Kinetic characterization of Bub1 complexes.

<p>(<b>A</b>-<b>C</b>) Bub1^kinase and Bub1:Bub3 complex exhibit similar catalytic activity toward H2A (<b>A</b>) and Bor:Sur substrates (<b>B</b>) and hydrolyze ATP at similar rates (<b>C</b>). The mutation D917N abrogates ATPase activity (<b>C</b>). The kinase activity was determined using the ADP-Glo^TM Kinase Assay and is plotted as a function of substrate concentration to allow fitting according to the Michaelis-Menten equation with R² = 0.99 (Bub1^kinase on Bor:Sur R² = 0.97). KD–kinase dead. Error bars represent SD of a mean of at least 2 independent experiments. (<b>D</b>) Kinetic parameters of the Michaelis-Menten fits as determined in (<b>A</b>-<b>C</b>). (<b>E</b>) H2A contained in H3- or CENP-A nucleosomes is efficiently phosphorylated by Bub1^kinase and Bub1:Bub3. (<b>F</b>) The kinase activity, plotted as a function of substrate concentration, allows fitting with the Michaelis-Menten equation with R² = 0.95 (H3) and 0.99 (CENP-A). Error bars represent SD of a mean of at least two independent experiments. (<b>G-H</b>) EMSA assays showing DNA and nucleosome binding of Bub1^kinase and H3- or CENP-A nucleosomes. MN–mononucleosomes.</p

Reconstitution of Bub1 kinase and BubR1 pseudo-kinase.

<p>(<b>A</b>) Bub1 and BubR1 share a similar domain organization. Schematic description of the domains and constructs used in this manuscript: TPR–tetratricopeptide repeat, BD–binding domain, WD40 –an approximately 40-residue sequence repeat often terminating with a tryptophan (W) and aspartate (D) dipeptide. (<b>B</b>) Purified Bub1^kinase, Bub1:Bub3, BubR1^kinase, and BubR1:Bub3 were separated by SDS-PAGE. Their respective expected molecular mass is indicated. (<b>C</b>) Both Bub1^kinase and BubR1^kinase bind to mant-ATP. The change in fluorescence emission at 450 nm is plotted as a function of total mant-ATP concentration. The data were fitted with a one site binding equation using Origin 9.0, with R² = 0.99 for both curves. Error bars represent SD of a mean of at least 2 independent experiments. a.u.–arbitrary units. (<b>D</b>-<b>E</b>) ESI-MS spectra of purified Bub1^kinase (<b>D</b>) or BubR1^kinase (<b>E</b>) before and after incubation with ATP for auto-phosphorylation. Theoretical calculated masses are given in brackets under the measured masses. (<b>F</b>) BubR1 is not an active kinase. Maltose binding protein (MBP), H1, and the Borealin:Survivin complex (Bor:Sur) were incubated with 50 nM BubR1 constructs and ATP and analyzed on SDS PAGE visualizing phosphates using the Pro-Q® Diamond Phosphoprotein Gel Stain. 10 nM Bub1:Bub3 was used as a positive control. BR1^kinase–BubR1^kinase, BR1B3 –BubR1:Bub3.</p

Bub1^kinase possesses conserved residues that recognize a substrate consensus sequence.

<p>(<b>A</b>) Alignment of phosphorylation sites found in Bub1-dependent phosphorylation reactions using the ClustalX algorithm in Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref054" target="_blank">54</a>]. Numbers indicate boundaries of the protein segments shown; phosphorylated residues are denoted with an asterisk, the conservation of residues is highlighted using the ClustalX coloring scheme. (<b>B</b>) The structure of Bub1^kinase (PDB ID 4QPM) was superimposed onto the structure of PKA bound to a pseudo-substrate inhibitor [PDB ID 1APM, reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref055" target="_blank">55</a>]]. The image shows only Bub1 and the position of the pseudo-substrate peptide (yellow) after alignment of PKA to Bub1. Bub1 residues putatively involved in substrate recognition are depicted as sticks. (<b>C</b>) Bub1^kinase sequence and structure conservation. The same residues as in (<b>B</b>) are shown in sticks. Conservation was determined by aligning Bub1^kinase from 14 organisms with ConSurf [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref056" target="_blank">56</a>], the scoring legend is depicted on the upper left. Conservation scores obtained for positions in the alignment that had less than 6 un-gapped amino acids were considered to be unreliable and colored light yellow in the graphic visualization output. Images were created with CCP4MG [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref053" target="_blank">53</a>] and Pymol (Schrödinger LLC, Portland, OR). (<b>D</b>) Bub1^kinase-dependent H2A phosphorylation can be reduced by mutating V115D, L116N, L117N on H2A. GST-H2A constructs were incubated with Bub1^kinase and ATP, then analyzed by SDS PAGE, and phosphorylated proteins were specifically stained using Pro-Q® Diamond Phosphoprotein Gel Stain.</p

Biochemical characterization of Bub1 complexes.

