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. 2016 Sep 13;5:e17523. doi: 10.7554/eLife.17523

Figure 4. A conserved sequence motif of Spir binds to MyoV-GTD.

(A) A short highly conserved sequence motif within the central Spir linker region is responsible for myosin V binding. Alignment of vertebrate Spir-1 and Spir-2 sequence fragments. The short sequence motif of about 27 amino acids in the middle part of the Spir linker region shows high sequence homology (see also Figure 4—figure supplement 1). Species abbreviations: Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Xenopus tropicalis (Xt), Brachydanio rerio (Br). All sequence-related data are available through CyMoBase (http://www.cymobase.org). Hs-Spir-2 residues contacting MyoVa within 4 Å distance are labeled with '*'; the Spir-2 conformation is stabilized by two conserved residues (see panel D) indicated with '+'. (B) Fluorescence anisotropy measurements of the binding of the Fluorescein-Spir-2-GTBM peptide (human, amino acid residues 401–427) to the MyoVa and MyoVb GTDs. The equilibrium dissociation constants (Kd) for MyoVa and MyoVb GTDs were determined by fitting the titration curves as detailed in the Materials and Methods section. The experiments were repeated twice with two different protein preparations. (C) Crystal structure of the MyoVa-GTD:Spir-2-GTBM complex. Spir-2-GTBM (purple) binds to subdomain-1 (SD-1) of MyoVa-GTD (orange). (D) Close-up view of the Spir-2-GTBM bound to MyoVa-GTD. Residues forming the interaction sites are shown as sticks and are labeled. Spir-2 E416 and T413 form intramolecular hydrogen bonds (dashed lines). Spir-2 V407, L409 and MyoVa N1590 and D1592 backbone atoms form an intermolecular β-structure like hydrogen bond network. (E) MLPH-GTBM bound to MyoVa-GTD (PDB ID 4LX2). The N-terminal part of MLPH-GTBM (residues from K186 to L189) interacts with MyoVa in a similar manner to Spir-2 (residues 406–409). A similar hydrogen bond between MyoVa E1595 and the main chain nitrogen of MLPH F191 is also observed in the Spir-2:MyoVa interface. (F) Structure based sequence alignment of the Spir-2-GTBM and MLPH-GTBM fragments. Residues making similar contacts with MyoVa are highlighted with a black line. (G) Hydrophobic residues anchoring Spir-2-GTBM on the MyoVa-GTD surface. Spir-2 A411 is packed on the top of MyoVa Y1596 (green) side chain (see also panel D). (H) Spir-2 (purple) and MLPH (gray) GTBMs docked on the surface of apo-MyoVa-GTD (PDB ID 4LX1). The apo-MyoVa-GTD hydrophobic cleft between the H5 and H3 is compatible with Spir-2 binding, but not with MLPH, where the side chain of F191 clashes with MyoVa Y1596 (green). In Spir, the conserved L414 side chain anchors the C-terminal Spir-2 fragment extending the interacting hydrophobic surface compared to what is found for MLPH-GTBM binding. (I) Hydrophobic residues anchoring MLPH-GTBM in the MyoVa-GTD pocket (PDB ID 4LX2). The MyoVa Y1596 (green) side chain is rotated to bury the side chain within the protein core (see also panel E) to accommodate MLPH F191 in the binding pocket.

DOI: http://dx.doi.org/10.7554/eLife.17523.007

Figure 4.

Figure 4—figure supplement 1. Gene and protein sequence structures of vertebrate Spir.

Figure 4—figure supplement 1.

(A) Exon/intron structure of human Spir-1 and Spir-2. The location of the fourth WH2 domain, MyoV binding motif (GTBM), Spir-box (SB) and FYVE-type zinc-finger is indicated. (B) Alignment of vertebrate Spir-1 and Spir-2 sequences starting from the last WH2 domain to the FYVE-type zinc finger. High sequence homology between all sequences was only observed for the WH2 domain, the Spir-box, the FYVE-type zinc finger and the GTBM, but not for the rest of the linker region. Species abbreviations are: Homo sapiens (Hs), Mus musculus (Mm), Gallus gallus (Gg), Xenopus tropicalis (Xt), Brachydanio rerio (Br). All sequence_related data are available through CyMoBase (http://www.cymobase.org).
DOI: http://dx.doi.org/10.7554/eLife.17523.008
Figure 4—figure supplement 2. Mapping of the Spir-2 myosin binding domain.

Figure 4—figure supplement 2.

