Dimer formation of ΔH-domain protein in solution.

<p>(A) The purified ΔH-domain protein (0.2 mg/ml) was loaded onto native-PAGE. M, protein marker; lane 1, ΔH-domain protein with 2% SDS; lane 2, ΔH-domain protein only; lane 3, ΔH-domain protein with 10 mM DTT. (B) The gel filtration profile of ΔH-domain protein at the final purification step is shown. Two peaks were observed upon analysis with a Superdex S75 16/60 column (GE Healthcare). The elution volume is indicated on the top of each peak. The elution volume of 79.70 ml corresponds to a dimer size (30 kDa) and 62.57 ml to a tetramer size (60 kDa). (C) SEC-MALLS analysis of ΔH-domain and full-length forms of <i>Co</i>MsrA. The refractive index (RI) of the column eluate is plotted as a function of time. The weight-averaged molecular weight (MW) of the material in the eluate is calculated from light-scattering measurements. The calculated molecular masses are 35,010 Da for the ΔH-domain protein and 24,570 Da for the full-length protein.</p

Data collection and structure refinement statistics.

<p>Values in parentheses are for highest-resolution shell.</p><p>Data collection and structure refinement statistics.</p

Rear view of dimeric <i>Co</i>MsrA.

<p>(A) The residues near the dimer interface of backside of dimeric MsrA are displayed with stick models. The distance between Y141 residues from each monomer is indicated with a black dashed line and the distance between G177 residues with a red one. (B) The surface model is represented, with the helical domain colored in red. Two helical domains are distantly located and make a crevice between two molecules.</p

Dimeric structures of <i>Co</i>MsrA.

<p>(A) Overall structure of dimeric <i>Co</i>MsrA. Three molecules are included in the asymmetric unit. Molecules A (cyan) and B (pink) form a dimer, while molecule C (yellow) forms another dimer with its symmetric molecule C' (gray). The catalytic Cys16 residues are shown with stick models. (B) Surface model of homodimer structure formed by molecules A and B in the asymmetric unit. The loop regions L1 and L2 (yellow and orange) contribute to dimerization. The helical domain is shown in red. The dimeric MsrA, featuring the L1 and L2 loop regions, makes a hole at the center where the catalytic Cys16 residues are located. (C) The hole forming residues in the dimeric structure formed by molecules A and B. The residues are represented with stick models. The matching residues on the other monomer are indicated with the prime sign ('). The two residues on the L1 loop (Y47 and E55) and the two residues on L2 loop (Q89 and R88) play an important role in hole formation. Black dotted lines represent hydrogen bond interactions.</p

General catalytic mechanism of MsrA.

<p>The catalytic Cys^A (shown in red) attacks the sulfoxide of the substrate (MetO) to form a sulfenic acid intermediate, with concomitant release of Met (step 1). The resolving Cys^B (shown in blue) then reacts with the sulfenic acid intermediate on Cys^A to form an intramolecular disulfide bond (step 2), and the disulfide bond between Cys^A and Cys^B is further reduced by thioredoxin (Trx) (step 3). DTT can be used as the <i>in vitro</i> reductant.</p

Kinetics of dimerization of <i>Co</i>MsrA.

<p>(A) Dimerization in response to the concentration of MsrA protein. The assay was performed for 2 h in the presence of 0.2 mM free methionine sulfoxide. (B) Dimerization in response to the reaction time. The assay was performed in the presence of 0.2 mM free methionine sulfoxide and 84 μM MsrA protein. Equal quantities of protein were loaded onto a non-reducing SDS-PAGE gel (upper panels). The dimer ratios were analyzed using the ImageJ program (lower panels). D_max, maximal percent of dimer; D_1/2, concentration of MsrA or reaction time to reach a half D_max value.</p

Resolution of dimeric <i>Co</i>MsrA by Grx analyzed by non-reducing SDS-PAGE.

