Structural and functional insight into the plant unique multimodular triphosphosphate tunnel metalloenzymes of Arabidopsis thaliana

Abstract

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family already characterized have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, inorganic polyphosphate). TTM proteins in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane - mediated by the single transmembrane domain at the C-terminus, supporting earlier studies. Crystal structures of AtTTM1 in the presence and absence of a non-hydrolyzable ATP analog reveal a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Structural comparison reveals that a helical hairpin may facilitate movement of the TTM domain thereby activating the kinase. Genetic studies show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. Rational design of mutations based on the 3D structure demonstrates that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch.