Fig. 2: Biochemical and cryo-EM analyzes show that PneKC forms adimer.

a The size-exclusion chromatography (SEC) profiles of PneKC alone (blue) and in complex with PneA (black) show distinct peaks at 12.1 ml and 12.2 ml, respectively, corresponding to a mass of approximately 200 kDa, indicating that PneKC by itself likely forms a dimer. b Nucleotide hydrolysis of PneKC was assayed using various nucleotides as substrates, both in the absence of PneA as well as in the presence of PneA at a PneKC monomer to PneA ratio of 1:1. Catalytic residue Asp353 in the kinase domain was initially mutated to alanine as a negative control as it was expected to abolish nucleotide hydrolysis. However, this mutant displayed noticeable activity even in the absence of PneA. PneKC (5 mg/ml) with different NTPs in the absence of PneA, exhibited very low hydrolysis activity. However, in the presence of PneA, significant hydrolysis activity was observed with various NTPs, even at a much lower PneKC concentration of 0.5 mg/ml. Error bars represent the standard deviation (SD) of three independent experiments (n = 3). c GTPase activity of PneKC with different amounts of PneA. Error bars represent the standard deviation (SD) (n = 3), and the statistical significance was assessed using a two-tailed t-test (n = 3) where PneKC: PneA (1:1) p = 0.0037; PneKC: PneA (1:3) p = 0.004. The indicated ratio is for PneKC monomer to PneA. d Top and side views of the density map of the PneKC-PneA complex showing the two PneKC protomers (ligand-bound protomer: purple; ligand-free protomer: orange; bound PneA: blue; and GTPγS: red). The two protomers are arranged in a head-to-tail manner. e Model of dimeric PneKC showing the N-terminal lyase domain (green), the central kinase domain (sandy brown), and the C-terminal putative cyclase domain (purple). f The PneKC protomer forms a cashew shape with PneA bound to the major groove that is formed between the lyase and kinase domains. g Model of PneKC showing an expansive central groove. The catalytic sites of the three domains are oriented towards the central cavity. Source data are provided as a Source Data file.