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Contents 1 Structural highlights 2 Background 3 Structure 4 Catalytic site 5 Diseases

Structural highlights

NBD (nucleotide binding domain) is composed of 4 subdomains (IA, IB, IIA, IIB)= ATP binding pocket.

Chain:	GTX
Length:	388 acides aminés
Weight:	44 Da
Source organism:	Homo Sapiens
Expression system:	E.Coli
Interpro:	Heat shock protein 70
Ligands:	4 (ADP, CA, NA, PO4)
Gene:	HSPA1A, HSPA1, HSPA1B (human)

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 a 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 on this two proteins on their sequence suggests that the mechanism of ATP hydrolysis is universal among all HSP70 proteins in eukaryotes. Besides, 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 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 arangement may potentially detect a shift in the relative position of beta and gamma phosphate of ATP during hydrolysis and suggets as 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 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