User:Bostonalumn/GrpE

GrpE (Gro-P like protein E) is a bacterial nucleotide exchange factor that is important for regulation of protein folding machinery. 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 first discovered by researchers in 1977 as a protein necessary to propagate bacteriophage λ in Escherichia coli. Bacteriophage λ is a virus that infects bacteria by highjacking the bacteria's own replication machinery. GrpE assists phage λ replication through its role as a nucleotide exchange factor and significantly accelerating the exchange of nucleotides. By using a mutant screen, researchers knocked out certain genes in E''. coli'' and then tested whether the bacteria was able to replicate, GrpE was found to be crucial to propagation. Since this time GrpE has been identified in all bacteria and in archea 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. GrpeE's structure determination was important because it demonstrated the interaction of nucleotide exchange factors at the nucleotide binding domain of DnaK.

Functional domains
The GrpE homodimer has three distinct domains:


 * N-terminal disordered regions — 33 amino acids 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 — four α-helices, two short and two long, 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-sheet s — one β-sheet from each protein stick out from the helices like arms. The β-sheets are compact, the one 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.

Bacteriophage λ Replication
The discovery of GrpE demonstrated its critical role in phage λ replication. Mutations of GrpE, so that it is no longer functional, 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.

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 DnaJ•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
DnaK functions to fold proteins in the cytoplasm, it does so by binding the substrate, and using energy in the form of ATP hydrolysis, folds it. After ATP hydroylsis the DnaK•ADP complex relies on GrpE to induce a conformational change and release the nucleotide so that the cycle can continue. 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. Mutated GrpE, without its disordered N-terminal domain, is still able to bind to DnaK•ADP and induce a conformational change in the nucleotide binding cleft but the substrate will not be released.

Thermosensor
GrpE is a nucleotide exchange factor for DnaK, a heat shock protein, as a result GrpE is temperature sensitive and its activity is downregulated by 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 DnaK•ADP. 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.

Transcription
In the archeas' genome, the gene for GrpE is located upstream of the gene for DnaK which, is upstream of the gene for DnaJ. Out of these three proteins, only the promoter region of GrpE has a complete TATA binding box and upstream heat-responsive binding site. This suggests that, in Archea, these three genes are transcribed at the same time.

In E. coli, GrpE's transcription is regulated by binding of the heat-shock specific subunit of RNA polymerase, σ32. Under physiological conditions, σ32 is kept at low levels through inactivation by interacting with DnaK and DnaJ, then subsequent degradation by proteases. However, during heat shock these proteins are unable to interact with σ32 and target it for degradation. Therefore, during heat shock, σ32 binds to the promoter region of heat shock proteins and causes rapid induction of these genes.

Eukaryote Homologues
In Saccharomyces cerevisiae, the GrpE homologue, Mge1, is found in mitochondria. Mge1 is a nucleotide exchange factor important for shuttling proteins across mitochondrial membranes and in protein folding, it interacts with a yeast homologue of DnaK. Mge1 has a similar role as a thermosensor. Yeast have additional GrpE homologues including Sil1p and Fes1p. In humans, mitochondrial organelles have GrpE-like 1 (GRPEL1) protein.

In eukaryotic cells, there any many additional eukaryotic GrpE homologues. Members of the BAG family specifically, BAG1 are the main nucleotide exchange factors for heat shock protein 70kDa (Hsp70), which is the eukaryotic equivalent of DnaK. Other nucleotide exchange factors that interact with heat-shock proteins in eukaryotes include, Sse1p, Sil1p, Hip, and HspBP1. These eukaryotic nucleotide exchange factors are all heat-shock inducible meaning that they serve a similar function as GrpE, to protect the cell from unfolded protein aggregation. These nucleotide exchange factors always interact with subdomain IIB of the nucleotide binding cleft of their respective heat-shock proteins. The binding of the nucleotide exchange factor to a nucleotide binding cleft and the shift to an "open" conformation is conserved from prokaryotes to eukaryotes.

Plant Homologues
In plants, GrpE homologues, CGE1 and CGE2, are found in chloroplasts. CGE1 has two splice isoforms that differ in 6 amino acids in the N-terminal, with isoform CGE1b being 6 nucleotides longer than CGE1a. This N-terminal domain is important in substrate release through competitive binding to the heat-shock protein. All of these plant nucleotide exchange factors interact directly with the cpHsc70, the plant homologue of DnaK. They are heat-inducible however, at 43°C, they are not as effective as GrpE at protecting the cell from unfolded protein accumulation.

Bacterial Pathogenesis
GrpE has an important role in bacteria's infection of host species. Enterococci are bacteria that are commonly found in the guts of humans and other animals, they are pH, temperature, and salt insensitive. These bacteria are antibiotic resistant and are dangerous when they produce an infectious biofilm. Enterococcal biofilm is prevalent in hospital and surgical settings, it is responsible for 25% of catheter-related infections, is found in 50% of root-filled teeth with apical periodontitis , and can be isolated from other wounds. GrpE is found in the genome of Enterococcus faecilis and Enterococcus faecium and is critical for enterococcal biofilm attachment to polystyrene tubes, a plastic polymer commonly used in hospital settings.

Group A Streptococcus pyogenes is a bacteria that can lead to common infections, including strep throat and impetigo, but is also responsible for life-threatening infections. During infection, GrpE helps streptococcus bacteria adhere to pharyngeal epithelial cells. GrpE in Streptococcus binds to endogenous proline-rich proteins in saliva, allowing adhesion of the bacteria to the host.