CHAPERONES

         The primary mechanism for catalysis was brought about by RNA molecule. During evolution, a great transition occurred between the first peptide formed and the modern enzyme. The first peptides formed had no defined structure and performed simple functions like permeability to membrane. The protein-folding problem was taken care of by the chaperones and made the proteins more flexible.

   I.      Definition: Chaperones are proteins that mediate correct assembly or folding of a target protein by causing the target protein to acquire one conformation instead of others.

II.                Function:

A.    Chaperones function by binding to reactive surfaces (hydrophobic side chains) that are exposed during the assembly process and prevent those surfaces from interacting with other regions of the protein to form the wrong folded conformation.                    

B.     Chaperones may also use the energy released by ATP hydrolysis to keep precursor proteins in an unfolded state so that they can be taken up by  the mitochondria, or to enable their passage through other  membrane channels. Chaperones can also participate in the degradation of misfolded proteins.

III.             Chaperone Families

A.    Hsp70 system contains Hsp70 (Dna K in bacteria), Hsp40 (Dna J) and GrpE (GrpE). These are 70,000MW proteins and this system targets newly made proteins, proteins transported through membranes, and denatured proteins (due to stress).  

1.      Properties: Hsp70 chaperones are ubiquitous. They are found in bacteria, eukaryotic cytosol, ER, chloroplasts and mitochondria.  

2.      Hsp70 and a related protein called Hsc70 act on nasent proteins on ribosomes. Similar proteins in the ER, BIP or Grp78 in higher eukaryotes, or Kar in S. cerevisiae, function on proteins as they emerge into the interior of the organelle and pass through the membrane.

3.       When nasent secretory or membrane proteins cross into the ER lumen they are bound by Bip. Bip means heavy chain binding protein and was discovered by the interaction of the heavy chain IgG antibodies in the ER. Bip binds to hydrophobic sections of  unfolded  proteins that have just been translocated into the ER and prevents the denaturation or nonspecific aggregation (misfolding) of the polypeptide. Many newly made proteins bind to Bip as they enter the ER lumen, and Bip also is required for the translocation of secretory proteins into the ER.

                  B    Chaperonins

    This system consists of a large oligometic organization. These proteins form  a structure into which unfolded proteins are inserted. Chaperonins are divided into Group I or Group II based on the presence or absence of a co-chaperonin. This system includes Hsp60 (GroEL) and Hsp10 (GroES). 

1.  Group I chaperonins include Hsp60 protiens (GroEL  in bacteria, RBP in plants) And all consist of  14 identical 60,000MW subunits arranged in two stacked rings of seven subunits   each.  Hsp10 (GroES in bacteria) is called a co- chaperonin and it  contains seven copies of a 10,000 MW protein. GroEl and GroES for a complex into which substrates (proteins) are inserted in order to facilitate their correct folding.

2. Group II chaperonins are found in Archaebacteria and eukaryotes. They include eukaryotic T-complex-related polypeptide (TCP-1). Group II chaperonins have two stacked octameric rings and have a larger central cavity than group one. Group two chaperonins have six to nine different subunits of 55-60kDa and they have a helical protrusion that takes the place of GroES.

References

1. Slovotinek, Anne and Leslie G. Biesecker. Unfolding the role of chaperones and chaperonins in human disease. Trends in Genetics. 17:528-535.

2. Lodish, Harvey et al. Molecular Cell Biology 3rd Edition. 1995 Scientific American Books Inc.

3. Lewin, Benjamin. Genes VII. 2000 Oxford Press Inc.

Localization and function of chaperones in a cell:

Protein Folding

The importance of protein folding has been recognized for many years. Almost a half-century ago, Linus Pauling discovered two quite simple, regular arrangements of amino acids—the a-helix and the b-sheet (see Fundamental Patterns of Protein Structure) - that are found in almost every protein. And in the early 1960s, Christian Anfinsen showed that the proteins actually tie themselves: If proteins become unfolded, they fold back into proper shape of their own accord; no shaper or folder is needed.

