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Research Article
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Systematic identification of recognition motifs for the hub protein LC8

View ORCID ProfileNathan Jespersen, Aidan Estelle, View ORCID ProfileNathan Waugh, Norman E Davey, View ORCID ProfileCecilia Blikstad, York-Christoph Ammon, Anna Akhmanova, Ylva Ivarsson, David A Hendrix, View ORCID ProfileElisar Barbar  Correspondence email
Nathan Jespersen
1Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA
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  • ORCID record for Nathan Jespersen
Aidan Estelle
1Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA
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Nathan Waugh
1Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA
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Norman E Davey
2Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Ireland
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Cecilia Blikstad
3Department of Chemistry - Biomedical Centre, Uppsala University, Uppsala, Sweden
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York-Christoph Ammon
4Department of Biology, Utrecht University, Utrecht, The Netherlands
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Anna Akhmanova
4Department of Biology, Utrecht University, Utrecht, The Netherlands
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Ylva Ivarsson
3Department of Chemistry - Biomedical Centre, Uppsala University, Uppsala, Sweden
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David A Hendrix
1Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA
5School of Electrical Engineering and Computer Science, Oregon State University, Corvallis, OR, USA
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Elisar Barbar
1Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA
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  • ORCID record for Elisar Barbar
  • For correspondence: barbare@oregonstate.edu
Published 2 July 2019. DOI: 10.26508/lsa.201900366
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  • Figure 1.
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    Figure 1. Motif sequence logo and surface analysis of LC8.

    (A) Crystal structure of a representative LC8 dimer (protomers shown in shades of green) bound to a peptide (shades of red). (B) Electrostatic charge potential for the LC8 pocket structure using PyMOL’s charge-smoothed potential calculator, with positive potentials shown in blue, negative in red, and neutral in white. Peptides from available crystal structures of bound LC8 are shown, and colored based upon amino acid chemical characteristics (right). Amino acid enrichment is shown below each position within the LC8-binding motif, calculated from 79 known binder motifs listed on the LC8 database (http://lc8hub.cgrb.oregonstate.edu). Amino acid letter heights represent relative enrichment of that amino acid. (C) Solvent accessible surface area depiction of the same LC8/peptide pair shown in (A). Color scheme was defined at the atomic level using the GetArea program (Fraczkiewicz & Braun, 1998), with magenta representing more solvent exposed and orange regions more buried atoms.

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    Figure 2. LC8 and its binding partners display broad cellular localization.

    (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.

  • Figure 3.
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    Figure 3. Thermodynamic analysis of binding peptides.

    (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 (Kd 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. Kds 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.

  • Figure 4.
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    Figure 4. Analysis of LC8-binding and nonbinding motifs reveals distinct positional preferences.

    (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 (Kd < 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.

  • Figure 5.
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    Figure 5. LC8 is structurally variable but conserved in sequence.

    (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).

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    Figure 6. Generation and testing of The LC8Pred algorithm.

    (A) PSSMs for amino acids (A, top), bins by chemical property—positively charged, negatively charged, polar, or nonpolar (middle), and bins by volume—less than 106 A3, 122 to 142 A3, 155 to 171 A3, and greater than 200 A3 (bottom). Values correspond to the combined weight at a given position for the binder-only matrix and the nonbinder-normalized matrix. (B) Scatterplot of available sequences scored using a leave-one-out method of cross validation. For binders with a known Kd, the size of the bubble was varied inversely with the Kd, with binders with a Kd below 0.5 μM represented as the maximum possible dot size. Binder sequences with an unknown binding affinity were plotted as hollow circles and nonbinders as red triangles. The light grey box denotes predicted binding sequences using this scoring system. A second threshold for the volume and polarity axis indicates the very high confidence region, above which the specificity is unity. Outliers are noted in the tables (inset) and numbered in figure. (C) Normalized scores from matrices used to evaluate known LC8-binding protein Chica, where a score of one equates to the ideal amino acids of physicochemical properties at all positions. A sliding window to evaluate Chica for predicted binding sites across the protein was used, with the “0” position within the motif plotted (i.e., at 400, the 0 position is the 400th amino acid within Chica). A diagram of Chica showing secondary structure prediction (grey) and LC8 binding sites (purple) is above, and sequences predicted to bind are on the right, along with their corresponding scores. (D) Venn diagram of human proteins in the LC8Hub database, proteins that contain at least one LC8-binding sequence as determined by LC8Pred, and proteins reported to bind LC8 in the protein–protein interaction database Mentha (Calderone et al, 2013).

