snap md  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: Pf K5ΔL6-MD is a slow MT-stimulated ATPase. A , left , domains of full-length Pf K5 and Pf K5ΔL6-MD, displaying the N-terminus (Nt), motor domain (MD), neck linker (NL), stalk (S), C-terminus (Ct) aa = amino acids; right , Coomassie-stained SDS-PAGE of Pf K5ΔL6-MD after purification. B , Pf K5ΔL6-MD ATPase rate in the absence of MTs. Technical replicates = 12 ( circles ), experimental replicates = 4 ( triangles ), biological replicates ( i.e. , number of different protein purifications used in the experiments) = 2. The mean of experimental replicates (0.039 ATP s −1 ) and 95% confidence interval are plotted. C , Pf K5ΔL6-MD MT stimulated ATPase activity. The mean and standard deviation of three experimental replicates (no technical replicates) are plotted. Biological replicates = 2. The fit is plotted as a blue line , with corresponding 95% confidence interval plotted as black lines . Inset displays an example of raw data (A = ATP, P = Pf K5ΔL6-MD, and M = MTs). D , Pf K5ΔL6-MD MT stimulated ATPase activity as in ( C ), except with ATP as the substrate variable, and using a constant of 1 μM MT.

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques: Staining, SDS Page, Purification, Activity Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: Pf K5ΔL6-MD drives plus-end directed MT gliding and its MT interactions are nucleotide modulated. A , schematic of the Pf K5ΔL6-MD-SNAP fusion protein used for TIRFM experiments, and SDS-PAGE of Pf K5ΔL6-MD-SNAP after purification. ∗ indicates the band for Pf K5ΔL6-MD-SNAP. B , Pf K5ΔL6-MD-SNAP driven MT gliding velocity. Technical replicates = 75 ( circles ), experimental replicates = 5 ( triangles ), biological replicates = 3. The mean of experimental replicates (5.4 nm/s) and 95% confidence interval are plotted. Grayscale images on the right are snapshots of a single bright plus-end labeled MT over time. C , example kymographs from Pf K5ΔL6-MD-SNAP single molecule MT-binding experiments, with each vertical white streak corresponding to a single Pf K5ΔL6-MD-SNAP binding event. Concentrations refer to the amount of Pf K5ΔL6-MD-SNAP required to see single molecule binding for each nucleotide. D , Pf K5ΔL6-MD-SNAP MT association rates in different nucleotide states (NN = no nucleotide). For each nucleotide condition, technical replicates ( circles ) and experimental replicates ( triangles ) are plotted, in addition to the mean and 95% confidence interval of experimental replicates. Number of MTs = 34, 22, 46, 28, 29 for the background, ATP, ADP, no nucleotide, and AMPPNP states, respectively. E , frequency distribution of Pf K5ΔL6-MD-SNAP MT dwell times, in different nucleotide states. Number of experimental replicates = 3; however, frequency distributions are calculated from pooled experimental data. Number of biological replicates = 2. The fit for one-phase exponential decay models is shown, with corresponding decay constant (k off ). Number of events = 1065, 1084, 1799, 1717 for ATP, ADP, no nucleotide, and AMPPNP states, respectively. F , mean MT association (k MTlanding ) as a function of MT dissociation (k off ), plotted with 95% confidence intervals.

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques: SDS Page, Purification, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: Cryo-EM data collection, 3D image processing and model refinement statistics

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: Cryo-EM 3D reconstruction of Pf K5ΔL6-MD MT complexes . A , example micrograph of the Pf K5ΔL6-MD bound MTs with 5 mM AMPPNP. White arrows indicate Pf K5ΔL6-MD decoration present every 8 nm (1 αβ-tubulin dimer). B–E , 3D reconstructions have been locally low-pass filtered according to local resolution. B , the unsymmetrized (C1) reconstruction of the Pf K5ΔL6-MD no nucleotide state bound to MTs, depicting the central portion of the MT reconstruction. C , as in ( B ), for the AMPPNP state. D , reconstruction of the Pf K5ΔL6-MD no nucleotide state bound to αβ-tubulin after asymmetric unit refinement. E , as in ( D ), for the AMPPNP state. F , Ribbon depiction of the no nucleotide state model in corresponding cryo-EM density. G , as in ( F ), for the AMPPNP state. In ( F ) and ( G ), key components of the Pf K5ΔL6-MD are labeled and color-coded as indicated.

