Control of Substrate Specificity by a Single Active Site Residue of the KsgA Methyltransferase

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and <i>N</i>⁶-methyladenosine as substrates to catalyze formation of the final product <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of <i>S</i>-adenosyl-l-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the <i>N</i>⁶-methyladenosine intermediate to produce <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of <i>N</i>⁶-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA’s multiple catalytic steps

Control of Substrate Specificity by a Single Active Site Residue of the KsgA Methyltransferase

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and <i>N</i>⁶-methyladenosine as substrates to catalyze formation of the final product <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of <i>S</i>-adenosyl-l-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the <i>N</i>⁶-methyladenosine intermediate to produce <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of <i>N</i>⁶-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA’s multiple catalytic steps

Control of Substrate Specificity by a Single Active Site Residue of the KsgA Methyltransferase

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and <i>N</i>⁶-methyladenosine as substrates to catalyze formation of the final product <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of <i>S</i>-adenosyl-l-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the <i>N</i>⁶-methyladenosine intermediate to produce <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of <i>N</i>⁶-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA’s multiple catalytic steps

Crystallographic Trapping of Heme Loss Intermediates during the Nitrite-Induced Degradation of Human Hemoglobin

Heme is an important cofactor in a large number of essential proteins and is often involved in small molecule binding and activation. Loss of heme from proteins thus negatively affects the function of these proteins but is also an important component of iron recycling. The characterization of intermediates that form during the loss of heme from proteins has been problematic, in a large part, because of the instability of such intermediates. We have characterized, by X-ray crystallography, three compounds that form during the nitrite-induced degradation of human α2β2 hemoglobin (Hb). The first is an unprecedented complex that exhibits a large β heme displacement of 4.8 Å toward the protein exterior; the heme displacement is stabilized by the binding of the distal His residue to the heme Fe, which in turn allows for the unusual binding of an exogenous ligand on the proximal face of the heme. We have also structurally characterized complexes that display regiospecific nitration of the heme at the 2-vinyl position; we show that heme nitration is not a prerequisite for heme loss. Our results provide structural insight into a possible pathway for nitrite-induced loss of heme from human Hb

Comparison of 4PUS with other truncated N^1-165 domain M1 structures (in ribbon diagrams).

<p>Unless noted otherwise, monomers A and B are colored grey and orange, respectively. <b>A</b>. Superposition of monomers A of 4PUS (grey), 1AA7 (yellow), and 1EA3 (red). <b>B</b>. Dimeric structure of 1AA7 showing face-to-face interaction between the two monomers. <b>C</b>. Relative positions of the non-superposed monomers B of 4PUS (grey), 1AA7 (yellow) and 3MD2 (red) after superposition of the corresponding monomers A. For clarity, only parts of the non-superposed monomers B are shown. <b>D</b>. Two monomers of 1EA3 interacting in a face-to-back fashion with the positively charged residues facing the negatively charged residues. For clarity not all positive or negative residues at the monomer faces are shown.</p

Data collection and refinement statistics of Safo-4PUS-pH4.7.

a<p>R_mege = Σ_hklΣ_i/I_hkli–hkli>/Σ_hklΣ_ihkli>.</p>b<p>R_free was calculated with 5% excluded reflection from the refinement.</p><p>Data collection and refinement statistics of Safo-4PUS-pH4.7.</p

Schematic representation of the proposed multiple conformational transitions in M1 structures.

<p><b>A</b>. Cartoon diagrams illustrating the oligomeric state of the M1 crystal structures, including 4PUS (grey), 1AA7 (blue), 3MD2 (red), 1EA3 (magenta) and 2Z16 (yellowish-green). Structures at neutral pH (top) are monomeric and arranged face-to-back; those at acidic pH (bottom) are dimeric and arranged face-to-face. Disorder in the Safo (4PUS) structure is indicated by a light-colored region bounded by a dashed line. <b>B</b>. Cartoon illustrating the interfacial regions of 4PUS (grey), 1AA7 (blue), 3MD2 (red), 1EA3 (magenta) and 2Z16 (yellowish-green). Individual M1 chains (consisting of two four-helix bundles separated by a linker) are represented as rectangles. Each crystal structure is represented by an ‘A’ chain (leftmost rectangle; grey border) and a ‘B’ chain (rightmost rectangle). The acidic (red) and basic (blue) residues at the interfacial regions are labeled and may be grouped into five clusters (K21; E8, E29 and D30; R76, R77 and R78; D89, D94, K95, K98, R101 and K104; R134) based on their proximity to one another on the M1 surface. Salt bridges are represented by yellow rectangles joining opposite charges. Note that the salt-bridge interactions depicted in Liu (3MD2) are>3.7 Å. Green transparent boxes within the rectangles represent the relative amount of shared interfacial surface area (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109510#pone-0109510-t002" target="_blank">Table 2</a>).</p

Packing (stacking) arrangement of the M1 molecules (in ribbon diagrams). Monomers A and B are colored grey and orange, respectively.

<p><b>A.</b> A dimer of 1AA7 stacked on top of another dimer to form a pseudo-tetramer. A similar arrangement is observed in 3MD2. <b>B.</b> A pseudo-tetramer appears to form similarly in 4PUS, however, the two stacked dimers do not interact with each other. <b>C.</b> Arrangement of monomers in 1EA3 to form a pseudo-tetramer.</p

Hydrogen bond interactions (<3.8 Å) and solvent accessible buried surface area of the monomer–monomer interface of M1 structures.

<p>Hydrogen bond interactions (<3.8 Å) and solvent accessible buried surface area of the monomer–monomer interface of M1 structures.</p

Structure of truncated N^1-165 domain of M1 (PDB code 4PUS) at pH 4.7.

<p>A. Monomer A of 4PUS. The N-terminal and C-terminal domains are shown in grey ribbons. Shown in yellow sticks are the basic NLS motif residues (Arg101, Lys102, Lys104, Arg105), Lys95 and Lys98 located on the right face of the molecules; the negatively charged residues Glu8, Glu23, Glu29, Asp30, Asp38, and Glu44 located on the left face of the molecule; and representative hydrophobic core residues on H1 and H4 buried between the N-terminal and C-terminal domains. For clarity not all residues of interest are shown. The nine helices are labelled <b>B</b>. Dimeric structure showing the positively charged residues including the NLS (yellow sticks) on the surface of the molecule. Monomers A and B are colored in grey and orange, respectively. Note the disorder in the N-terminal domain of monomer B. <b>C</b>. Same as figure B, but rotated by ∼90°. <b>D and E</b>. Final refined 2Fo-Fc electron density maps (contoured at 0.9α) of analogous regions (residues 45–50) of monomers A and B, respectively. The region in monomer B is clearly disordered.</p