Tables

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    Table 1.

    Peptides synthesized based on their binding in phage display experiments.

    UniProtGeneSequenceStartEndBinder?Citation
    O43521BCL2L11APMSCDKSTQTPSP108117YPuthalakath et al (1999)
    Q9UPA5BSNIIPRATAEFSTQTPSP1,4981,511YFejtova et al (2009)
    Q86VQ1GLCCI1SSSTRSIDTQTPSV340353YHutchins et al (2010)
    Q96R06SPAG5HPETQDSSTQTDTS463476YSchmidt et al (2010)
    Q6IMN6CAPRIN2NQSFTTASTQTPPQ791804Y
    O75665OFD1AKESCNMETQTSST153166YBoldt et al (2016)
    Q02505MUC3APVLTSATGTQTSPA1,8001,813Y
    Q9UBY0SLC9A2DDHSREKGTQTSGD749804Y
    Q9Y2F5ICE1EKELRHIGTQISSD181194Y
    Q9ULV3CIZ1ARAGRSVSTQTGSM1326Yden Hollander & Kumar (2006)
    Q99102MUC4SQNHWTRSTQTTRE200213Y
    Q99102MUC4DDNHWTRSTQTTRE200213Y
    P07359GP1BAGQGAALTTATQTTHLE566581Weaka
    Q9Y4F4TOGARAM1SKTQQTFGSQTECT788801Weaka
    Q8WWN8ARAP3SPSPTGLPTQTPGF1,5141,527Weaka
    Q01973ROR1DDSGGNATTQTTSD760774Weaka
    Q8NEZ4KMT2CIVSCVSVSTQTASD205218N
    Q9UPA5BSN-shiftbSTQTPSPAPASDMP1,5051,518N
    Q7Z2Z2EFL1DERLMCTGSQTFDD375386N
    Q02817MUC2TPTPTPTGTQTPTT2,0002,013N
    Q9HC84MUC5B-3SMATPSSSTQTSGT2,6732,686N
    Q8TEC5SH3RF2TLVSTASGTQTVFP714727N
    Q9P2G1ANKIB1RGDGSDVSSQTPQT1,0651,078N
    O43526KCNQ2DDPMYSSQTQTYGD370380N
    P14859POU2F1ESGDGNTGTQTNGL1326N
    P35568IRS1LPRKVDTAAQTNSR841854N
    Q2KHR3QSER1KTLTFSGSSQTVTP374387N
    Q99814EPAS1TEAKDQCSTQTDFN509522N
    Q9Y4K1CRYBG1RSFVLPVESTQDVSSQ550565N
    P49862KLK7SFRHPGYSTQTHVN98111N
    Q92904DAZLTQDDYFKDKRVHHFRRS272288N
    Q96FV2SCRN2VRTLPRFQTQVDRR342355N
    Q96FV2SCRN2DDTLPRFQTQVDRR344355N
    Q7Z589EMSYKITFTKPSTQTTNT261274N
    Q13952NFYCCLKETLQITQTEVQ289302N
    Q9HC84MUC5B-1TTLPVLTSTATKST3,0493,062N
    P53350PLK1AASLIQKMLQTDPTAR278293N
    Q92499DDX1DDHSGNAQVTQTKFD271282N
    Q8NBH2KYITSYNSQGTQLTVE8194N
    Q06190PPP2R3ALQETLTTSSQANLS625638N
    Q13618CUL3KHSGRQLTLQHHMG542555N
    Q9H4B6SAV1NQSFLRTPIQRTPH7083N
    Q2TV78MSTL1EGYRGTANTTTAAYLA259274N
    Q6ZU65UBN2PLQATISKSQTNPV942955N
    Q96SC8DMRTA2SSRSAFSPLQPNAS433446N
    Q6ZRI0OTOGTLQQPLELTASQLPAG1,5411,556N
    Q96JG9ZNF469RAAALPEETRSSRR1,0141,027N
    • Anchor motifs are underlined.

