by
The complex is recognized by cell-surface receptor Causing RBP to release retinol; and RBP will be degraded
Structure:
The eight anti-parallel β strands twist and curl
Two β sheets packed against each other.
(green and blue, with red participating both).
Open end
RETINOL TAIL

6. A retinol molecule is bound inside the barrel, between the two β sheets, such that its only hydrophilic part (an OH tail) is at the surface of the molecule. The binding site is lined with hydrophobic residues, which provide a hydrophobic surrounding for the hydrophobic part of the retinol molecule. OH

7. RBP: amino acid sequence reflects β structure
A large part (blue) of RBP beta strands are exposed to solvent. This is amazingly achieved by alternating hydrophobic and hydrophilic residues in those beta strands.
Strands 1,2,3,4,5,6 form one sheet; strands 1,8,7,6,5 form another sheet; 1,5,6 are shared.
The amino acid sequence of strands of RBP clearly illustrate this arrangement.

8. Membrane spanning β-structures
Porin channels are made by up and down β- barrels. Sixteen β strands form an antiparallel β barrel that traverses the membrane. The loops at the top of the picture are extracellular, the short turns at the bottom face the periplasm.

9. A 2nd example of up-and-down b barrel: neuraminidase
Influenza virus –
1. an RNA virus with an outer lipid envelope;
2. two viral proteins anchored in the membrane:
neuraminidase & hemagglutinin (both transmembrane)
Inside membrane region few residues;
transmembrane region;
Outside membrane: a stalk and a head piece
antigenic determinants
Neuraminidase enzymes are glycoside hydrolase enzymes (EC  ) that cleave the glycosidic linkages of neuraminic acids. Neuraminidase enzymes are a large family, found in a range of organisms. The best-known neuraminidase is the viral neuraminidase, a drug target for the prevention of the spread of influenza infection
 The viral neuraminidases are frequently used as antigenic determinants found on the surface of the Influenza virus. Some variants of the influenza neuraminidase confer more virulence to the virus than others. Other homologs are found in mammalian cells, which have a range of functions. 
Role of hemagglutinin , will be mentioned.
Role of neuraminidase
facilitate the release of progeny virions from infected cells by cleaving sialic acid residues from the carbohydrate side chains of the viral hemagglutinin and of the glycosylated cellular membrane proteins

12. Neuraminidase Homotetramer
Here, up-and-down β strands form six small sheets, instead of a barrel.
Each sheet has 4 strands.
The 6 sheets arranged like the blades of a 6-bladed propeller.
Loop regions between the β strands form the active site in the middle of one side of propeller.
These are on top and will form binding site
Yellow loop connect

13. Active sites
The 12 loops
6 connecting 6 propeller,
6 within each blade connecting 2nd and 3rd strands, form the active site.
This is similar to the active site in the α/β barrel active site (on top of the barrels formed by loops on top)

14. http://home. cc. umanit oba
oba.ca/~hultin/chem2 220/Support/CoolStuff /Influenza/Neuramini dase.htm
uare.org/flash/Tamiflu .html#Start

15. 2) Greek key motif
Only (a) is observed in proteins
Notice that this is not up-and-down barrel since the red strands connects from n to n+3. They are not connected by hairpin loops. Red and green form a Greek key motif

16. γ-crystallin as an example for greek key
Transparency and refractive power of the lenses of our eyes
Smootly changing concentration gradient of Lens specific proteins-crystallins

17. 3) The Jelly Role The top right picture is jelly roll motif
Hydrogen bonding
The top right picture is jelly roll motif
Then imagine you have this antiparallel beta strand, interrupted by loops.
Roll according to the jelly roll motif around the barrel, you get a jelly roll barrel.
This is generalized Greek key

18. Hemagglutinin of Influenza
Like neuraminidase, hemagglutinin is another protein anchored in influenza virus lipid envelope. (E.g. the virus caused the “Asian flu”)
Role of hemagglutinin (glycosylated)
mediate virus binding to host cells by recognizing and binding to sialic acid residues on glycoproteins of the cell membrane
The polypeptide chain is proteolytically cleaved to yield 2 chains of 328 (HA1) and 221 (HA2) a.a. residues;
HA1 and HA2 are held together by –S-S- bonds.
Three such subunits together form the whole protein

19. One subunit of hemagglutinin
HA1: the first 63 residues extends 100A long, then (residues ) at the top, it forms an 8-stranded distorted jelly roll barrel. The remaining 70 residues return to the stem region, running nearly antiparallel to the initial stretch of 63 residues.
HA2: a hairpin loop of two alpha helices packed together. The second alpha helix is 50 residue long and 76A long, goes towards the membrane.
At the bottom, there is a beta sheet of 5 antiparallel strands, with one strand from HA1. The last 20 residues (called fusion peptide) at the amino end of HA2 are associated with the activity by which the virus penetrates the host cell membrane to initiate infection.

