GrpE

GrpE (Gro-P like protein E) is a bacterial nucleotide exchange factor that is important for regulation of protein folding machinery, as well as the heat shock response. It is a heat-inducible protein and during stress it prevents unfolded proteins from accumulating in the cytoplasm. Accumulation of unfolded proteins in the cytoplasm can lead to cell death.

Discovery GrpE is a nucleotide exchange factor that was discovered by researchers in 1977 as a protein necessary to propagate bacteriophage λ, a virus that infects bacteria by hijacking the bacteria's replication machinery, in Escherichia coli. By using a genetic screen, researchers knocked out certain genes in E. coli and then tested whether the bacteria were able to replicate, GrpE was found to be crucial to propagation. Since that time, GrpE has been identified in all bacteria and in Archaea where DnaK and DnaJ are present. The crystal structure of GrpE was determined in 1997 at 2.8 Angstrom and identified GrpE as a homodimer that binds DnaK, a heat-shock protein involved in de novo protein folding. GrpE's structure determination was important because it demonstrated the interaction of nucleotide exchange factors at the nucleotide-binding domain of DnaK.

Structure

Functional domains The GrpE homodimer has three distinct domains:

N-terminal disordered regions — Amino acids 1-33 in the N-terminal domain can compete for binding to the substrate binding cleft of DnaK. Amino acids 34-39 have not been visualized because they are either too disordered or too unstructured to be crystallized. α-helices — There are four α-helices, two short and two long, these are stalk-like and parallel to each other. These helices come together to form a helical bundle however, there is no superhelical twisting due to the heptad-hendecad (7-11-7-11) spacing of hydrophobic residues in these helices. Portions of this helical bundle are able to bind to Domain IIB of DnaK. These helices also act as thermosensors. C-terminal β-sheets — There are two compact β-sheets which stick out from the helices like arms. The β-sheet proximal to DnaK interacts with its ATP binding cleft directly by inserting itself into the cleft and causing a conformational shift in Domain IIB causing the release of ADP. The distal β-sheet does not interact with DnaK.

Binding induces a conformational change Binding of GrpE's proximal β-sheet to Domain IIB of DnaK causes a 14° outward rotation of the nucleotide binding cleft, disrupting the binding of three side chains to the adenine and ribose rings of the nucleotide. This conformational change shifts DnaK from a closed to an open conformation and allows the release of ADP from the binding cleft.

Function

Nucleotide exchange factor Nucleotide exchange factors are proteins that catalyze the release of adenosine diphosphate (ADP) to facilitate binding of adenosine triphosphate (ATP). ATP has three phosphate groups and the removal of one of the phosphate groups releases energy which is used to fuel a reaction. This removal of a phosphate group reduces ATP to ADP. GrpE is a nucleotide exchange factor that causes the release of bound ADP from DnaK, a heat shock protein important in de novo protein folding. DnaK, in its open conformation, binds ATP with low affinity and has a fast exchange rate for unfolded proteins. Once DnaJ, a co-chaperone, brings an unfolded protein to DnaK ATP is hydrolyzed to ADP to facilitate folding of the protein. At this point, the DnaK•ADP complex is in a stable conformation and requires GrpE to bind DnaK, change its conformation, and release ADP from the N-terminal ATPase domain of DnaK. Once ADP is released from the cycle is able to continue.

Kinetics The interaction between GrpE and the nucleotide binding cleft of DnaK is strong with a Kd between 1 nM (assessed during active conformation using transient kinetics) and a Kd of 30 nM (based on inactive conformation through surface plasmon resonance). This low dissociation constant indicates that GrpE readily binds to DnaK. Binding of GrpE to DnaK•ADP greatly reduces the affinity of ADP for DnaK by 200-fold and accelerates the rate of nucleotide release by 5000-fold. This process facilitates the de novo folding of unfolded protein by DnaK.

Protein Folding GrpE also has an important role in substrate release from DnaK. The disordered N-terminal region of GrpE competes for binding to DnaK's substrate binding cleft. Researchers mutated GrpE to identify the function of its structural domains. Mutated GrpE, without its disordered N-terminal domain, is still able to bind to DnaK's nucleotide binding cleft and induce a conformational change however, the substrate will not be released.

Thermosensor GrpE is a nucleotide exchange factor for DnaK, a heat shock protein, its activity is downregulated with increasing temperature. In biology, reversible unfolding of α-helices begins at 35 °C with a midpoint Tm of 50 °C, this unfolding affects the structural integrity of GrpE and prevents binding of GrpE to the nucleotide binding cleft of DnaK This has an important physiological role to limit the substrate cycling and subsequent ATP expenditure during heat stress. The thermal regulation of DnaK slows protein folding and prevents unfolded proteins from accumulating in the cytoplasm at high temperatures.

Bacteriophage λ replication GrpE was first identified for its role in phage λ replication. GrpE that has been mutated so that it is nonfunctional prevents phage λ replication in vivo and greatly decreases replication in vitro. In vitro overexpression of DnaK can recover phage λ replication without GrpE. GrpE's pivotal role in phage λ replication is at the origin of replication, after assembly of DnaB and other replication factors, GrpE facilitates bidirectional DNA unwinding through interaction with DnaK.

Regulation

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