The role of individual amino acids in the dimerization of CR4 and ACR4 transmembrane domains

CRINKLY4 is a growth factor-like plant receptor kinase designated as CR4 in Zea mays and ACR4 in Arabidopsis. Using the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment, we have previously demonstrated that the dimerization potential of the ACR4 transmembrane (TM) domain is significantly weaker than that of CR4 TM domain, even though 13 of the 24 residues are identical. Neither of the TM domains contain the GxxxG motif that has been shown to be important for the dimerization of the TM segments of several receptors. To further investigate the relationship between protein sequence and dimerization potential, we (a) mutated each of the 11 differing residues in the CR4 TM domain to the corresponding residue of ACR4 (b) made reciprocal mutations in ACR4 and (c) made hybrids consisting of half CR4 and half ACR4 TM domains. Our results suggest that most mutations in ACR4 or CR4 TM domains have low to moderate effects on the dimerization potential and that residues in the N-terminal half of the CR4 TM domain are important for dimerization.

Research highlights
► TOXCAT assay indicates that the transmembrane domain (TM) of the CRINKLY4 receptor-like kinase (RLK) from maize (CR4) has a higher dimerization potential than the TM domain of the RLK from Arabidopsis (ACR4).
► Replacing the first 12 residues of CR4 TM with residues from the ACR4 TM domain leads to significantly weaker dimerization (ACR4/CR4 hybrid).
► On the other hand, engineering the first 12 residues of CR4 TM sequence in place of the first 12 amino acids of ACR4 TM domain (CR4/ACR4 hybrid) confers an enhanced dimerization propensity.
► The N-terminal region of CR4 TM harbors two motifs, VAEIV and VFAVV, that contribute to enhanced dimerization.