Fungal Kti12 proteins display unusual linker regions and unique ATPase p-loops

Abstract

Kti12 (Kluyveromyces lactis toxin insensitive 12) is an evolutionary highly conserved ATPase, crucial for the tRNA-modification activity of the eukaryotic Elongator complex. The protein consists of an N-terminal ATPase and a C-terminal tRNA-binding domain, which are connected by a flexible linker. The precise role of the linker region and its involvement in the communication between the two domains and their activities remain elusive. Here, we analyzed all available Kti12 protein sequences and report the discovery of a subset of Kti12 proteins with abnormally long linker regions. These Kti12 proteins are characterized by a co-occurring lysine to leucine substitution in their Walker A motif, previously thought to be invariable. We show that the K14L substitution lowers the affinity to ATP, but does not affect the catalytic activity of Kti12 at high ATP concentrations. We compare the activity of mutated variants of Kti12 in vitro with complementation assays in vivo in yeast. Ultimately, we compared Kti12 to other known p-loop ATPase family members known to carry a similar deviant Walker A motif. Our data establish Kti12 of Eurotiomycetes as an example of eukaryotic ATPase harboring a significantly deviating but still functional Walker A motif.

Keywords: ATPase; Active site; Flexible linker; Kti12; P-loop; PSTK.

Fig. 1

Identification of Kti12 proteins with elongated linker and their characterization. a Architecture of Kti12 protein. ATPase domain (grey) and tRNA-tethering domain (yellow) are connected by a flexible linker. Structural overview of Chaetomium thermophilum Kti12 ATPase domain (PDB ID: 6QP0) with a particular emphasis on nucleotide binding pocket and K14 located in a p-loop. b Distribution of linker length across fungal Kti12 proteins. Numbers on O_x indicate middle values for particular baskets, for instance, 18 ± 4. Black triangles indicate groups to which ScKti12 and CtKti12 belong to. c Scheme of supervised a machine-learning experiment that pinpointed coincidence of L14 with elongated linkers. Sequences with linkers of a regular length (grey) always carry lysine at position 14 (K14), whereas Kti12 proteins with elongated linkers (pink) can be distinguished by the presence of leucine in a p-loop (L14). d Violin plots represents distribution of Kti12 protein sequences in a two-dimensional feature space. Linker length is on O_x whereas presence of leucine or lysine qualifies sample to one of the groups on O_y axis. Number of sequences within each group is indicated next the O_y axis

Fig. 2

Phenotypic characterization of Kti12 K14L mutation and elongated linker in yeast. a Phylogenetic tree based on Kti12 sequence alignment displays distribution of different residues at position 14 among species of different divisions [Ascomycota (orange)/Basidiomycota (green)]. Kti12 proteins carrying L14 and long linker (purple squares) belong to Eurothiomycetes. Multiple sequence alignment of Kti12 proteins from 25 model fungi species was performed using JalView software (Waterhouse et al. 2009) and Muscle server (Edgar 2004). The phylogenetic tree was built using the neighbor-Joining method. Individual branch lengths are shown above the branches and indicate the number of amino acid substitutions per site. The fraction of replicate trees in which the associated taxa clustered together in the bootstrap test [1000 replicates (Felsenstein 1985)] are shown beneath the branching points. The evolutionary distances used to calculate the tree were calculated using the Poisson correction method with the pairwise deletion of ambiguous positions. Final dataset contained 610 positions. All evolutionary analyses were conducted in MEGA X (Kumar et al. 2018). The tree was visualized using iTOL (Letunic and Bork 2007). b Zymocin assay assessing performance of the Elongator complex and presence of U₃₄ modifications. WT yeast strain (ScKTI12) served as a growth control, Scelp3Δ and Sckti12Δ pose negative controls. Tenfold dilutions were spotted on YEPD media with the addition of 50% Zymocin or YNB media replacing arginine by 40 µg/ml canavanine or lacking adenine (-adenine). In the different strains KTI12 was mutated (K14A/L), parts or the whole gene were exchanged with the corresponding sequences from Aspergillus niger (An knot + linker, AnKTI12) or parts of it were deleted (marked with ∆ and the deleted residues in brackets). Sckti12-NTD codes for the first 184 amino acids

Fig. 3

Biochemical properties of Kti12 mutants. a Thermostability profiles of CtKti12 K14 point mutants. Thermal shift assay, curves represent an average of three independent experiments. b Fluorescent polarization study of MANT-ATP bound by Kti12 mutants. CtKti12 WT Kd = 1.5 μM ± 0.2 μM, K14L Kd = 25.0 μM ± 2.4 μM. In case of CtKti12 K14A and K14V we were unable to determine K_d in a measured concentration range. c Relative tRNA^Sec-induced ATPase activity of CtKti12 and AnKti12 mutants, compared to the respective WT versions. Free phosphate concentration was measured using malachite green assay. Bars represent an average of three independent experiments ± standard deviation. Proteins carrying different amino acids at position 14 are color coded (K14 grey, A14 orange, L14 purple and V14 green)

Fig. 4

Influence of K14L on the nucleotide binding pocket of Kti12 protein. a Hydrophobicity of nucleotide binding pocket of Kti12 mutants. PDB ID: 6QP0. Nucleotide binding pocket consists of positively charged (blue), negatively charged (red) and hydrophobic (yellow) residues. Only surface of the residues within 5 Å from the phosphor atoms of ADP is shown. Kti12 is shown bound to ADP (standard coloring), AlF3 (cyan and pink) and magnesium (green). K14A mutation creates a relatively big gap, leaving the hydrophobic core unprotected. Subsequent K14V and K14L substitutions gradually repair the breach. b A homology-based model of gp28 terminase (based on PDB ID 3EZK) from mu phage with previously described deviant Walker-A motif shown in ball and stick representation. c Multiple sequence alignment and structural comparison of Walker A motifs in CtKti12, AnKti12 (based on 6QP0) and mu phage gp28. Shades of violet indicate conservation score. Please note that AnKti12 Walker A motif does not have additional lysine which is present in case of mu gp28, but has L14 residue (pink) which corresponds to L70 residue of mu gp28. d An overview of lysines (dark blue) present in the ATPase domain of CtKti12 (PDB ID 6QP0, top) and AnKti12 (based on PDB ID 6QP0; bottom). Walker A motif is highlighted in pink