Of course, neither Pauling nor Anfinsen nor the committees that awarded them their respective Nobel prizes knew at the time that these discoveries would be so important for understanding Alzheimer's disease or cystic fibrosis. And when Pauling, at least, was doing his breakthrough studies, he could hardly have imagined the enormity of today's biotechnology industry. What scientists did know is that any process that was so fundamental to life as protein folding would have to be of the utmost practical importance.

But research did not stop with Pauling and Anfinsen. Indeed, we now know that Anfinsen's conclusions needed expansion: Sometimes a protein will fold into a wrong shape. And some proteins, aptly named chaperones, keep their target proteins from getting off the right folding path). These two small but important additions to Anfinsen's theory hold the keys to protein folding diseases.

In vivo folding of proteins

Partially folded intermediates at the junction between productive and off-pathway folding: Generalized pathways showing an inclusion body derived from an intermediate on the folding pathway. This illustration shows a speculative intermediate in the formation of an a/b protein in which a helical domain is docking against a sheet. In the inclusion body pathway, the same interaction proceeds between intermediates, resulting in a polymeric aggregate. [Redrawn from FASEB J. 10, 58 (1997)]

But misfolding of proteins may cause the formation of aggregates or abnormalities that finally leads to disease.

Many diseases are associated with misfolding of proteins. For example, Alzheimers, cystic fibrosis, mad cow disease are a few examples of the diseases resulted from misfolding of proteins.

Cystic fibrosis

Cystic fibrosis (CF) is a common and fatal recessive disease, which is caused by dysfunction of a chloride ion channel, termed the CF transmembrane conductance regulator (CFTR). The CF gene was cloned in 1989; subsequently, several mouse models have been created using gene targeting within embryonic stem cells. The current models encompass mice with a complete knockout of CFTR function, with residual CFTR function, and with precise mutations corresponding to those in humans that precipitate CF. Lung disease in human CF is the major cause of death in early adulthood. Recent research has clearly shown that the many, previously mysterious symptoms of this disorder all derive from lack of a protein that regulates the transport of the chloride ion across the cell membrane. More recently scientists have shown that by far the most common mutation underlying cystic fibrosis hinders the dissociation of the transport-regulator protein from one of its chaperones. Thus, the final steps in normal folding cannot occur, and normal amounts of active protein are not produced.

(http://www.faseb.org/opar/protfold/protein.html )

(Courtesy: www.people.virginia.edu/`rjh9u/cfsciam.html )

http://www.people.virginia.edu/~rjh9u/cfsciam.html

Alzheimer’s disease

The body processes amyloid precursor protein into a soluble peptide (small protein) known as Ab; under certain circumstances, Ab then aggregates into long filaments that cannot be cleared by the body's usual scavenger mechanisms. These aggregates then form the b-amyloid, which make up the neuritic plaque in Alzheimer patients. So the consistent association of amyloid precursor protein mutations with early-onset Alzheimer's has finally answered a long-debated question: the deposition of neuritic plaque is part of the pathway leading to the disease, not a late consequence of it.

Exciting recent studies suggest that over-expression of chaperones results in a protective effect in several models of degenerative disease including Huntington's disease, Amyotrophic Lateral Sclerosis and cardiomyopathies. Recent studies at University of Pennsylvania showed in Drosophila that the progression of neurodegenerative disorders like Parkinson’s disease can be blocked using molecular chaperones. How chaperones mediate protection against disease at the molecular and cellular levels, and whether over-expression of chaperones can induce a protective effect in other protein misfolding diseases remain unresolved questions.

                                              Useful websites

1.  Web page that describes the molecuar basis of cystic fibrosis

      http://www.people.virginia.edu/~rjh9u/cfsciam.html

2.  A good web page that describes about the diseases associated with misfolding of specific proteins and also gives suggested readings

                   http://www.faseb.org/opar/protfold/protein.html

3.  This is a faculty web page that describes about the molecular basis of misfolding and occurance of disease

                    http://depts.washington.edu/phcol/fac/muchowski.html

4.   This is a research web page on protein misfolding and disease  

        http://www.astbury.leeds.ac.uk/People/SER.htm

5.     http://unisci.com/stories/20014/1220014.htm

6.    http://www.medicine.uiowa.edu/paulson/AJHG%201999.pdf

7.    http://www-ermm.cbcu.cam.ac.uk/discuss.htm

                This is the discussion group on medical advancements in biology

Journal References

1.    Slavotinek and Biesecken (2001); TIGS 17: 9,528: Unfolding the role of chaperones in human disease

          3.  Heat shock proteins in health and disease: therapeutic targets or therapeutic agents?
              by A. Graham Pockley. Web | PDF/reprint

In vitro Folding of proteins

Quality control in the cell 

Structure of GroEL and GroES

The structure of GroEl (hsp60) and GroES was determined through a series of EM, crystal, and mutagenesis studies. 