21. The hemagglutinin trimer
To start infection, hemagglutinin binds to sialic acid residues of glycosylated receptor proteins in the target cell surface.
The receptor sites formed by the jelly roll domain.
Receptor site
Globular Head
The virus gets into cell by endocytosis (plasma membrane folds inward to take substances into the cell)
Antibodies in our immune system bind to this receptor binding site to prevent the virus entering our cells
But the virus can mutate the receptor to escape this binding. This receptor is ideal drug target.
membrane

22. The binding site of hemagglutinin
The binding site is located at the tip of the subunit within the jelly roll structure.
Beta strand 1 contains a long insertion, causing a bulge in strand 8 at the corresponding place.
Yellow ball is binding site.
Alpha helix between beta strand 3 and 4 form one side of receptor binding site

23. Space filling model of the sialiac acid binding domain of hemagglutinin with a bound inhibitor (red)
Hydrophobic part of the sialic acid binds in a hydrophobic channel from central groove to the bottom of the domain.

24. hemagglutinin to change conformation due to low pH
A 2nd function of Hemagglutinin in the infection of host cells: aid in endosome and viral membrane fusion
First function: binding to sialic acid residue
It binds to the plasma membrane via the receptor, and is taken into the cells by endocytosis.
vesicles containing bound viruses causes an accumulation of protons and a consequent lowering of the pH value inside the vesicles
hemagglutinin to change conformation due to low pH
Induce the fusion of the viral envelope membrane with the membrane of the endosome.
THIS EXPELS THE VIRAL RNA INTO THE CYTOPLASM, WHERE IT REPLICATES.

26. Structural change at lower pH value
Hemagglutinin undergoes a massive conformational change at lower pH value (<6).
The most obvious: HA2 straightens to form a long helix of 100A.

27. The large conformational change is not reversible.
At lower pH value:
B becomes part of helix of 100A long, it also straightens up.
An alpha-helix region between C and D becomes loop.
The beta-hairpin E-F, and helix G all are in different places.
The large conformational change is not reversible.
The low pH form is actually more thermo-stable
Disulfide bond
Between HA1 and HA2.

28. Hemagglutinin in Action
Hemagglutinin is a deadly molecular machine that targets and attacks cells. This occurs in several steps. First, the three binding sites at the top of the spike bind to sugars on cellular proteins, shown in green at the top left (PDB entry 1hge ). Then, the whole virus is carried inside the cell into the endosome and the cell adds acid, which normally digests the stuff inside the endosome. But in the case of the virus, the acidic environment serves to arm the attack mechanism. In acid, hemagglutinin unfolds and then refolds into an entirely different shape. The portions shown in orange and red are normally folded against the protein, but in acid, they pop out and point upward, as shown in the center illustration (PDB entries 1htm , 1ibn  and 2vir ). The red portion, termed the fusion peptide, has a strong affinity for membranes, so it inserts into the cell membrane and locks the virus to the cell. Then, as shown on the left (PDB entry 1qu1 ), the yellow portions zip up the side of the protein, pulling the two membranes close together. Finally, the new conformation of hemagglutinin somehow causes the two membranes to fuse--that part is still not well understood--and the viral RNA flows into the cell, starting the process of infection.

29. http://cbm.msoe.edu/teachRes/palm/influenza. html
ations/content/influenza.html

30. 4) The parallel β-helix asp not antiparallel
Two sheet β-helix: each turn, contains 2 beta strands and 2 loop regions. Repeated 3 times in extracellular bacterial proteinases, to form a right-handed coiled structure: two beta sheets and hydrophobic core in between.
Each unit has 18 residues: 6 in each loop and 3 in each beta strand. Formed by repeat of 9 residue pattern:
Gly-Gly-X-Gly-X-Asp-X-U-X
where X is any a.a., U is large & hydrophobic, often leucine.
First 6 are in loop, and later 3 are in beta strand. U from both strands are packed inside. (remember X’s face the other side)
The loops are stabilized by having Asp binding calcium (Ca).
This 9 a.a. pattern can be used to search for such patterns.
asp

31. Comparison of all those b-barrels
Up-and-down
Greek key (g-crystallin-like)
jelly-roll

32. Conclusion The barrels act as container for diverse ligands
Diversity is due to differences in the size of the barrel and in the amino acids that participate in formation of common core.

33. End of the Lecture