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. 2012 Aug 17;287(34):28227-42.
doi: 10.1074/jbc.M112.373928. Epub 2012 Jun 13.

Sequence Determinants of a Microtubule Tip Localization Signal (MtLS)

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Free PMC article

Sequence Determinants of a Microtubule Tip Localization Signal (MtLS)

Rubén M Buey et al. J Biol Chem. .
Free PMC article

Abstract

Microtubule plus-end-tracking proteins (+TIPs) specifically localize to the growing plus-ends of microtubules to regulate microtubule dynamics and functions. A large group of +TIPs contain a short linear motif, SXIP, which is essential for them to bind to end-binding proteins (EBs) and target microtubule ends. The SXIP sequence site thus acts as a widespread microtubule tip localization signal (MtLS). Here we have analyzed the sequence-function relationship of a canonical MtLS. Using synthetic peptide arrays on membrane supports, we identified the residue preferences at each amino acid position of the SXIP motif and its surrounding sequence with respect to EB binding. We further developed an assay based on fluorescence polarization to assess the mechanism of the EB-SXIP interaction and to correlate EB binding and microtubule tip tracking of MtLS sequences from different +TIPs. Finally, we investigated the role of phosphorylation in regulating the EB-SXIP interaction. Together, our results define the sequence determinants of a canonical MtLS and provide the experimental data for bioinformatics approaches to carry out genome-wide predictions of novel +TIPs in multiple organisms.

