Opening image: transthyretin dimer. The two subunits are identical. The functional form of transthyretin is a homotetramer.
Outline
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- Subunit Interface
Introduction
None of the previous tutorials on quaternary structure discuss protein symmetry or protein misfolding diseases. I intend to change that fact with an in-depth discussion of the secreted protein, transthyretin, along with a brief look at superoxide dismutase. The former is a very prominent plasma protein (20-40 mg/dL) with a lifetime in the plasma of only 1-2 days, while the latter is a homodimer expressed in all cells but is not secreted. Both proteins are associated with fatal neurological disorders.
Transthyretin is a homotetramer synthesized in the liver and secreted into the plasma. This protein has special binding and transport functions that will be considered in the next tutorial, which also deals briefly with two protein misfolding diseases, transthyretin amyloidosis and Lou Gehrig's disease.
By studying transthyretin you can gain a deeper understanding of the following key concepts of protein-protein interactions:
- A globular protein's 4° structure
- Rotational symmetry
- Identical, complementary contact surfaces in homomeres
- Paired, symmetrical interactions
Symmetry in Proteins
One of the delights in life is the discovery and rediscovery of symmetry in nature – the symmetry in the petals of a daisy, in the wings of a butterfly, or in a snowflake. Although symmetry in multimeric proteins may be unfamiliar to many people, protein symmetry is also beautiful.
Many multimeric proteins are symmetric when around an axis. Such proteins are said to have cyclic or rotational symmetry. A protein with cyclic symmetry, designated Cn, has n identical units related by a single n-fold rotational axis. As the multimeric protein is rotated 360/n degrees, the view of the protein remains the same. For instance, rotation of a homotrimer with C3 symmetry by 360/3, or 120°, around its axis of symmetry brings the second out of three identical subunits into view. In molecular animations, we often color the subunits . But structurally and functionally, they are the same. This is clear when we use a and around the axis of symmetry. Dihedral symmetry and spiral symmetry, two other kinds of symmetry found in proteins, are not covered here.
Symmetry in Proteins
C2 Symmetry of the Transthyretin Dimer
Transthyretin (TTR) has four identical subunits, labeled A through D. The tetramer is a dimer of two identical dimers, AB and CD. Each dimer has C2 symmetry – a 180° rotation of one subunit about the rotation axis will bring it into coincidence with the other. Stated another way, the two subunits are related by a single axis of rotation known as a twofold rotation axis, hence the designation C2 symmetry.
down the Y axis, i.e., position the C2 axis is perpendicular to the screen.
Proteins with cyclic symmetry have a special property that relates the atoms of one subunit to their counterparts in the other subunits. This means that, for example, the Cα atoms of lysine residues K-80A and K-80B in the TTR dimer are related in space by the twofold axis of symmetry.
of the Cα atoms of K-80A and K-80B from the center of rotation. by 180° around the twofold rotation axis simply interchanges the positions of the atoms in space.
Return to the model. The subunits are now colored differently.
Symmetry in Proteins
C11 Symmetry of TRAP
Now that you know how cyclic symmetry looks, you can recognize other cyclic symmetries, C3, C4, and so on. Here's a mind-boggling example of C11 symmetry.
TRAP (Trp RNA-binding attenuation protein) from Bacillus subtilis. [Note: Several lysine side chains on the surface of the protein point in different directions from that expected by symmetry. That's because of charge-charge interactions with carboxyl groups of neighboring protein molecules in the crystal lattice.]
Each TRAP subunit binds a . All 11 tryptophan molecules are related by reflection across the 11-fold axis of rotation. How many degrees of rotation around the axis are required to move the model to a new position that is coincident with the original position?
Subunit Interface
Buried Water Molecules
We return to a spacefilling model of the transthyretin dimer. Note the tight fit between the two subunits. The contact surfaces are complementary. This precise fitting of nonpolar and polar groups at the dimer interface is why the two subunits stick to each other and not to other proteins.
What is puzzling at first glance is how an odd number of water molecules can be bound inside the dimer yet still be related by a twofold axis. The answer, of course, is that one of the waters must be at the center. The way that the protein binds the five waters is a stunning example of symmetry and molecular complementarity.
Examine the model from different angles. See any red atoms? [Note: At most resolutions the positions of the hydrogen atoms of immobilized water molecules cannot be determined, i.e., only the oxygen atoms (red) of water molecules can be seen.]
view of dimer interface. Note that dimer formation entraps five water molecules at the interface.
Before we look at the noncovalent interactions binding the waters, let's see how the five are related to the twofold axis of rotation.
water. Water #3 (W3) lies exactly on the rotation axis which lies in the plane of the screen and is oriented vertically. 180° around the axis of symmetry
Subunit Interface
Buried Water Molecules
Exploded view of dimer interface, with one subunit removed. Each bound water molecule is to one or more hydrogen bond donors or acceptors from chain A. [Note: the position of the hydrogens of water could not be located in the model.]
