GroEL and GroESGroEL and GroES

By: Jim, Alan, JohnBy: Jim, Alan, John

BackgroundBackground Proteins fold spontaneously to Proteins fold spontaneously to

their native states, based on their native states, based on information in their amino acid information in their amino acid sequencesequence

Sometimes proteins fail to fold Sometimes proteins fail to fold and need helpand need help

Cells have developed Cells have developed molecules that catalyse protein molecules that catalyse protein folding called folding called chaperoneschaperones..

Molecular chaperones Molecular chaperones supervise the state of newly supervise the state of newly formed proteins, hold them to formed proteins, hold them to the proper pathway of folding, the proper pathway of folding, and keep them from improper and keep them from improper influences that might lead to influences that might lead to incorrect assembly.incorrect assembly.

BackgroundBackground

““Chaperones act Chaperones act catalytically to speed catalytically to speed up the process of up the process of protein folding by protein folding by lowering the lowering the activation barrier activation barrier between misfolded between misfolded and native states” and native states” (Lesk 297)(Lesk 297)

Chaperones Chaperones themselves contain themselves contain no information about no information about particular folding particular folding patterns, rather they patterns, rather they anneal misfolded anneal misfolded proteins and allow proteins and allow them to find their them to find their native state.native state.

GroEL-GroESGroEL-GroES GroEL-GroES contains 2 products of the GroE operonGroEL-GroES contains 2 products of the GroE operon

GroEL—L for largeGroEL—L for large GroES—S for smallGroES—S for small

The active complex contains 14 copies of GroEL and 7 The active complex contains 14 copies of GroEL and 7 copies of GroEScopies of GroES

In the presence of a nucleotide, GroEL and GroES form a In the presence of a nucleotide, GroEL and GroES form a symmetrical GroESsymmetrical GroES77-GroEL-GroEL1414-GroES-GroES7 7 complexcomplex

GroEL StructureGroEL Structure

14 GroEL form 2 seven-fold 14 GroEL form 2 seven-fold rings, packed back to backrings, packed back to back

Each ring surrounds an open Each ring surrounds an open cavity to receive unfolded cavity to receive unfolded proteinsproteins

The cavity is closed at the The cavity is closed at the bottom (with a wall between bottom (with a wall between the two rings) so the protein the two rings) so the protein cannot pass internally between cannot pass internally between cavitiescavities

The 2 rings communicate with The 2 rings communicate with allosteric structural changesallosteric structural changes

GroES StructureGroES Structure

GroES subunits form another 7-membered ring GroES subunits form another 7-membered ring that caps one of the GroEL ringsthat caps one of the GroEL rings

The GroEL ring capped by GroES is called the The GroEL ring capped by GroES is called the cis cis ringring and the non-capped ring is the and the non-capped ring is the trans ringtrans ring

GroEL-GroES ComplexGroEL-GroES Complex ““Formation of the GroEL-Formation of the GroEL-

GroES complex requires a GroES complex requires a large and remarkable large and remarkable conformational change in the conformational change in the cis GroEL ring, changing the cis GroEL ring, changing the interior surface of the cavity interior surface of the cavity from hydrophobic to from hydrophobic to hydrophilic, and breaking the hydrophilic, and breaking the symmetry between the two symmetry between the two GroEL rings.GroEL rings.

ATP is bound and broken ATP is bound and broken down to ADP+P for the down to ADP+P for the energy to make this changeenergy to make this change

GroEL-GroES ComplexGroEL-GroES Complex The enclosed cavity is the site The enclosed cavity is the site

of protein foldingof protein folding Misfolded proteins in the cavity Misfolded proteins in the cavity

are given about 20 seconds to are given about 20 seconds to refold. After 20 seconds, they refold. After 20 seconds, they are expelled either refolded are expelled either refolded successfully, or given another successfully, or given another chance to enter the same or a chance to enter the same or a different chaperon complex to different chaperon complex to try againtry again

A misfolded protein will be kept A misfolded protein will be kept in “solitary confinement” until it in “solitary confinement” until it has reformed correctly.has reformed correctly.

Swinging or Hinging MotionSwinging or Hinging Motion In order to do it’s work, In order to do it’s work,

GroEL undergoes GroEL undergoes conformation changes to its conformation changes to its domains: twisting and domains: twisting and bending along multiple axes bending along multiple axes

The apical domain “swings” The apical domain “swings” about 60º in a hinge motion, about 60º in a hinge motion, hiding hydrophobic residues hiding hydrophobic residues inside the cavity. inside the cavity.