Figures

FIGURE 1.
FIGURE 1.
X-ray crystal structure of the EB1c-MACFp1 complex. A, representation of the complex between MACFp1 (cyan sticks) and EB1c (transparent surface and schematic representation). The transparent surface of EB1c is color-coded with its electrostatic potential (from −69.74 to 69.74 KbT; red and blue depict negative and positive electrostatic potentials, respectively). Hydrogen bonds are represented with dashed orange lines. The crystal structure of the EB1c-MACFp1 (Protein Data Bank entry 3GJO) complex has been relaxed by running a 1-ns molecular dynamics simulation. The figure was prepared using PyMOL (Schrödinger, LLC). B, schematic diagram of the EB1c-MACFp1 complex. Hydrogen bonds are indicated by dashed lines between atoms, including their distance in Å. Residue labels are shown in black for EB1 and blue for MACFp1. Hydrophobic contacts are represented by arcs with spokes radiating toward the ligand atoms, which are shown with spokes radiating back. The figure has been generated using LIGPLOT (48). The chain identifiers are shown in parentheses for EB1 residue labels.
FIGURE 2.
FIGURE 2.
Sequence profiling of a canonical MtLS. A, complete single-point substitution analysis of the peptide MACFp1. Black spots indicate interactions between EB1 and membrane-bound MACFp1 variants. Each spot corresponds to a variant in which one residue of the MACFp1 sequence (given on the left) was replaced by one of the 20 gene-encoded amino acids (shown at the top). Spot SI values were determined by densitometry. The spots in the first column and the ones marked by white crosses represent replicas of the wild type MACFp1 sequence and were used to define the threshold between binding and non-binding (binding spot: SI ≥ ½(mean SI of wild type spots)). The box highlights the sequence 12PSKIPTPQRKSP23, which is the region of MACFp1 most sensitive to substitutions. The SKIP motif (i.e. which corresponds to the SXIP motif of MACFp1) is indicated by a vertical bar on the left. B, percentage of replacement variability (V) at each position of the MACFp1 sequence. The V value was calculated as V = (number of binding spots/20) × 100 and plotted against the MACFp1 sequence. The dashed lines divide sequence regions into low (V ≤ 25%), medium (75% ≥ V ≥ 25%), and high residue variability (V ≥ 75%). C, correlation between the SPOT signal (ln(SI/SIWT) and the predicted binding free energy differences. The dashed line represents the lineal regression of the different data points. The two crossed squares (P16A and K14A) indicate outliers for which the predictions failed.
FIGURE 3.
FIGURE 3.
Binding of FC-MACFp1 to EB1. A, binding isotherms obtained by FP at 30 °C. FC-MACFp1 was titrated into a solution containing either wild type EB1 (circles) or EB1-Y217A/E225A (triangles). Fractional saturation values were calculated from the FP data using equation 1 and fitted to the simple ligand binding model described by Equation 2 (see “Experimental Procedures”). B, binding isotherms obtained by ITC at 32 °C. MACFp1 (circles) or FC-MACFp1 (squares) were titrated into a solution containing EB1. Data points were fitted by using the “one set of sites” binding model described by Equation 7 (see “Experimental Procedures”). C, displacement isotherms obtained by FP at 30 °C. Shown is the displacement of EB1-bound FC-MACFp1 by increasing amounts of MACFp1 (circles) or MACFp1-NN (triangles). FP displacement data points were fitted using Equation 4 (see “Experimental Procedures”). D, binding isotherms obtained by FP at 32 °C. FC-MACFp1 was titrated into a solution containing either human EB1 (circles), S. pombe Mal3p (diamonds), or A. thaliana AtEB1A (triangles). When shown, error bars represent S.E. Symbols represent the measured data points; solid lines represent the fit to the data.
FIGURE 4.
FIGURE 4.
Thermodynamic characterization of the EB1-MACFp1 interaction. A and B, Van't Hoff (A) and Gibb's (B) plots of the EB1-MACFp1 interaction. The binding constants were determined by FP measurements at different temperatures. Shown are the experimental data points with S.D. values (error bars) and the linear regressions used to derive ΔHapp, following the van't Hoff equation, ln Kb = −ΔH/RT + ΔS/R, and ΔSapp, following the Gibbs equation, ΔG = ΔHTΔS. C, binding isotherms obtained at 30 °C for the EB1-MACFp1 interaction in the presence of different NaCl concentrations: 50 mm (circles), 150 mm (squares), 300 mm (diamonds), and 500 mm (triangles). Symbols represent the measured fractional saturation values calculated from the FP data using Equation 1. Error bars, S.E. values; solid lines, best fit to the data according to a simple ligand binding model described by Equation 2 (see “Experimental Procedures”).
FIGURE 5.
FIGURE 5.
Effects of phosphorylation of MACFp1 on EB1 binding. The sequence 2–28 of MACFp1 is shown at the top. The serine and threonine residues that were systematically phosphorylated are underlined, and the corresponding residue number is shown below. The SKIP motif is highlighted by a line above the sequence. The bottom panel displays schematic representations of the phosphorylated MACFp1 variants tested. Phosphorylated residues are indicated. For simplicity, double numbering of sites (e.g. pS5/8) refers to single modified residues (i.e. phospho-Ser-5 or -8 (pS5 or pS8)). Phosphorylated MACFp1 variants are classified as EB1 binders (right) and non-binders (left). See also supplemental Fig. 2.
FIGURE 6.
FIGURE 6.
EB1 binding affinities of different MtLS-containing +TIPs peptides. A, sequence alignment of +TIP fragments encompassing an MtLS: MACF2, accession number NP_899236 (6); APC, accession number P25054 (49); melanophilin, accession number AAH01653 (50); MCAK, accession number NP_006836 (51); Ipl1, accession number P38991 (24, 25); CLASP2, accession number NP_055912 (19); STIM1, accession number Q13586 (20); DDA3, accession number AAN73431 (52); FILIP, accession number NP_056502 (Jiang et al.)4; SLAIN2, accession number NP_065897 (21); Navigator, accession number NP_065176 (53); TIP150 (Tip-interacting protein of 150 kDa), accession number Q5JR59 (54); p140Cap, accession number P30622 (23); and CLIP170, accession number P30622 (55). The sequences are grouped into EB binders (Kd in the range between 1 and 140 μm) and non-binders (Kd in the millimolar range or higher). The residues proposed to compensate for the unfavorable residue at the X position of the SXIP motif in melanophilin, and DDA3 are underlined. APC residues encompassing the SXIP motif that were shown to interact with EB1c by NMR (15) are underlined with a discontinuous line (see “Results” for more details). B, displacement isotherms obtained by FP at 30 °C. Shown is the displacement of EB1-bound FC-MACFp1 by increasing amounts of MACFp1 (circles), APCp1 (squares), TrxMACF (diamonds), TrxAPC (triangles), and Trx alone (inverted triangles). C, binding isotherms obtained by ITC at 25 °C. MACFp1 (circles) or TrxMACF (diamonds) were titrated into a solution containing EB1. Data points (symbols) were fitted (solid line) by using the “one set of sites” binding model, following Equation 7, as described under “Experimental Procedures.” D, displacement isotherms obtained by FP at 25 °C. Shown is the displacement of EB1-bound FC-MACFp1 by increasing amounts of TrxMACF (diamonds), TrxCLASP (triangles), and TrxSLAIN (squares). E, displacement isotherms obtained by FP at 25 °C. Shown is the displacement of EB1-bound FC-MACFp1 by increasing amounts of unlabeled TrxMACF (diamonds), TrxIpl1-p1 (inverted triangles), TrxIpl1-p2 (circles), TrxMACF-GCN4 (triangles), and tandem TrxIpl1-p12 (squares). When shown, error bars represent S.E. values. FP-derived fractional saturation values (symbols) were calculated from the FP data, according to Equation 1, and fitted (solid line) to Equation 4, as described under “Experimental Procedures.”
FIGURE 7.
FIGURE 7.
Live cell imaging of monomeric and dimeric MtLS-containing peptides fused to GFP. Live images of COS-7 cells transiently transfected with the indicated GFP fusions are shown in two different ways. Panels on the left show single frames obtained by averaging of five consecutive images acquired with a 0.5-s interval (2.5 s in total). Panels on the right show maximum intensity projections of six consecutive and averaged frames, displayed in different colors. The first of the six frames is shown as grayscale (microtubule plus-ends are white), frames 2, 4, and 6 are shown in green (plus-ends appear green), and frames 3 and 5 are shown in red (plus-ends appear red). This representation facilitates the visualization of microtubule tip tracking events over time. Insets, enlargements of small parts of the images to illustrate the presence or absence of comet “tracks” reflecting +TIP accumulation at growing microtubule ends.

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