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Structural highlights

Background

The 1S3X is a 44kDa domain of the NDB (nucleotide binding domain) of the 70kDa heat shock protein (Hsp70). It is composed of 388 amino acids. This protein, encoded by HSPA1, is composed of two important domains that are linked with its function : the N-terminal ATPasse domain that is 45kDa (NDB) and the C-terminal polypeptide-binding domain (SBD). HSP70 is a huge molecular chaperone in eukaryotes. The N-terminal domain plays a role in the protein synthesis, folding, translocation, degradation and modulation of protein expression. This protein is ATPasique, its activity is linked with ATP hydrolysis and phosphate release. That is why 1S3X represents a key in this process. Moreover, the structure of this chaperone is similar to the Hsc70, the homologue ATPase of bovine. The similarity of these two proteins on their sequence suggests that the mechanism of ATP hydrolysis is universal among all HSP70 proteins in eukaryotes. Moreover, the C-terminal domain ensures the communication between the two domains which is really important for its activity.

Structure

The 1S3X domain takes part of the binding of ADP, ADP+Pi, and ATP in the full protein. Its structure allows the communication between the active site and the binding site. The conformational changing is linked to the protein's folding. Thr 13, Thr 14 and Asp 366 are three polar residues that transmit the information from the active site to the peptide-binding domain. Thr 13 and 14 interacts directly with the inorganic phosphate released by ATP hydrolysis. The C terminal alpha helix crosses over this beta strand and contracts the beta phosphatte of ADP through the Asp 366 residue.Two amino acids taking part of the beta strand of the 1S3X domain, Ala 2 and Lys 3, can control the peptide domain by extend itself away from the ATPase surface. Such an arrangement may potentially detect a shift in the relative position of the beta and gamma phosphate of ATP during hydrolysis and suggests a possible way of transmitting information. Moreover, hydrophobic side chains located on the surface of the 1S3X domain, interact closely with the ATPase domain.

Catalytic site

ADP is bound in the cleft between two sub-domains of ATPase and is located within the protein's body except for the edge of adenine that is solvent exposed in vitro. The adenine base is caught between the hydrophobic segment of two arginine residues (Arg 272 and 342. The arginine guanidinium group stabilizes the solvent molecules in vitro, which are linked by hydrogen bonds. In the ADP binding site of the ATPase, the ribose hydrogen bonds to Asp 268 and Lys 271 and the phophates project into the 1S3X domain which contains metal ions and a number of well ordered water molecules. This cavity contains one calcium and two sodium ions.The sodium ions may are responsible of the rotation of the beta phosphate.The inorganic phosphate group is coordinated by a salt bridge with Lys71, an hydrogen bonds from Thr13 to Thr204 and then it interacts immediately with the calcium ion. The exit of the Pi creates a channel potential and involves a conformational changing transition of the Hsp70 molecular chaperone.

Two calcium sites have been identified in the crystal structure of the 1S3X domain. The first calcium site binds within the catalytic pocket and bridges ADP and inorganic phosphate. It is octahedrally coordinated by the oxygen of 𝞫 phosphate , two oxygen atoms of Pi and four water molecules in vitro. Moreover, thanks to the presence of this calcium ion, the activated 𝝲 phosphate can be transferred to a conserved threonine (Thr204). This structurally conserved residue is suggested as a phosphate acceptor.

The second calcium is tightly coordinated on the hATPase protein surface by Glu231, Asp232 and carbonyl of His227. This new metal-binding motif is formed at the junction between a β sheet (residues 190–225) and ɑ helix (230–250) and is in close proximity to the catalytic site. Moreover, the amide groups of 3 residues (202, 203 and 204) point out toward phosphate groups (Pi and 𝞫 phosphate) of ADP. All three amide groups are in position to form a hydrogen bond with a nested water molecule (in vitro) that bridges the 𝞫 phosphate of ADP with the Pi group.

The calcium-bound structure of hATPase represents a state in which phosphorylation can occur. Protein sidechains in the catalytic site, in particular threonine sidechains, can serve as an acceptor of the phosphate group during ATP hydrolysis. Moreover, small but important movements of ions and sidechains have been observed. Potentially, phosphorylation in the presence of calcium may serve as a regulatory function, because at high calcium concentrations a fraction of the Hsp70 chaperone molecules could become phosphorylated and thereby arrested in one state or inactive.

Diseases

Heat shock proteins (HSPs) have the ability to fold proteins and reconstitute them once they have deformed. It is thus a key element in the cure of cancer and some neurological diseases. Indeed, HSP70 functions as a chaperone and protects neurons from protein aggregation and toxicity (Parkinson's disease, Alzheime disease ...), protects cells from apoptosis (Parkinson's disease), is a marker stress (epilepsy), protects the cells from inflammation (brain injury), plays an adjuvant role in the presentation of the antigen and participates in the immune response in autoimmune diseases (multiple sclerosis) However, it has been shown that an overabundance of HSP70 may have a deleterious role in some diseases. Indeed, HSP70 can cause continuous protein folding in cancer cells, which exacerbates cell growth. These proteins are also involved in viral diseases such as Zika virus (ZIKV). ZIKV has been associated with serious symptoms, including infant microcephaly and Guillain-Barré syndrome. HSP70 has been shown to be an infection factor for several viruses. In the ZIKV infection process, inducing and suppressing the expression of HSP70 has been shown to increase and decrease the production of ZIKV, respectively. HSP70 thus appears to be an important factor in the ZIKV infection process. To conclude, if we can not survive without Hsp70, its activity must still be balanced. When we know more about the structure and activity of Hsp70, we may be able to design molecules, drugs, to modulate its activity to prevent or slow the growth of certain diseases.