■ EM analysis showed three possible shapes

-         Coffee can (GroEL only)

-         Bullet (GroEL with GroES)

-         American football (GroEL capped on both ends by GroES)

The figure above is from the Tulane webpage that is listed below under webpage references. 

GroEL consists of two stacked rings each made of 7 subunits (14 subunits total).  The structure exhibits seven-fold symmetry.

■ Crystal Structure Analysis of GroEL by Hartl in 1994 Revealed Three Domains

Below is a figure 1 from Sigler’s article in Cell (vol: 99, pg757)

Figure A represents the structure of GroEL (on the right) and GroEL with the GroES cap (on the left).  The subunits colored pink represent the apical domain, green for the intermediate domain, and blue for the equatorial domain. 

The structure of the chaperonin GroEL (hsp60) Left, a low resolution view of the 14-mer, from the X-ray crystal structure filtered to 25 A resolution. There are 2 contacts (numbered) between the two back-to-back heptameric rings. Right, a single subunit (60 kDa) shown as an alpha-carbon trace. There are three domains, separated by hinge regions (marked H1 and H2). Bound ATP is shown in space filling form, and the yellow residues are hydrophobic sites of substrate (non-native polypeptide) binding. These residues are also required for GroES (hsp10) binding, in addition to the blue residues. The charged residues in the inter-ring contacts are shown in red and blue. The structure was determined by Braig et al (1994) Nature 371, 578-586; Braig et al (1995) Nature Structural Biology 2, 1083-1094

■ Crystal Structure of GroES

-GroES is 35Ǻ in length with a 10Ǻ central cavity. 

-7 Flexible loop structures (made of aromatic residues) point inward towards the central region of the cavity.

The figure above is a top view of GroES with the 7 flexible loops which mediate binding to GroEL.  This figure was obtained from Tulane’s webpage.

When GroES binds to GroEL the cavity size of GroEL increases.  ATP binding also increases cavity size.  “Windows” in the region of the GroEL cavity where the peptide binds begin to form and increase in size when ATP and GroES bind.  These windows allow for the diffusion of water and ATP.

Above is a figure taken from the Chaperonin webpage (also listed under webpage references).  You can see cavity expansion as well as window structure size increase with the addition of GroES and ATP.

■ Mutagenesis Studies were used to Map the Functional Domains of GroEL

-ATPase assays determined that the equatorial domain was responsible for ATP binding.          

-Polypeptide binding occurs in the apical domain.

-GroES binding occurs in the apical domain. 

-All three domains are required for polypeptide folding.

-The basal domain consists of a hinge that plays a role in subunit oligomerization and protein folding 

Ribbon diagram of the GroEL subunit that is shown hatched in the diagram in Fig. 1a. In the apical domain, residues that have been shown to be involved in binding of unfolded polypeptide are highlighted. They include the hydrophobic residues Tyr-199, Ser-201, Tyr-203, Phe-204, Leu-234, 237, 259, Val-263, 264. The diagram was constructed from the coordinate file pdb1der.ent [10] using Rasmol.

■ Crystal Structure of GroEL with strongly bound peptide (SBP).  See Cell vol. 99 pg757.

Paul Sigler (1999) studied peptide binding to GroEL using phage display, fluorescence anisotropy, and crystallography studies.  Below is figure 3 from his article in Cell. 

The apical domain of GroEL consists of three helices H, I, and J as well as seven β strands.  In figure 3A the strong binding protein is in yellow.  And helices H and I are labeled in red.  As you can see SBP interacts in a cleft between helices H and I.  Those residues of the peptide that have van der Waals contacts with the helices are shown with their side chains. 

In figure 3B hydrogen bonding interactions between the SBP and the helices are shown. 

