Molecular Playground/DnaK

From Proteopedia

The E. coli Hsp70, DnaK 2kho

Drag the structure with the mouse to rotate

One of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground.

Molecular Playground banner: DnaK, a central hub in maintaining proteostasis in E. coli

The E. coli Hsp70, DnaK, is a protein chaperone whose function is to bind exposed hydrohpobic residues of unfolded proteins. This binding event prevents aggregation and rescues the nascent chain from kinetic traps along the folding pathway. Hsp70 protein chaperones switch between an , low-substrate affinity form and an , high substrate affinity form during their allosteric cycle.1 Hsp70 protein chaperones are ubiquitously found in almost all known organisms and cell types and represent a potential target for anti-cancer and neurodegenerative therapies.2

1 Structure
2 Function
3 Drug Target
4 3D structures of Hsp70
5 See Also
6 References

Structure

DnaK is a 638 residue protein of approximately 70 kDa. The protein can be thought to be made up of two domains, the N-terminal nucleotide-binding domain () (residues 1-388), the C-terminal substrate-binding domain () (residues 393-638), which are divided by the (residues 389-392, shown in violet). The NBD is further divided into four subdomains: (residues 1-37, 112-184, 363-383, shown in red), (residues 38-111, shown in green), (residues 185-227, 310-362, shown in gold), and (residues 228-309, shown in purple). The SBD of DnaK consists of a (residues 393-507), an (residues 508-605), and a disordered C-terminal tail (not shown due to lack of available crystal structure).3

Function

DnaK binds stretches (7-8 residues in length) of exposed hydrophobic residues of its client proteins, in the beta-basket of the SBD, in order to prevent their aggregation.4 Upon ATP binding, the NBD subdomains rotate relative to each other and induce a conformational change in the NBD (compare the NBD to the form in Figure 1.5

Figure 1. A superimposition of the ATP-bound and ADP-bound NBDs. The ATP-bound form is shown in lighter colors.

When ATP binds the NBD, the interdomain linker communicates the allosteric signal to the SBD that induces a conformational change in the SBD, causing the alpha-helical lid to dock onto the NBD.6 Compare the form of the SBD (when the NBD binds ATP) and the form of the SBD (when the NBD binds ADP) in Figure 2.

Figure 2. A superimposition of the 'open' and 'closed' forms of the SBD. The 'open' form is shown in lighter colors.

The alpha-helical lid of the SBD rotates 180 degrees during the closing motion.7 The result is that the same residues that dock with the NBD are responsible for binding the nascent chain of the client protein. The SBD has low substrate affinity in the 'open' conformation, but high substrate affinity in the 'closed' conformation.8 Upon ATP hydrolysis, the NBD reverts to the ADP-bound state, undocking the alpha-helical lid from the NBD and regenerating the 'closed' conformation of the SBD. Alternating cycles of binding and release allow DnaK to unfold kinetically trapped intermediates and allow the client protein to refold to its native state.9 Recently, a third 'allosterically active' state of DnaK has been discovered. In this state, the two domains remain undocked, with the SBD retaining high substrate affinity, but the interdomain linker is bound to the NBD. This state occurs when both ATP and substrate are bound simultaneously and has not yet been crystallized.10

Drug Target

Since Hsp70s are critically positioned in the proteostasis systems of many organisms, including humans, they represent a tempting target for anti-cancer and neurodegenerative disease therapies. Recent efforts to develop a competitive inhibitor for Hsp70s, using DnaK as a model system, have met with only marginal success.11 Recently, interactions between Hsp70s and its co-chaperones have been targeted for drug development, but with no leads currently in clinical trials.12