<p>(<b>A</b>) Bub1:Bub3 (green) and BubR1:Bub3 (blue) co-elute in a stable complex from a size exclusion chromatography column (black) and can be assembled as a complex onto phosphorylated MBP-Knl1^138-225 (purple). A bar of the respective color indicates fractions analyzed on SDS PAGE (below). (<b>B</b>) Analytical ultracentrifugation. Normalized c(s) distribution curves for Bub1:Bub3 (green) and BubR1:Bub3 (blue). A predominant peak at 4.5 S (s_20,w = 4.7) is apparent for Bub1:Bub3 and at 4.3 S (s_20,w = 4.5) for BubR1:Bub3 indicating a single dominant sedimentation species that corresponds to a theoretical molecular weight of 150 kDa and 144 kDa, respectively. The theoretical mass of a 1:1 complex is indicated for both in brackets. Frictional ratios were determined as 2.16 for Bub1:Bub3 and 2.21 for BubR1:Bub3. (<b>C</b>) Complexes of Bub1 exhibit similar kinase activity toward a GST-H2A substrate. The kinase activity is plotted as a function of substrate concentration to allow fitting according to the Michaelis-Menten equation with R² = 0.99. Error bars represent SD of a mean of at least two independent experiments. (<b>D</b>) Kinetic parameters of the Michaelis-Menten fits as determined in (<b>C</b>). a.u.-arbitrary units.</p

S969 is a major phosphorylation site in Bub1^kinase.

<p>(<b>A</b>) Ribbon diagram showing the model of phosphorylated Bub1^kinase. N and C indicate the N- and C-terminus, respectively. The P+1 loop harboring the phosphorylated S969 is highlighted in green, the activation loop is in blue, and the catalytic loop in red. Side chains of residues D946, D917, S969 and ADP are shown as sticks, an Mg²⁺ atom is represented by a gray sphere. (<b>B</b>) Detailed view of the active site of Bub1 showing P-S969 pointing towards the catalytic aspartate at position 917 (D917) and ADP. The electron density around the P+1 loop represents a 2F_o-F_c map contoured at 1.5σ as a blue mesh. (<b>C</b>-<b>D</b>) Overlay of phosphorylated Bub1 with unphosphorylated Bub1 [4R8Q, (<b>C</b>)] and phosphorylated Bub1 [4QPM, (<b>D</b>)]. The P+1 loop of 4R8Q and 4QPM is colored in shades of blue. All images were created with CCP4MG [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref053" target="_blank">53</a>]. (<b>E</b>) Amino acid sequence of the Bub1 P+1 loop with the mutations of S969 highlighted in red (S969D) and blue (S969A). (<b>F</b>) Bub1^kinase wild-type (WT) and S969A, S969D, S969E were incubated with ATP or λ-phosphatase (λ-pp) and analyzed on SDS PAGE using Phos-tag to detect a phosphorylation-specific shift. (<b>G</b>) P-S969 does not interfere with ATP binding of Bub1^kinase. The change in fluorescence emission at 450 nm is plotted as a function of total mant-ATPγS concentration. The data were fitted with a one site binding equation using Origin 9.0 and R² = 0.99. Error bars represent SD of a mean of at least two independent experiments. a.u.-arbitrary units. (<b>H</b>) The phosphomimetic S969D is able to restore kinase activity while S969A is catalytically inactive. The kinase activity is plotted as a function of substrate concentration to allow fitting according to Michaelis-Menten kinetics with R² = 0.99 (WT), R² = 0.97 (S969D). Error bars represent SD of a mean of at least 2 independent experiments.</p

Data Collection and Refinement Statistics.

<p>Data for the highest-resolution shell are given in parentheses.</p><p><i>R</i>_<i>merge</i> = Σ_<i>h</i>Σ_<i>i</i>|<i>I</i>_{<i>h</i>,<i>i</i>} − ⟨<i>I</i>_<i>h</i>⟩|/Σ_<i>h</i>Σ_<i>i</i><i>I</i>_{<i>h</i>,<i>i</i>}, where the outer sum (h) is over the unique reflections and the inner sum (i) is over the set of independent observations of each unique reflection.</p><p><i>R</i>_<i>work</i> = ∑_<i>hkl</i>||<i>F</i>_<i>obs</i>| − |<i>F</i>_<i>calc</i>||/∑<i>F</i>_<i>obs</i>, where F_obs and F_calc are the observed and calculated structure factors of the respective reflections hkl.</p><p>R_free is equivalent to R_work but is calculated on a random set of reflections corresponding to 5% of all reflections and that are excluded from refinement.</p><p>*As defined by MolProbity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144673#pone.0144673.ref052" target="_blank">52</a>]</p><p>Data Collection and Refinement Statistics.</p

Sensorimotor behavioural tests after CSP-TTK21 treatment in chronic SCI with severe disability.