Figure 3C is a table that shows the specific residues involved in van der Waals and hydrogen bonding interactions.

From his work Sigler was able to conclude that the SBP was stabilized within the apical domain by hydrophobic interactions, van der Waals contacts, and hydrogen bonding. 

Binding of the peptide to the apical domain occurs in an ordered preference (N to C terminal orientation) and results in the formation of a β hairpin turn.  So substrate binding to the apical domain requires flexibility from both the peptide and GroEL.

MECHANISM OF CHAPERONIN-ASSISTED PROTEIN FOLDING

The GroEL/ GroES Reaction Cycle

1.   The GroEL protein participates in an asymmetric complex (the cis configuration) with one heptameric ring of GroES and 14 ADP's (one per GroEL subunit). GroEL binds to a denatured polypeptide in its central cavity and releases all 14 ADP's and the bound GroES molecule (Voet, D. and Voet, J., 1995).
2. GroEL binds to 14 ATP's. The binding of ATP reduces GroEL's affinity for the substrate protein. The interaction between GroEL and the substrate weaken thereby allowing the GroES protein to bind to the opposite face of GroEL (a trans configuration) (Weissman, et al., 1995).
3. All 14 ATP's are hydrolyzed simultaneously thus releasing the bound polypeptide from the GroEL cavity. This release allows the polypeptide, which is probably in the molten globule state, to fold in a protected microenvironment and out of contact with other denatured proteins with which it would otherwise aggregate.
4. If the polypeptide has completely folded into its native conformation, it is completely released from GroEL.

5.  If the polypeptide is only partially folded, it will rebind to the chaperonin, thus perpetuating the cycle until   folding is complete. GroES binds but does not hydrolyze ATP. In addition to facilitating the binding of ATP, GroES coordinates the 14 ATP's simultaneous hydrolysis and prevents the escape of a partially folded polypeptide from the GroEL cavity (Voet, D. and Voet, J., 1995). The cycle is mechanistically coupled so that the reaction is self-perpetuating until protein folding has finished. Eventually, the folded protein will have lost its affinity for GroEL (Weissman, et al., 1995).

 

The Bullet and Football Structures

Binding of the GroES to one side of the GroEL double toroid confers a structural asymmetry to GroEL. This conformation is commonly known as the bullet. This GroEL/GroES complex acts as the substrate-protein acceptor state because its asymmetric nature ensures that one of the two GroEL rings has its peptide binding site available. Nevertheless, there can exist a symmetric complex where GroES is bound to both ends of the GroEL protein. This structure has been termed the football and its function remains unclear. There is a possibility that the football conformation is an intermediate structure during the transition from the trans to the cis complexes. The formation of the football structure ensures that the rebinding of GroES will occur on the same side as the the polypeptide and would account for the observed flip-flopping of GroES between the two GroEL rings (Weissman, et al., 1995).

CHAPERONIN ACTION AND FOLDING PATHWAYS in vivo

Pathway of protein folding in the bacterial cytosol. Nascent polypeptide chains are bound by the molecular chaperones DnaJ and DnaK. A ternary complex is formed, consisting of nascent chain, DnaJ, and DnaK in its high-affinity ADP-state. Binding of GrpE to DnaK results in dissociation of the complex and release of substrate protein into solution. Single or multiple rounds of interaction of the folding protein with the DnaK/DnaJ/GrpE system lead to the native state. A subset of folding intermediates, 5-10% of all newly-synthesized cytosolic proteins, requires further assistance and is transferred to the GroEL/GroES system to complete folding

Conformational Changes in the GroEl Folding Cage

Journal References

1.      Braig K, Otwinowski Z, Hedge R, Boisvert DC, Joachimiak A, et al. 1994.  The crystal structure of the bacterial chaperonin at 2.8Ǻ.  Nature 371:578-86

2.      Buckle A.M., Zahn R., and Fersht A.R. 1997.  A structure model for GroEL-polypeptide recognition.  Proc Natl Acad Sci USA 94:3571-3575.  Online.  http://www.pnas.org/cgi/content/full/94/8/3571

3.      Chen L and Sigler P.B.  1999.  The crystal structure of a GroEL/Peptide Complex:  Plasticity as a Basis for Substrate Diversity.  Cell 99:757-768.  Online.  http://www.cell.com/cgi/content/full/99/7/757/

4.      Fenton W.A., Kashi Y, Furtak K, Horwich A.L.  1994.  Residues in chaperonin GroEL required for polypeptide binding and release.  Nature 371:614-619.