A second hinge in the A second hinge in the equatorial domain swings to equatorial domain swings to lock in ATP and expose a lock in ATP and expose a critical residue for hydrolysis.critical residue for hydrolysis.

Rotational MotionRotational Motion The most important motion of GroEL is The most important motion of GroEL is

the rotational motion of the apical the rotational motion of the apical domain in order to expose a different domain in order to expose a different bounded surface to the inner GroEL bounded surface to the inner GroEL tube.tube.

In its unbound form, the residues that In its unbound form, the residues that line the interior of the GroEL tube are line the interior of the GroEL tube are hydrophobic, or not willing to combine hydrophobic, or not willing to combine with water molecules. In the bounded with water molecules. In the bounded form, they readily accept water form, they readily accept water molecules.molecules.

The hydrophobic residues that formed The hydrophobic residues that formed the lining become part of the inter-the lining become part of the inter-subunit contacts, while the hydrophilic subunit contacts, while the hydrophilic residue surfaces are exposed.residue surfaces are exposed.

Why is the Apical Domain Rotation Why is the Apical Domain Rotation Important?Important?

As stated previously, GroEL attempts to As stated previously, GroEL attempts to allow misfolded proteins to refold allow misfolded proteins to refold themselves. themselves.

““The characteristic of misfolded proteins, The characteristic of misfolded proteins, that renders them subject to non-specific that renders them subject to non-specific aggregation, is the surface exposure of aggregation, is the surface exposure of hydrophobic residues that are buried in the hydrophobic residues that are buried in the native state.” (Lesk, 299) native state.” (Lesk, 299)

No, Really, Why is that Rotation So No, Really, Why is that Rotation So Great?Great?

Proteins with this negative property bind to the Proteins with this negative property bind to the open form of GroEL. (Recall that in the open, open form of GroEL. (Recall that in the open, unbounded form, GroEL’s cavity is also unbounded form, GroEL’s cavity is also hydrophobic.) Then once the misfolded protein is hydrophobic.) Then once the misfolded protein is within GroEL’s inner tube, GroEL’s inner residues within GroEL’s inner tube, GroEL’s inner residues become hydrophilic (having water affinity), become hydrophilic (having water affinity), coaxing the protein to refold itself correctly. coaxing the protein to refold itself correctly.

The GroEL-GroESThe GroEL-GroESOperational CycleOperational Cycle

•The operational cycle of the complex GroEL-GroES can best be described through individual points.

•When capped by GroES, the GroEL rings have two different states. The first is open, where the ring is available for the reception of misfolded proteins. The second is closed, and actually contains a misfolded protein.

When seen separate from GroES, the GroEL ring is in an open state, allowing for the entry of proteins. The inside of the Gro-EL ring has a flexible hydrophobic lining. This allows for the binding of misfolded proteins, through hydrophobic and van der Waals interactions. Throughout these processes, however, it is possible for the protein to become unfolded partially, when in an incorrect state.

With the binding of ATP and GroES, the cap (GroES) is ready for the ring (GroEL), however, there must first be a conformational change in the cis ring. The ending result is a closed cavity where the “substrate protein” can refold, once it is released from the apical domains. It must also be taken away from potential aggregation partners.

When the cis ring undergoes the conformational change, it more than doubles the available volume of the cavity. This allows for larger unfolding/refolding transition states to exist.

The inside of the GroEL cavity also changes from hydrophobic to hydrophilic. This peels the bound misfolded protein off of the surface and unfolds it even further.

“The burial of the original interior GroEL surface in intersubunit contacts within the GroEL-GroES complex itself breaks the binding of the protein to the original hydrophobic internal surface, leaving a macroclathrate complex.” This new complex (cavity) is composed of a crystal lattice, housing the protein in the cavity.

Finally, hydrolysis of ATP in the cis ring allows for the weakening of the structure. Binding of the ATP in the trans ring causes the disassembly of the cis assembly, allowing for the release of the GroES and substrate protein. In the end, the ring is left in original state.

It needs to be noted that through the process, there is a necessity of seven or fourteen ATP molecules. In the end, the cost is much larger than the energy required to unfold a protein. However, it needs to be stated that it this cost is infinitely smaller than the death of a cell, and is also quite smaller than the synthesis of the polypeptide chain.

GroEL-GroESGroEL-GroES

““The interaction The interaction between GroEL and between GroEL and GroES is necessary GroES is necessary

for certain proteins to for certain proteins to fold under otherwise fold under otherwise

non-permissive non-permissive conditions” (Lorimer conditions” (Lorimer

720).720).