Systematic identification of recognition motifs for the hub protein LC8

(A) Live HeLa cells stably expressing LC8-GFP (green) were transiently transfected with the focal adhesion marker TagBFP-paxillin (blue) and the nucleus marker dsRed-tagged histone H2B (red). The top view images shown on the left represent an optical section located next to the coverslip. LC8-GFP is present throughout the cell but forms puncta at the cell cortex. (B) HeLa cells stably expressing LC8-GFP were fixed with PFA and stained for the endogenous α-tubulin (red) and with DAPI (blue) to visualize the DNA. LC8 accumulates at the kinetochores (box 1) and at the spindle poles (box 2). In both (A) and (B), images were acquired from a single cell each, using confocal spinning disc microscopy. (C) Localization information derived from the COMPARTMENTS program demonstrates that LC8 binding partners are localized to all cellular compartments. High confidence localization data were available for 59 of the 73 eukaryotic proteins listed on the LC8Hub database.

(A) Representative ITC thermograms of LC8 bound to 14–amino acid–long synthetic peptides. The data were collected at 25°C in 50 mM NaCl, 50 mM NaPO4, and 1 mM NaN₃, pH 7.5. Experiments were performed in triplicate. We categorized peptides as strong binders (K_d reliably determined; SPAG5), weak binders (heat generated, but unable to fit the data; TOGARAM1), and nonbinders (QSER1). Weak binders are those with affinities >25 μM. (B) Binding affinities and thermodynamic parameters for strong LC8 binders identified in this study. Thermodynamic parameters for all binding peptides at 25°C are shown. ΔG (black), ΔH (orange), and TΔS (blue) kcal/mol values are the average of two to three independent ITC experiments. K_ds are shown in μM. 8 amino acid motifs are shown, with residues capable of making conserved hydrogen bonding interactions highlighted in grey. Sequences are ordered by descending ΔH values.

(A) Motif preferences for LC8 binding partners. “Ф” denotes hydrophobic residues; “X” signifies any residue (unless certain residues are disfavored); underlined “X” signifies any residue but with strong preferences for particular residues; “+” denotes positively charged amino acids. Physiochemical properties beneficial for binding are colored dark blue or light blue, based on magnitude, and deleterious properties are colored in red. (B) All known tightly binding sequences (K_d < 10 μM) are cropped to 8 amino acid motifs and built using the Chimera molecular modeling software. This includes LC8 sequences found on the LC8Hub database, and those determined in this article. (C) Overlay of all nonbinding peptides used in this study. Residues are colored based upon whether they are beneficial (blue), deleterious (red), or neutral (white) for binding, using the amino acid enrichment and depletion in known motifs (Fig 6A). (D) Categories of nonbinding sequences. Residues highlighted in red depict the reason the sequence is placed within a given category. *Denotes sequences placed in multiple categories.

(A) Surface representation of LC8 colored by sequence conservation using ConSurf. More sequence-conserved regions are shown in magenta, less sequence-conserved regions are shown in cyan. Highly conserved residues map to those within the LC8 binding site. (B) Surface representation of LC8 colored by structural conservation in the free protein using the Ensemblator. Regions that are more structurally variable are shown in red, whereas more structurally conserved regions are shown in blue. An overlay of NMR and crystal structure protomers used for the structural analysis is shown as a cut-out in (B). (C) 2D depiction of the binding interface between an example peptide (orange) and the binding β-strand within LC8 (Teal). (D, E) Polar bonds between LC8 and peptides from crystal structures are shown in (D) (top down view, only backbone interactions) and (E) (pocket view). Colors of polar contacts are based on whether the polar contacts stem from backbone (yellow) or side chain (purple) residues on the peptide. Peptide residues with frequent side chain interactions are labeled in red. (C, E) Residues outside of the binding β-strand that are important interaction sites shown in (C) are labeled in (E).