Homotypic dimerization of a maltose kinase for molecular scaffolding

Abstract

Mycobacterium tuberculosis (Mtb) uses maltose-1-phosphate to synthesize α-glucans that make up the major component of its outer capsular layer. Maltose kinase (MaK) catalyzes phosphorylation of maltose. The molecular basis for this phosphorylation is currently not understood. Here, we describe the first crystal structure of MtbMaK refined to 2.4 Å resolution. The bi-modular architecture of MtbMaK reveals a remarkably unique N-lobe. An extended sheet protrudes into ligand binding pocket of an adjacent monomer and contributes residues critical for kinase activity. Structure of the complex of MtbMaK bound with maltose reveals that maltose binds in a shallow cavity of the C-lobe. Structural constraints permit phosphorylation of α-maltose only. Surprisingly, instead of a Gly-rich loop, MtbMaK employs 'EQS' loop to tether ATP. Notably, this loop is conserved across all MaK homologues. Structures of MtbMaK presented here unveil features that are markedly different from other kinases and support the scaffolding role proposed for this kinase.

Figure 1. Structure of MtbMaK.

(a). Cartoon representation of a monomer of MtbMaK. The N-lobe contains two β-sheets with strands β8 and β9 protruding out of the protein. The N- and C-terminals of the protein are marked as N and C, respectively. (b). Cartoon representation of a homotypic dimer of MtbMaK. Strands β8 and β9 from each monomer within a dimer mutually insert into each other's N-lobe to assemble sheet 2. (c). Structural homologues of MtbMaK. A Dali analysis of MtbMaK (left) retrieved low structural matches like methylthioribose (MTR) kinase from Bacillus subtilis (right; PDB code 2PUN) that could not be superimposed over MtbMaK. (d). Location of conserved motifs. The α-C helix (shown in red), activation loop (black color) and catalytic loop (magenta color) of MtbMaK are located at the junction of N- and C-lobes. A ‘¹⁴²EQS¹⁴⁴’ loop (cyan color) connecting two strands of an adjacent monomer (blue color) contributes residues critical for catalysis. DFE and HGD motifs of MtbMaK as well as residues of ‘¹⁴²EQS¹⁴⁴’ loop are shown as sticks (inset).

Figure 2. Homotypic dimerization of MtbMaK.

(a). A dimer of MtbMaK where one molecule is depicted as cartoon (blue color), while the second molecule is shown in a surface representation (salmon color). Mutual insertion of strands β8 and β9 results in the formation of a tight dimer. (b). Strand β9 of each monomer (green and blue) run anti-parallel with the backbone atoms interacting along the entire length of the strand. (c). Dimer interface of MtbMaK. Interfacing residues are shown in sticks. Region of molecule 2 participating in dimerization is marked with red color on the cartoon. Side chains as well as backbone atoms participate in dimerization.

Figure 3. Maltose binding site of MtbMaK.

(a). 2Fo-Fc electron density for maltose contoured at 1.5σ is shown. (b). Conformational changes upon binding of maltose. The structure of maltose bound MtbMaK (green color) was superimposed over the unliganded structure (blue color). Main and side chains of several residues move as a result of substrate binding. (c). Maltose binds in the C-lobe and is in proximity to the conserved motifs essential for catalysis. (d). A surface electrostatic potential representation of the region around the maltose binding site. Blue represents positive potential; red, negative potential. (e). Residues interacting with maltose. Residues from the catalytic loop interacting with maltose are shown as blue sticks, while P344 from the activation loop is shown as magenta sticks. Maltose is shown as sticks in panels A-E. (f). Alanine scanning mutagenesis of amino acids interacting with maltose. Relative activity of mutants (%) with respect to the wild type is plotted as a bar graph. Error bars represent s.d. (n = 3).

Figure 4. Nucleotide binding site of MtbMaK.

(a). Sequence alignment of nucleotide binding site (NBS) of MtbMaK and its homologues. SSE of MtbMaK are labeled on top of the aligned sequences. Identical residues are highlighted in red, and other conserved residues are highlighted in yellow. (b). Stereo view of AMPPNP-Mg (green sticks and spheres, PDB code 1J7U) superimposed on the NBS of MtbMaK. The C atoms of ‘¹⁴²EQS¹⁴⁴’ loop, HGD motif and DFE motif are colored in cyan, magenta and light green, respectively. Strands β8′ and β9′ from the other subunit of MtbMaK are colored in blue. The side chains of residues potentially interacting with AMPPNP-Mg are shown as sticks. The O and N atoms are colored in red and blue. (c). Mutagenesis of residues from the NBS. A bar graph of relative activity (%) of mutants compared to the wild type enzyme is shown. Error bars represent s.d. (n = 3). (d). Model of hetero-octameric complex of TreS with MaK. Potential interaction of a tetramer of TreS (green color, PDB code 4LXF) with two dimers of MtbMaK (purple color) is shown. Active sites are marked with a red star; putative path of product marked in yellow.