    • Aspartates shown in italics were added to increase solubility.

    • ↵a Peptides that displayed an interaction with LC8 via ITC, but the data were not of sufficient quality to obtain reliable Kd measurements.

    • ↵b BSN-ProP-PD is the Bassoon sequence pulled down by phage display, without shifting the TQT sequence into the correct position.

    • View popup
    Table 2.

    Peptides synthesized based on favorable sequence comparisons.

    UniProtGeneSequenceStartEndBinder?Citation
    Q6LCS3E4 (HPV)YLQGRQEDKQTQTPPP1630Y
    Q8IX07ZFPM1PAPPSYSDKGVQTPSK947962Y
    P11193VP4 (Rotavirus A)YVTNSLNDISTQTSTI600614Y
    P13500CCL2YDSMDHLDKQTQTPKT8599Y
    P18583SONYSRKSRCVSVQTDPT87100Y
    Q9Y2H9MAST1YGCTRHQSVQTEDG1,3871,399YNavarro-Lérida et al (2004)
    P80098CCL7QDFMKHLDKKTQTPKL8499Y
    Q5K4E3PRSS36YGPDGEETETQTCPP468581N
    P20702ITGAXYGQIAPENGTQTPSP1,1461,159N
    P03586MT/HEL (TMV)AQPKQKLDTSIQTEYP1,3051,320N
    Q8IYH5ZZZ3KSVAENGDTDTQTSMF237252N
    Q5DMI6DNLJ2 (phage T5)YKIEIPTQCPSCGSK215N
    Q92904DAZLYPQKKSVDRSIQTVVS243257N
    Q9NZ56FMN2YHHRILEAKSIQTSPT735749N
    Q13418ILKMDDIFTQCREGN112N
    O43432EIF4G3DFTPAFADFGRQTPGG676691N
    Q99613EIF3CYELMASLDQPTQTVVM830844N
    O15444CCL25NKVFAKLHHNTQTFQA94109N
    P20042EIF2S2KPFMLDEEGDTQTEET2136N
    • Anchor motifs are underlined.

    • Tyrosines shown in italics were added for accurate concentration determination.

Supplementary Materials

  • Figures
  • Tables
  • Table S1 Binding enriched peptides from ProP-PD.

  • Table S2 LC8Pred scan of the human proteome reveals 378 likely binding sequences.

  • Table S3 LC8 binding sequences identified and verified in this study.

  • Table S4 Binding sequences used for algorithm generation. Nonbinding sequences used for algorithm generation.

  • Supplemental Data 1.

    [LSA-2019-00366_Supplemental_Data_1.docx]

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Motif recognition of the dynamic hub LC8
Nathan Jespersen, Aidan Estelle, Nathan Waugh, Norman E Davey, Cecilia Blikstad, York-Christoph Ammon, Anna Akhmanova, Ylva Ivarsson, David A Hendrix, Elisar Barbar
Life Science Alliance Jul 2019, 2 (4) e201900366; DOI: 10.26508/lsa.201900366

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Motif recognition of the dynamic hub LC8
Nathan Jespersen, Aidan Estelle, Nathan Waugh, Norman E Davey, Cecilia Blikstad, York-Christoph Ammon, Anna Akhmanova, Ylva Ivarsson, David A Hendrix, Elisar Barbar
Life Science Alliance Jul 2019, 2 (4) e201900366; DOI: 10.26508/lsa.201900366
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