Immunoglobulin binding protein (BiP) forms two types of dimers.

Immunoglobulin binding protein (BiP) is a chaperone protein that plays crucial roles in protein folding and transport by binding unfolded proteins in the substrate binding domain (SBD), an interaction allosterically linked to the nucleotide occupancy of the nucleotide binding domain (NBD). BiP also forms oligomers that influence its activity, particularly in binding polypeptide clients. Using single-molecule approaches and mechanical and enzymatic activity assays, BiP monomer stability and oligomerization were analyzed both alone and in response to nucleotides and peptides. We find that the formation of BiP dimers exhibits biphasic behavior as a function of BiP concentration, suggesting concentration-dependent changes in dimer dissociation constants. At low BiP concentrations, dimers were disrupted by peptide substrate and adenosine triphosphate (ATP) but remained unaffected by ADP or Adenosine 5'-O-(3-thio)triphosphate (ATPγS). At high concentrations, dimers are unaffected by peptides, but their assembly is inhibited by ATP and ATPγS to the same degree. These results suggest the formation of two distinct BiP dimers exhibiting unique binding affinities, kinetics, and potentially structures. We propose the existence of a high-affinity dimer binding site within the SBD of BiP, while the formation of a low-affinity dimer involves interactions between the lid and NBD of each protomer. Our findings demonstrate the importance of considering single-molecule characteristics when interpreting bulk studies on protein function and regulation.