Heat Shock Protein 70 the Molecular Chaperone

Wesley Kamau

Dr. Leslie Bruce

Eng 363-01

7 May 2020
 Heat shock protein 70 (Hsp70) are proteins that respond to environmental stresses to

assist in protein folding, protein refolding and prevent protein aggregation. Maintaining proper

protein structure is important for all living organisms as cells rely on proteins to shuttle material

in and out, hold the cell together, and perform critical reactions for making energy and

replication (making a copy of itself). The elements responsible for these proteins are called

molecular chaperones. Having a system that monitors and responds to any changes in protein

performance will assure that an organism is able to survive and reproduce. In all eukaryotic and

prokaryotic organisms Hsp70 is the system used making it highly conserved (gene sequences

across species are similar) (Heat Shock Protein, 70). In both these organisms it is found in the

cytosol (inside the cell) where it has efficient access to newly synthesized proteins and area with

high protein density.

Activation

Hsp70 unique role in assisting proteins to gain back their functional conformational

identity makes it a member of the molecular chaperone family. Its name emerged from high

expression rates when exposing cell to heat shock treatments (Bascos and Landry, 2019). Their

binding is triggered when cellular stresses such as cancer, UV light, heat shock, and etc are

introduced causing proteins to be denatured, misfolded or begin to aggregate (Boshoff et al.

2004). Exposure to high stressors will facilitate a cellular response of many heat shock proteins

which are classified according to their observed molecular weight in kilodaltons (kDa) (Bascos

and Landry, 2019). Proteins under stress will lose secondary and tertiary structure (partially

unfold) or aggregate activating Hsp70 to bind the problematic protein as seen in Figure 1. Once

this protein is bound to Hsp70 it can refold in correct orientation to have cellular function.

Recently Hsp70 has been found to be a helping partner in systems of protein unfolding, complex
dissociation (protein-protein/protein-DNA/protein-RNA interactions), and membrane

translocation (moving from cytosol to organelles) (Bascos and Landry, 2019).

Structure

Hsp70 is comprised of three key functional sites called domains represented in Figure 2

the ATPase domain for nucleotide binding, substrate binding domain for binding of target

protein, and C-terminal domain (Boshoff et al., 2004). Connection of the domains together is

done by interdomain linker (Bascos and Landry, 2019). The sequences of amino acids in these

domains and interdomain linker are important for protein-protein interaction to bind problematic
protein. Resolution back to proper protein is fast and efficient for cellular processes to continue

which

can only be done by a mechanism that binds tightly to problematic proteins then loose for proper

protein to unbind.

The ATPase domain is where the nucleotide adenosine triphosphate (ATP) or adenosine

diphosphate (ADP) will bind to Hsp70 to control how tight or loose the substrate will be bound.

The ATPase domain site is comprised of two identical subdomains that will bind ADP or ATP to

maintain substrate affinity levels (tightness of binding) (Boshoff et al., 2004). Maintaining

binding stability of ADP or ATP is critical because when ADP is bound to Hsp70 then substrate

binding affinity is high and when ATP is bound to Hsp70 then substrate binding affinity is low

(Boshoff et al., 2004). ATP and ADP are similar molecules with a difference of an additional

phosphate molecule on ATP. Cellular functions that use these molecules convert between the

two by adding energy (ADP to ATP) or removing energy (ATP to ADP). For the conversion

between ATP and ADP binding to Hsp70 ATP hydrolysis takes place, which is the addition of a

water molecule to ATP to convert it into ADP and an inorganic phosphate.

Presence of interdomain linkage (amino acids between domains) has resulted in not only

functioning as a domain connector but a regulatory region that is target protein dependent based

on unique properties, sequences of amino acids (Bascos and Landry, 2019). Interdomain linkage

role in target protein binding mechanism is a new discovery as these amino acids were

previously thought to be only structural for Hsp70.