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques: Cryo-EM Sample Prep, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: AMPNPP binding causes Pf K5ΔL6-MD subdomain rearrangement and switch loop closure. A , rearrangements of Pf K5ΔL6-MD switch loops upon AMPPNP binding, with a schematic showing connectivity of the switch loops to motor domain secondary structure elements on the left. Models and corresponding density are displayed on the right, with nucleotide-binding loops colored according to the schematic. The brown arrow indicates where there is missing density for loop11, and the gray arrow indicates connecting density between loop9 and 11. B , comparison of Pf K5ΔL6-MD no nucleotide and AMPPNP nucleotide binding loops, demonstrating AMPPNP induced conformational changes. C , the Pf K5ΔL6-MD no nucleotide state model, coloured according to kinesin subdomain, with α and β-tubulin depicted in light and dark gray surface rendering, respectively. D , Pf K5ΔL6-MD subdomain rearrangement, showing no overall movement in the tubulin-binding subdomain, with rotations in the P-loop subdomain shown on the left , and the switch-I/II subdomain on the right .

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: AMPNPP binding supports Pf K5ΔL6-MD cover neck bundle formation. A , no nucleotide Pf K5ΔL6-MD model and corresponding cryo-EM density of the cover neck bundle region. Helixα6/neck linker, the N-terminus, and loop13 are colored according to the schematic. Note the lack of density for the neck linker at the terminus of helixα6. +/− symbols denote the MT polarity. Pink arrow indicates the lack of density for an ordered neck linker. B , as in ( A ) for the AMPPNP state (at equivalent density thresholds), showing density corresponding to the neck linker docked along the motor domain and cover neck bundle formation (CNB), which is highlighted by the dotted black circle .

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: Pf K5ΔL6-MD has an altered MT interface. A , The Pf K5ΔL6-MD no nucleotide state model in ribbon and coloured light blue with secondary structure elements partaking in the MT interface colored according to the key below. B , as for in ( A ), for the Pf K5ΔL6-MD AMMPNP state model, colored in light orange . C , upper image , no nucleotide Pf K5ΔL6-MD density, rotated 90° from ( A ), and colored according to αβ-tubulin when a particularly area of density is <7 Å away. Lower image , αβ-tubulin density from the no nucleotide state, colored according to different Pf K5ΔL6-MD secondary structure elements, as outlined in the key. D , as in ( C ), for the Pf K5ΔL6-MD AMPPNP state.

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

doi: 10.1016/j.jbc.2021.101063

Figure Lengend Snippet: Pf K5ΔL6-MD loop5 alters the environment of the kinesin-5 drug-binding site. A , primary sequence alignment of loop5 from Hs K5 (UniProt ID: P52732), Pf K5 (O77382) and various other Plasmodium species ( Pv = vivax (A0A564ZV10), Pk = knowlesi (A0A1Y3DTC2), Pb = berghei (A0A122I4M3)). Conserved positions are colored according to the ClustalX scheme, and a conservation score as calculated in Jalview is given below. B , Loop5 density ( gray ) in the no nucleotide state, compared with other Pf K5ΔL6-MD density ( blue ). C , Loop5 density ( gray ) in the AMPPNP state, compared with other Pf K5ΔL6-MD density ( left ), and the Pf K5ΔL6-MD model ( right ). D , the STLC bound Hs K5 crystal structure in lime (PDB ID: 2WOG ), which was rigid body fitted into the Pf K5ΔL6-MD AMPPNP state map. STLC is colored purple , and Hs K5 loop5 is colored blue . Pf K5ΔL6-MD loop5 cryo-EM density is depicted in gray . E , conservation of residues partaking in STLC binding between Pf K5ΔL6-MD and Hs K5, based on primary sequence alignment in ( A ). Using PDB ID: 2WOG , Hs K5 residues contacting STLC were found using Chimera, and are displayed, with loop5 residues colored blue , and residues from helicesα2-3 colored green . Equivalent Pf K5 residues are shown in gray with Hs K5 residue labels. Nonconserved residues are displayed in red boxes , with conserved ones shown in black boxes . F , ATPase rates of Pf K5ΔL6-MD and Hs K5 rate in the absence of MTs, with either no treatment, + 20 μM STLC, or DMSO (with the same % v/v as the % v/v of STLC). Statistical relationships were tested using a one-way ANOVA, followed by a post-hoc Tukey's multiple comparison test.

Article Snippet: Pf K5ΔL6-MD-SNAP was biotinylated or fluorescently labeled by overnight incubation at 4 °C with SNAP-Biotin or SNAP-Surface Alex Fluor 647 (New England BioLabs) with at least a 3:1 M excess of these labels to Pf K5ΔL6-MD-SNAP.

Techniques: Binding Assay, Sequencing, Cryo-EM Sample Prep