(A) BMS quantification of CSP (n = 15) or CSP-TTK21 (n = 14) treated mice after chronic SCI (Treatment: f(1) = 0.002, p = 0.96 (TOST: given equivalence bounds of −0.21 and 0.21, 90% confidence intervals fall −0.37 and 0.32 and p = 0.307, thus H0 is undecided); Time: f(12) = 192.1, p p = 0.215, thus H0 is rejected); Interaction (treatment × time): f(12) = 6.6, p = 0.82 (TOST: given equivalence bounds of −0.21 and 0.21, 90% confidence intervals fall 4.19 and 4.99 and p > 0.999, thus H0 is rejected). Two-way repeated measures ANOVA with Sidak post hoc test. (B) Gridwalk quantification of the percentage of slips per total number of steps per run in CSP (n = 4) or CSP-TTK21 (n = 6) treated mice with a BMS score greater than 3 after chronic SCI (Treatment: f(1) = 0.2, p = 0.69 (TOST: given equivalence bounds of −1.78 and 1.78, 90% confidence intervals fall −7.96 and 2.63 and p = 0.693, thus H0 is undecided); Time: f(12) = 7.1, p p p = 0.82 (TOST: given equivalence bounds of −1.78 and 1.78, 90% confidence intervals fall −28.59 and 41.29 and p > 0.999, thus H0 is rejected). Two-way repeated measures ANOVA with Sidak post hoc test). (C) Bar graph indicating the number of mice with a BMS score greater than 3 across postinjury time points. (D) Hargreaves test indicating average paw withdrawal latency in CSP or CSP-TTK21 (Welch two-tailed t test: CSP: 6.7 ± 1.1, n = 4; CSP-TTK21: 7.6 ± 0.5, n = 6, p = 0.51, TOST: t(8) = −0.3, p = 0.63 given equivalence bounds of −0.5 and 0.5 on a raw scale and an alpha of 0.05) treated mice with a BMS score greater than 3. (E) Von Frey test indicating average paw withdrawal threshold in CSP or CSP-TTK21 (Welch two-tailed t test: CSP: 5.5 ± 1.6, n = 4; CSP-TTK21: 6.4 ± 0.7, n = 6, p = 0.64, TOST: t(4.3) = −0.1, p = 0.52 given equivalence bounds of −0.8 and 0.8 on a raw scale and an alpha of 0.05) treated mice with a BMS score greater than 3. All data are given as mean ± SEM. n = biologically independent animals. The data can be found in S1 Data. BMS, Basso Mouse Scale; CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests.</p

S4 Fig -

(A) Representative micrographs of GFAP intensity (red) around the SCI site (white asterisks) and cavity size (white dotted line) in CSP or CSP-TTK21 mice. (B) Quantification of cavity size in CSP or CSP-TTK21-treated mice (CSP: 471,574.0 ± 76,631.0, n = 8; CSP-TTK21: 486,466.0 ± 45,491.0, n = 14; p = 0.87; TOST: t(20.0) = 0.5, p = 0.31 given equivalence bounds of −56,344.2 and 56,344.2 on a raw scale and an alpha of 0.05). (C) Quantification of GFAP intensity in CSP or CSP-TTK21-treated mice (CSP: 1,272.0 ± 31.3, n = 3; CSP-TTK21: 1,339.0 ± 20.8, n = 4, p = 0.12, TOST: t(3.7) = −1.4, p = 0.88 given equivalence bounds of −14.5 and 14.5 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test or Welch t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests. (PNG)</p

Graphical diagram summarizing the experimental design. <i>Made with BioRender</i>.

(PNG)</p

S5 Fig -

(A) Representative micrographs of CD68 immunofluorescence (red) and DAPI (blue) around the SCI site (white asterisks) in CSP or CSP-TTK21-treated mice. Lesion site (white dotted line). (B) Quantification of CD68 intensity in CSP or CSP-TTK21-treated mice (CSP: 472.0 ± 8.1, n = 3 CSP-TTK21: 434.5 ± 37.8 n = 4, p = 0.44, TOST: t(3.3) = 0.5, p = 0.69 given equivalence bounds of −16.3 and 16.3 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests. (PNG)</p