(A) Co-immunoprecipitation experiments employing AcGFP-tagged MyoVb-GTD (GFP-MyoVb-GTD) as well as N-terminal and C-terminal Spir-2 deletion mutants show myosin V co-precipitation with the C-terminal Myc-Spir-2-398-714 protein and the N-terminal Myc-Spir-2-2-423 protein, but not with C-terminal Myc-Spir-2-411-714 protein and the N-terminal Myc-Spir-2-2-410 protein. Numbers indicate amino acids. N = 2–4 experimental repeats. (B) Overview of the Spir-2 deletion mutants used in (A) compared to full-length Spir-2 and their capacity to bind (+) or not bind (−) to MyoVb-GTD. (C) The minimal mapped Spir-MyoV interaction sequence of human Spir-2 contains amino acids 398 to 423.
DOI: http://dx.doi.org/10.7554/eLife.17523.009
Figure 4—figure supplement 3. Comparison of myosin V isoforms Spir/MLPH binding sites.

Figure 4—figure supplement 3.

(A) Spir-2-GTBM (purple), represented in sticks, bound to MyoVa-GTD (orange). Shown in orange sticks and labeled are the side chains of the MyoVa-GTD residues whose solvent accessible area is reduced upon complex formation. Hydrogen bonds are represented as dashed lines. (B) MyoVa-GTD undergoes minor conformational change upon Spir-2-GTBM binding. MyoVa bound to Spir-2-GTBM is shown in orange while apo-MyoVa (PDB ID 4LX1) is white. Upon Spir-2 binding MyoVa, H3 is shifted along the helix axis by 0.9Å and the side chains of Y1596, F1591, K1539, R1528 change slightly their orientations to accommodate the partner. (C) The MyoVb structure is compatible with Spir-GTBM binding. The Spir-2-GTBM binding site is almost identical in MyoVa-GTD (orange) and MyoVb-GTD (PDB ID 4J5M) (white) (Nascimento et al., 2013). Residue labeling corresponds to the MyoVb sequence. Residues that are not identical in the MyoVb and MyoVa sequences are labeled in red. (D) MyoVc binds Spir-GTBM. The Spir-2-GTBM binding site is similar in MyoVa-GTD (orange) and MyoVc-GTD (PDB ID 4L8T) (white) (Nascimento et al., 2013). Residue labeling corresponds to the MyoVc sequence, the labels for identical residues are in black and for homologous residues in red. (E) Fluorescin-Spir-2-GTBM peptide (human, amino acids 401–427) binding to MyoVc-GTD measured by microscale thermophoresis. N = 3 experimental repeats.
DOI: http://dx.doi.org/10.7554/eLife.17523.010
Figure 4—figure supplement 4. MyoVa conformational change upon MLPH binding.

Figure 4—figure supplement 4.

(A) MLPH-GTBM (white), represented in sticks, bound to MyoVa-GTD (orange). Shown in orange sticks and labeled are the side chains of MyoVa-GTD residues whose solvent accessible area is reduced upon complex formation. Hydrogen bonds are represented as dashed lines. (B) MyoVa-GTD conformational change upon MLPH-GTBM binding. MLPH-GTBM bound MyoVa is orange, apo-MyoVa-GTD (PDB ID 4LX1) is white. MLPH-GTBM binding requires a small (0.5 Å) shift of MyoVa H3 along the helix axis; and conformational changes of Y1596, I1535 and R1528 side chains.
DOI: http://dx.doi.org/10.7554/eLife.17523.011
Figure 4—figure supplement 5. Structural similarities of Spir and melanophilin.

Figure 4—figure supplement 5.

(A) Spir-GTBM sequence logo indicates the sequence conservation in Spir-1 and Spir-2 proteins; MLPH-GTBM sequence logo demonstrates the sequence conservation in MLPH proteins. WebLogos (Crooks et al., 2004 ) were generated from respective parts of the alignment of 223 Spir sequences and 44 MLPH sequences after reducing redundancy with CDhit (Li and Godzik, 2006) applying a 90% sequence similarity cut-off. The Spir and MLPH motifs involved in binding the MyoV globular tail domain have a similar charge distribution (the N-terminal sequence is positively charged and the C-terminal sequence is negatively charged). Both fragments have two conserved leucine residues in the N-terminal part of the binding motif. (B) Comparison of the Spir-2 and MLPH domain composition. Both MyoV interacting proteins share a GTBM which is linked to a putative small GTPase interacting Spir-Box (SB) in Spir-2 or synaptotagmin-like protein homology domains (H1 and H2) in MLPH and FYVE-type membrane binding units. The MLPH N-terminal region encompassing the H1 FYVE-type H2 region interacts with the Rab27 GTPase (Kukimoto-Niino et al., 2008). Despite its structural similarity to membrane binding domains, a direct interaction of the MLPH FYVE-type domain has not yet been addressed. MLPH also contains a second MyoVa exon-F binding domain (EFBD), and an F-actin binding domain (ABD) at the C-terminus.
DOI: http://dx.doi.org/10.7554/eLife.17523.012