Fig. 5: Identifying His35 as a hot spot for PneKC dimerization.

a The PneKC dimer is stabilized by two symmetric interaction interfaces between the lyase and cyclase domains. b The PneKC dimerization interface is formed by the N-terminal loop of the lyase ___domain and the KC linker and β-hairpin 2 of the cyclase ___domain. Residue His35 from the lyase ___domain of one protomer interacts with three motifs from the cyclase ___domain of the other protomer. c Size-exclusion chromatography profile of PneKC with mutations of interface residues, showing that mutation of His35 to alanine and aspartate disrupt dimerization. d 2D-classs averages of wild-type PneKC forming a dimer and H35A mutant PneKC forming a monomer. e DSF melting curves for the thermal unfolding of wild-type and H35A mutant PneKC, showing that dimeric wild-type PneKC has higher thermostability. f GTPase activity of dimeric wild-type and monomeric H35A mutant PneKC in the presence of equal amounts of PneA, with a PneKC monomer to PneA ratio of 1:1. The monomeric H35A mutant PneKC exhibits approximately twice the level of GTPase activity as the dimeric wild-type protein. Error bars represent the standard deviation (SD) (n = 3), and the statistical significance was assessed using a two-tailed t-test (p = 0.02), suggesting negative cooperativity for GTP hydrolysis in the dimer. To ensure that the same amount of protein and peptide were used in these assays, the protein concentrations were assessed by quantitative SDS-PAGE. Source data are provided as a Source Data file.