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Insights into dimerization and four‐helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli

The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long α‐helices at the NH2‐terminal end from each monomer (forming a “tail”), which lead into a small four‐helix bundle from which each monomer contributes two short sequential α‐helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four‐helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long α‐helical tail portion (GrpE1–88) is unable to form a dimer, most likely due to a decrease in α‐helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short α‐helices (GrpE1–138 and GrpE88–197) are able to form a dimer and presumably the four‐helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer–dimer equilibrium that are very similar to the full‐length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short α‐helices (GrpE1–112) exists as a tetrameric species, which presumably is forming a four‐helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four‐helix bundle formation.