Hsp70

Overview

Chaperon proteins are important to almost all organisms. Their function is to assist in the folding of newly translated proteins unable to fold on their own and even refold proteins that have become nonfunctional due to some type of misfolding. Misfolding can be caused by several different types of stressors such as high temperature, starvation, inflammation, water deprivation, or nitrogen deficiency. Heat shock proteins, primarily the Hsp70 family, partially bind to the protein’s exposed hydrophobic surfaces, to promote protein refolding and prevent interactions that might lead to aggregation ^[1].

There are even forms of Hsp70 that are specialized for protein transport into mitochondria and chloroplasts. The proteins transferred into these organelles are not in their mature states yet. Hsp70’s actually bind to the newly translated polypeptides keeping them unfolded until they are in the organelle. Once they are in the designated organelle, their interiors contain specialized Hsp70’s (mitochondrial or chloroplast Hsp70) that fold the polypeptides into a stable, functional state ^[2].

Hsp70 function is critical to homeostasis. Protein aggregation is not typically beneficial to most organisms. For example, protein aggregation in different areas of the brain can lead to certain neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Hsp70’s promotion of refolding is key to make the misfolded protein functional once again. The many members of the Hsp70 family are highly conserved ^[3]. This reiterates the fact that these proteins are vital to most organisms. When something is widely expressed and highly conserved it tells us it has been beneficial to cells and organisms for a very long time and there was no need for change.

Structure

The uncovering structure of the various proteins in the Hsp70 family is still underway, but for the most part the is known. The 70 in its name refers to its molecular mass. All members of the Hsp70 family have an N-terminal nucleotide binding domain (NBD)(~40 kDa) and a C-terminal (~25 kDa) connected by a short linker. The NBD consists of two subdomains, I and II, which are further divided into regions a and b. The Ia and IIa subdomains interact with ATP through a nucleotide-binding cassette related to those of hexokinase, actin and glycerol kinase. The SBD consists of a 10-kD α-helix subdomain and a 15-kDa β-sandwich. Crystal structures suggest that substrate peptides are bound in an extended conformation between loops of the β-sandwich and that the α-helix subdomain acts as a “lid.” Numerous functions of this protein rely on the communication between the ATPase domain activity within the NBD and the SBD ^[3]. This makes sense because the energy rendered from ATP hydrolysis in the ATPase Domain can be used by the substrate binding domain to perform its function. This is allosterically controlled by ATP binding.

Domain Interaction

When bound to ADP both the NBD and SBD domains are independent of one another. The allosteric model further explains that the ADP bound state is characterized by “noncommunication” between the domains ^[4]. For proper communication between the two they must contact each other in the ATP bound state of the protein. Upon the binding of ATP, the two NBD lobes rotate against one another by approximately 25° to expose binding sites for the interdomain linker and SBDβ ^[5]. This process is also known as the ATPase Cycle and it controls substrate binding. The ATP-bound state has low affinity for substrates and fast substrate exchange with the substrate binding pocket open, whereas the ADP-bound state has high substrate affinity and slow substrate exchange rates with the substrate binding pocket closed ^[6]. This is explained further in the Mechanism of Substrate Binding Domain and ATPase section of this article.

Mechanism of Substrate Binding Domain and ATPase when ATP Binds (Allostery)

Chaperons require energy do to their job. The structure within the chaperon in which ATP hydrolysis occurs in the Nucleotide Binding Domain (NBD) is directly attached to the Substrate Binding Domain (SBD). This is a favorable set up as the energy produced by ATP hydrolysis can be directly coupled with a change in shape of the substrate binding domain that allows for substrate folding/refolding. The interaction between the protein’s function and ATP binding as well as peptide binding is known to be allosteric, or the binding of a molecule to the protein regulates, or transmits a signal, to another area of the protein either enhancing or inhibiting function in that area/domain.

A has been discovered in position 147 of human Hsp70. A study done to understand this mechanism used E. coli’s DnaK which is a homolog of human Hsp70. Proline 143 is the corresponding residue in DnaK for Proline 147 in the Human ATPase domain. This proline is universally conserved and undertakes alternate conformations in response to ATP binding and hydrolysis. This proline is directly involved in catalytic residue positioning by facilitating the contact between Lys70 (Lys71 in humans)… and/or Glu171 (Glu175 in humans). Changing the Proline to an Alanine or Glycine residue affected Lysine 70’s positioning in the catalytic domain. Furthermore, lack of an extra amide hydrogen in proline, as opposed to Alanine and Glycine’s extra hydrogen on their amide group, seems to be beneficial for interaction with the Glutamine 171 residue. Both findings show how critical the proline residue is for catalytic domain function because without it the rate of ATP hydrolysis is greatly reduced. The Lys70 and Glu171 are positioned ideally for nucleophilic attack by water to hydrolyze the bound ATP. This process then sends a signal to the SBD to open its pocket which finally allows substrate binding. ^[7]

An image of the residues in the NBD contact with the ADP can be viewed .

ATP is not the only means of allosteric control on this enzyme. When a polypeptide binds to the SBD it actually decreases the stabilization between the SBD and NBD domains. The SBD change caused by polypeptide binding is transmitted to the NBD which increases the rate of ATP Hydrolysis in that domain ^[8].