W1 is hydrogen-bonded to the imidazole ring of His-88A.
W3 is hydrogen-bonded to the –OH group of Tyr-116A and the carbonyl oxygen of Glu-92A.
However, we have not accounted for all the hydrogen bonds to the five waters. For instance, since chain B was omitted, we cannot see the hydrogen bond between His-88B and W5. The next button takes us in for a closer view of the five waters and some -- but not all -- of their hydrogen bond partners.
Subunit Interface
Buried Water Molecules
Since the twofold axis passes through W3, W1 is related to W5 by reflection through the axis of rotation. That means that W5 is hydrogen-bonded to His-88B.
Likewise, W2 is directly opposite W4. All the interactions are paired. W1 is hydrogen-bonded to His-88A and W5 is hydrogen-bonded to His-88B, while W3 is hydrogen-bonded to both Tyr-116A and Tyr-116B. Together, His-88, Glu-92 and Tyr-116 account for six hydrogen-bonds to the trapped water molecules.
Have we accounted for all the hydrogen-bonds to the five waters? The answer is no. In particular, the hydrogen-bonds to W2 and W4 are not illustrated here. To understand the entire network of hydrogen-bonds we must first explore the secondary structure in the next section to find the missing hydrogen bonds.
Interactions between the edges of protein β-sheets occur widely in the formation of protein quaternary structures and in protein-protein interactions. In this mode of molecular recognition between proteins, hydrogen bonds form between the edges of two β-sheets, which stabilizes the partnership in conjunction with many other noncovalent forces (hydrophobic, van der Waals, salt bridges).
Subunit Interface
Alignment of β-sheets
Cartoon model of the transthyretin monomer. The structural scaffold is a β-sandwich.
In the monomer the peptide groups along the outside edge of each β-sheet in the sandwich are exposed and hydrogen bonded to water molecules in the solvent. However, the exposed edge of the β-sandwich is buried when the homodimer is formed. This requires that the peptide groups of the edge β-strand find new hydrogen bond partners. How the transthyretin dimer finds an elegant solution to this problem is explained below.
residues that are not part of the β-sandwich. is the C-terminus.
Model of the dimer looking along the twofold axis. Observe how the two three-stranded β-sheets are aligned. Is it possible that they can fit together to form a continuous β-sheet?
Let's take a closer look. As expected, the two innermost β-strands are joined by intermolecular hydrogen bonds to form an antiparallel β-sheet. Thr-123 is the C-terminal residue of each subunit. spacefill.
Return to the complete dimer. The interchain hydrogen bonds create a continuous six-stranded β-sheet across the dimer. There are other noncovalent interactions stabilizing the dimer.
spacefilling model. Van der Waals forces and hydrophobic interactions are indeed important, as indicated by the close fit between the side chains where the edge strands of the continuous β-sheet meet.
Subunit Interface
Role of Water at the Interface
Cartoon model, except that we've cut away everything except the edge β-strands at the interface. The two β-strands from each chain are colored blue and purple, respectively.
Two β strands are part of the intermolecular sheet.
The other two edge β-strands at the interface are too far apart to hydrogen bond to each other. Instead, both sheets are bonded to water molecules, which are effectively trapped between, forming a connection between the β-sheets of the homodimers.
the buried water molecules. Three water molecules are trapped between the two β-strands comprising the lower layer of each β sandwich.
Blow-up. Three water molecules form a hydrogen-bond network between the two β strands. Water molecule W3 lies on the twofold axis, where it can form two hydrogen bonds to the carbonyl oxygen atoms of the two β-strands. Water molecules W2 and W4 -- and their hydrogen bond partners -- are related by the twofold axis of rotation.
Altogether, W2, W3, and W4 form a hydrogen-bonded network of 12 hydrogen bonds at the subunit interface.
W3 is hydrogen-bonded to the hydroxyl group of Tyr-116 and the carbonyl oxygen of Glu-92. Because of the C2 symmetry, there are four hydrogen bonds to W3.
W2 and W4 are also hydrogen bonded to the carbonyl oxygen of His-90.
Those backbone carbonyls and amide groups not involved in the hydrogen bond network shown here are either buried (and therefore hydrogen bonded to various groups inside the monomer) or else they are still exposed in the dimer (and therefore still hydrogen bonded to water molecules in the solvent).
Spacefilling model, except for cartoon showing the two β-sheets that entrap W2, W3, and W4.
the β-sheets. Except for the water bridge, the two edge β-strands that form a trap for W2, W3, and W4 do not interact with each other.