GrpE

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. == 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). 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. == Regulation ==

Transcription In the Archaea 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 Archaea, these three genes are transcribed at the same time. 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. == Other biological systems ==

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. In humans, mitochondrial organelles have GrpE-like 1 (GRPEL1) protein. In eukaryotic cells, there any many additional eukaryotic GrpE homologues. 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. == Role in disease ==

Bacterial pathogenesis Enterococci are bacteria that are commonly found in the gastrointestinal tract of animals, including humans. These bacteria can form a biofilm, which is a layer of bacteria attached to a surface. a plastic polymer commonly used in hospital settings. Group A Streptococcus pyogenes is a bacterium 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. == References ==