The overall picture is this: In the ATP-bound state, the SBD pocket is open and ready for the substrate, a polypeptide, to bind. The binding of a polypeptide makes the ATP-bound state less stable and favor the ADP-bound state promoting hydrolysis of the ATP. This provides energy for the folding of the bound polypeptide. Once the energy is used up and the protein is folded, Hsp70 binds a new ATP. Because of the newly bound ATP, the chaperon will have less affinity for the substrate and release the newly folded protein so it can once again fulfill its role within the cell.

Coupling of ATPase Activity and the Substrate Binding Domain to fold/refold proteins

Although we know ATP is critical for conformational changes within the protein, so it may bind and release substrates, it is still not clear as to how exactly Hsp70 uses the free energy gained by ATP hydrolysis. It should also be noted that Hsp70s require cochaperones to do their job. These are called Hsp40’s or J proteins. Why these cochaperones are required is also not well understood, but we do know that these cochaperones radically enhance the rate of ATP hydrolysis ^[9].

Hsp70 as a Therapeutic target

Although most studies have been done experimentally with non-human modes of testing, in other words not necessarily useful in a clinical setting, the results shown in the two examples below provide promising evidence that the mechanism of this protein could possibly be modeled and recreated, or further research of the protein could allow it to actually be used clinically.

Stroke

A stroke is a very common, and often debilitating disease, that occurs in the brain typically caused by a narrowing of the vessels or blood clot in a vessel of the brain that cuts off blood supply to the area the vessel lies within. Without blood there is no oxygen supply to the brain which is known as ischemia. This causes immense damage to that area of the brain and often other parts of the body that part of the brain is responsible for controlling. During studies on cerebral ischemia, the regions of the brain found to be fairly resistant to ischemia induced Hsp70. Other studies have also displayed that Hsp70 can protect brain cells during experimental ischemia, models of neurodegenerative disease, and other forms of brain disorders. This is typically successful by inducing overexpression of the protein. During homeostasis Hsp70 is less likely to be expressed, however, following injury its expression is increased. This is why it can also be used as a biomarker to located cells under stress. The overexpression of these proteins in cells under ischemic stress indeed improved overall neuron and astrocyte survival ^[10].

Parkinson's Disease

Parkinson’s Disease is characterized by continuing loss of dopaminergic neurons in the substantia nigra pars compacta, with subsequent dopamine decline in the nigrostriatal pathway, and by intracytoplasmic fibrillar protein aggregates (Lewy Bodies, LB) in the remaining nigral neurons. Hsp70 overexpression demonstrated reduced α-Syn accumulation and toxicity in both mouse and Drosophila Parkinson’s Disease Models ^[11].

References

1. ↑ Sharma, D., & Masison, D. (2009). Hsp70 Structure, Function, Regulation and Influence on Yeast Prions. Protein & Peptide Letters, 16(6), 571-581. doi:10.2174/092986609788490230
2. ↑ Plopper, G. (2016). Cytosolic Proteins Targeted to the Mitochondria or Chloroplasts Contain an N-Terminal Signal Sequence. In Principles of Cell Biology (2nd ed.). Burlington, MA: Jones and Bartlett Learning
3. ↑ ^{3.0 3.1} Evans, C. G., Chang, L., & Gestwicki, J. E. (2010). Heat Shock Protein 70 (Hsp70) as an Emerging Drug Target. Journal of Medicinal Chemistry, 53(12), 4585-4602. doi:10.1021/jm100054f
4. ↑ Bertelsen, E. B., Chang, L., Gestwicki, J. E., & Zuiderweg, E. R. (2009). Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proceedings of the National Academy of Sciences of the United States of America, 106(21), 8471-6
5. ↑ Liu, Q., & Hendrickson, W. A. (2007). Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1. Cell, 131(1), 106-20
6. ↑ Bukau, B., & Horwich, A. L. (1998). The Hsp70 and Hsp60 Chaperone Machines. Cell, 92(3), 351-366. doi:10.1016/s0092-8674(00)80928-9
7. ↑ Vogel, M., Bukau, B., & Mayer, M. P. (2006). Allosteric Regulation of Hsp70 Chaperones by a Proline Switch. Molecular Cell, 21(3), 359-367. doi:10.1016/j.molcel.2005.12.017
8. ↑ Young J. C. (2010). Mechanisms of the Hsp70 chaperone system. Biochemistry and cell biology = Biochimie et biologie cellulaire, 88(2), 291-300
9. ↑ Xu H. (2018). Cochaperones enable Hsp70 to use ATP energy to stabilize native proteins out of the folding equilibrium. Scientific reports, 8(1), 13213. doi:10.1038/s41598-018-31641-w
10. ↑ Kim, J. Y., Han, Y., Lee, J. E., & Yenari, M. A. (2018). The 70-kDa heat shock protein (Hsp70) as a therapeutic target for stroke. Expert opinion on therapeutic targets, 22(3), 191-199
11. ↑ Turturici, G., Sconzo, G., & Geraci, F. (2011). Hsp70 and Its Molecular Role in Nervous System Diseases. Biochemistry Research International, 2011, 1-18. doi:10.1155/2011/618127