<p>A monothiol form of Grx1 (U13C/C16S) is able to break the intermolecular disulfide bond of the MsrA dimer. Purified Grx1 is predominantly in a dimeric form (lane 4) and a Grx1-MsrA complex is formed by the reaction with dimeric MsrA (lane 3). Lane 1, dimeric MsrA; lane 2, dimeric MsrA treated with 100 mM DTT; lane 3, dimeric MsrA exposed to Grx1 (in the absence of glutathione); lane 4, Grx1 alone.</p

Conformational changes in dimeric <i>Co</i>MsrA structure.

<p>(A) Superposition of monomeric sulfenic acid form <i>Co</i>MsrA (PDB ID: 4LWJ, cyan) to a dimeric form (gray). L1 and L2 regions are shown in red (dimeric form) and blue (monomeric form). The two residues on L1 (Y47 and Y48) and three residues on L2 (R88, Q89 and Y90) are represented with stick models. (B) The active site residues and residues contributing to hole formation are indicated with stick models. The R88, Q89, and Y90 residues on L2 in the monomeric form are indicated in blue letters. (C) Stereo view of B. The substrate position (yellow stick model) was built in the active site using the substrate-bound structure (PDB ID: 4LWM).</p

Dimerization of <i>Co</i>MsrA in the presence of substrate.

<p>(A) Dimerization of Sec-to-Cys <i>Co</i>MsrA and its variants. The dimerization was analyzed by non-reducing SDS-PAGE. A concentration of 0.2 mM free methionine sulfoxide (fMetO) or dabsyl-methionine sulfoxide (dMetO) was used. Lane 1, purified monomeric MsrA; lane 2, monomeric MsrA with fMetO; lane 3, monomeric MsrA with dMetO; lane 4, purified dimeric MsrA; lane 5, dimeric MsrA with 10 mM DTT; lane 6, purified E55A; lane 7, E55A with fMetO; lane 8, E55A with dMetO; lane 9, purified E55D; lane 10, E55D with fMetO; lane 11, E55D with dMetO. (B) Dimerization of native selenoprotein <i>Co</i>MsrA<b>.</b> The dimerization of natural Sec-containing MsrA was analyzed by non-reducing SDS-PAGE followed by Western blotting. Lane 1, purified selenoprotein MsrA; lane 2, selenoprotein MsrA with fMetO; lane 3, selenoprotein MsrA with dMetO; lane 4, the sample of lane 3 treated with 10 mM DTT.</p

Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA–MsrB Fusion Protein

Many bacteria, particularly pathogens, possess methionine sulfoxide reductase A (MsrA) and B (MsrB) as a fusion form (MsrAB). However, it is not clear why they possess a fusion MsrAB form rather than the separate enzymes that exist in most organisms. In this study, we performed biochemical and kinetic analyses of MsrAB from <i>Treponema denticola</i> (<i>Td</i>MsrAB), single-domain forms (<i>Td</i>MsrA and <i>Td</i>MsrB), and catalytic Cys mutants (<i>Td</i>MsrAB^C11S and <i>Td</i>MsrAB^C285S). We found that the catalytic efficiency of both MsrA and MsrB increased after fusion of the domains and that the linker region (<i>iloop</i>) that connects <i>Td</i>MsrA and <i>Td</i>MsrB is required for the higher catalytic efficiency of <i>Td</i>MsrAB. We also determined the crystal structure of <i>Td</i>MsrAB at 2.3 Å, showing that the <i>iloop</i> mainly interacts with <i>Td</i>MsrB via hydrogen bonds. Further kinetic analysis using the <i>iloop</i> mutants revealed that the <i>iloop</i>–<i>Td</i>MsrB interactions are critical to MsrB and MsrA activities. We also report the structure in which an oxidized form of dithiothreitol, an <i>in vitro</i> reductant for MsrA and MsrB, is present in the active site of <i>Td</i>MsrA. Collectively, the results of this study reveal an essential role of the <i>iloop</i> in maintaining the higher catalytic efficiency of the MsrAB fusion enzyme and provide a better understanding of why the MsrAB enzyme exists as a fused form