5.      Sigler P.B., Xu Z, Rye H.S., Burston S.G., Fenton W.A., Horwich A.L.  1998.  Structure and function of GroEL-mediated protein folding.  Annu Rev Biochem 67:581-608.  Online.  http://biochem.annualreviews.org/cgi/content/full/67/1/581

6.      Thirumalai D. and Lorimer G.H.  2001.  Chaperonin-Mediated Protein Folding.  Annu Rev Biochem 30:245-269.  Online.  http://biochem.annualreviews.org/cgi/content/full/30/1/245  

7.   Grantcharova, V., Alm, E.J., Baker, D., and Horwich, A.L. 2001. Current Opinion in Structural Biology 11:70–82. © 2001 from Elsevier Science.) Online

Useful Websites

1.      Structure and Dynamics of the Gro EL-Binding GroES Mobile Loop http://www.tulane.edu/~biochem/sam/billboard.htm

2.  Chaperone Group Crystallography Department Birkbeck College

     London  http://www.cryst.bbk.ac.uk/~ubcg16z/chaperone.html

3.     Chaperonin Structure and Conformational Changes http://www.biochemtech.uni-halle.de/PPS2/course/section12/ellis.html#intro

4.     The Chaperonin Home Page: Chaperonin Structure Gallery http://bioc02.uthscsa.edu/~seale/Chap/chap.html  

5.    Movies of the GroEL ATPase cycle

  http://www.cryst.bbk.ac.uk/~ubcg16z/elmovies.html

     6.    Movie for Model for a GroEL-GroES-Mediated Folding Reaction

  http://www.nottingham.ac.uk/biochemcourses/students/GroEL/Animations/Diagrammatic/Splitscreen2.html

Hsp 70 Structure

The hsp70 sequence shows two important domains the ATPase domain (close to the N terminus) and the Peptide binding domain (close to the C terminal).

The diagram above came from the hsp70 Chaperone System Webpage listed below. 

■ ATPase Domain

The ATPase region has a classic fold similar in structure to hexokinase and actin.  Below is a structural representation of the ATPase domain with an ATP molecule bound in green.  This figure also came from the hsp70 Chaperone System webpage

■ Peptide Binding Domain

The peptide-binding domain consists of two 4-stranded anti-parallel β sheets that form a sandwich around the peptide and an alpha helical lid that is closed when ADP is bound and open when ATP is bound.  This lid prevents the dissociation of the peptide from the hsp70 complex. 

The figure above is E. coli dnaK from the Tulane webpage for Biochemistry 601.  DnaK is in yellow and the peptide substrate is in black.  In the top right hand corner you can see how the beta sheets surround the peptide and the helical lid domain closes over the binding pocket.  The bottom half of the figure is a space-filling representation. 

Hsp 70 regulatory pathway

Journal References

Eluguindi S.B., Cwirla S.E., Dower W.J., Lipshutz R.J., Sprang S.R., Sambrook J.F., and Gething M.J.  1993.  Affinity panning of a library of peptides displayed on bacteriophages reveals binding specificity of BiP.  Cell 75:717-728.

Full text not online.

Rüdiger S., Germoth L., Scheider-Mergener J., Bukau B.  1997.  Substrate Specificity of the dnak chaperone determined by screening cellulose-bound peptide libraries.  EMBO 16:1501-1507.  Online.  http://emboj.oupjournals.org/cgi/content/full/66/7/1501

Zhu X., Zhao X., Burkholder W., Gragerov A., Ogata C.M., Gottesman M.E., Hendrickson W.A.  1996.  Structural analysis of sub-binding by the molecular chaperone dnak.  Science June 1996 Vol. 272

Useful Websites

Biochemistry 601 at Tulane

http://www.tulane.edu/~biochem/med/hsp.htm

The Hsp70 Chaperone System

http://pps98.cryst.bbk.ac.uk/assignment/projects/mak/main.htm