The substrate binding domain for target proteins has a clamping mechanism comprised of

two subdomains that are nonidentical allowing for target protein to be bound tightly and with
proper stability (Bascos and Landry, 2019). The benefits of a clamping mechanism are the

protection of hydrophobic regions (not attracted to water) from cytosol and increased efficiency

of refolding as it allows for a greater amount of refolding time. The substrate binding domain has

an open conformation when ATP or ADP is bound to the ATPase domain and will only bind

target protein during this time (Bascos and Landry, 2019). In this study changing the substrate

binding domain conformation to a closed state requires the binding of the target protein. In the

closed state the target protein is given time to refold by Hsp70 and Hsp70 is not able to bind to

other proteins as substrate binding domain is occupied. Once the protein is folded properly then

releasing occurs. Then open conformation of the substrate binding domain is gained for another

problematic protein to bind. Hsp70 has increased function (more target proteins are bound) and

expression (more amount of Hsp70 in cytosol) when cellular stress levels are high the ADP

bound state is mandated as it has higher affinity for target protein at the substrate binding

domain.

The C-terminal domain is the least understood and characterized as it is functionally

known as a lid for the substrate binding domain.

Cofactors

Hsp70 does not function independently when binding nucleotides (ATP or ADP) or target

protein. With the help of Hsp40 (J proteins) and nucleotide exchange factor (NEFs) the Hsp70

system is complete for efficient binding and release of target proteins and nucleotides.

Regulation of Hsp70 domains by these two cofactors helps to maintain function and binding

stability. These two groups are called co-chaperone as their role is to regulate the chaperone

activity of Hsp70 (Caplan, 2003).

 Hsp40s otherwise know as the J proteins are named because of the J domain they all have

which is conserved in all living organisms. Their abilities include to bind to target proteins to

facilitate their movement towards Hsp70’s substrate binding domain and hydrolysis of ATP

(Bascos and Landry, 2019). A problematic protein is freely swimming in the cytosol then Hsp40

binds to it first then carries it to Hsp70’s substrate binding domain. A complex comprising of a

target protein bound to Hsp70 and Hsp40 is formed. J proteins hydrolysis of ATP at the ATPase

domain results in Hsp70 affinity for substrates to change because ATPase activity is changed

(Kampinga and Craig, 2010).

Nucleotide exchange factors (NEFs) role is to release target proteins from the substrate

binding domain by binding to the ATPase domain. Binding to this domain changes the

conformation of the substrate binding domain from tight to loose facilitating the release of the

properly founded protein. When NEFs bind to the ATPase domain, they are involved in the

reaction that alternates ADP and ATP bound to Hsp70 (Kabani and Martineau, 2008). The

conformation changes with either J proteins or NEFs binding to Hsp70 are represented in Figure

3.
 References

Bascos, N. A. D., & Landry, S. J. (2019). A History of Molecular Chaperone Structures in the

Protein Data Bank. International Journal of Molecular Sciences, 20(24), 6195. doi:

10.3390/ijms20246195

Boshoff, Aileen & Nicoll, W.S. & Hennessy, Fritha & Ludewig, M.H. & Daniel, S. & Modisakeng,

K.W. & Shonhai, Addmore & McNamara, Caryn & Bradley, Graeme & Blatch, Gregory. (2004).

Molecular chaperones in biology, medicine and protein biotechnology. South African J Sci. 100.

Caplan, A. J. (2003). What is a co-chaperone? Cell Stress & Chaperones, 8(2), 105. doi:

10.1379/1466-1268(2003)008<0105:wiac>2.0.co;2

Heat Shock Protein 70. (n.d.). Retrieved from

https://www.sciencedirect.com/topics/neuroscience/heat-shock-protein-70

Kabani, M., & Martineau, C. (2008). Multiple Hsp70 Isoforms in the Eukaryotic Cytosol: Mere

Redundancy or Functional Specificity? Current Genomics, 9(5), 338–348. doi:

10.2174/138920208785133280

Kampinga, H. H., & Craig, E. A. (2010). The HSP70 chaperone machinery: J proteins as drivers of

functional specificity. Nature Reviews Molecular Cell Biology, 11(8), 579–592. doi:

10.1038/nrm2941