Hsp70: Difference between revisions
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Chaperons require energy do facilitate refolding. The structure within the chaperon in which ATP hydrolysis occurs in the NBD is directly attached to the 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. | Chaperons require energy do facilitate refolding. The structure within the chaperon in which ATP hydrolysis occurs in the NBD is directly attached to the 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 <scene name='81/813405/Scene_alex_4/2'>proline switch</scene> has been discovered in position 147 of human Hsp70 which resides in the NBD/ATPase domain. 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 | A <scene name='81/813405/Scene_alex_4/2'>proline switch</scene> has been discovered in position 147 of human Hsp70 which resides in the NBD/ATPase domain. 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 ideally positioned to direct nucleophilic attack by water and hydrolyze the bound ATP. This process then triggers the SBD to open its pocket which finally allows substrate binding. <ref name="Vogel">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</ref> | ||
An image of the residues in the NBD in contact with the ADP can be viewed <scene name='81/813405/Contact_to_adp/3'>here</scene>. | An image of the residues in the NBD in contact with the ADP can be viewed <scene name='81/813405/Contact_to_adp/3'>here</scene>. | ||
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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 <ref>Young J. C. (2010). Mechanisms of the Hsp70 chaperone system. Biochemistry and cell biology = Biochimie et biologie cellulaire, 88(2), 291-300</ref>. | 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 <ref>Young J. C. (2010). Mechanisms of the Hsp70 chaperone system. Biochemistry and cell biology = Biochimie et biologie cellulaire, 88(2), 291-300</ref>. | ||
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 | 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 carry out its role within the cell. | ||
=='''Coupling of ATPase Activity and the Substrate Binding Domain to fold/refold proteins'''== | =='''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 | 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 from ATP hydrolysis. It should also be noted that Hsp70s require cochaperones to facilitate refolding. These are called Hsp40’s or J proteins. Why these cochaperones are required is also not well understood, but it is known that these cochaperones radically enhance the rate of ATP hydrolysis <ref>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</ref>. | ||
=='''Hsp70 as a Therapeutic target'''== | =='''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. | Although most studies have been done experimentally with non-human modes of testing, such as in mice, 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''' | '''Stroke''' | ||