Levine TP (2020), Structural bioinformatics predicts that the Ret...

XB-ART-57478

F1000Res 2020 Jan 01;9:1052. doi: 10.12688/f1000research.25870.1.

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Structural bioinformatics predicts that the Retinitis Pigmentosa-28 protein of unknown function FAM161A is a homologue of the microtubule nucleation factor Tpx2.

Levine TP .

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Background: FAM161A is a microtubule-associated protein conserved widely across eukaryotes, which is mutated in the inherited blinding disease Retinitis Pigmentosa-28. FAM161A is also a centrosomal protein, being a core component of a complex that forms an internal skeleton of centrioles. Despite these observations about the importance of FAM161A, current techniques used to examine its sequence reveal no homologies to other proteins. Methods: Sequence profiles derived from multiple sequence alignments of FAM161A homologues were constructed by PSI-BLAST and HHblits, and then used by the profile-profile search tool HHsearch, implemented online as HHpred, to identify homologues. These in turn were used to create profiles for reverse searches and pair-wise searches. Multiple sequence alignments were also used to identify amino acid usage in functional elements. Results: FAM161A has a single homologue: the targeting protein for Xenopus kinesin-like protein-2 (Tpx2), which is a strong hit across more than 200 residues. Tpx2 is also a microtubule-associated protein, and it has been shown previously by a cryo-EM molecular structure to nucleate microtubules through two small elements: an extended loop and a short helix. The homology between FAM161A and Tpx2 includes these elements, as FAM161A has three copies of the loop, and one helix that has many, but not all, properties of the one in Tpx2. Conclusions: FAM161A and -its homologues are predicted to be a previously unknown variant of Tpx2, and hence bind microtubules in the same way. This prediction allows precise, testable molecular models to be made of FAM161A-microtubule complexes.

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Genes referenced: fam161a tpx2 ttn

???displayArticle.disOnts??? retinitis pigmentosa 28 [+]
???displayArticle.omims??? RETINITIS PIGMENTOSA; RP [+]

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	Figure 1. Alignment of human FAM161A/B/C with UPR0564/PF10595. Alignments include small gaps (up to 60 residues) that are not indicated. No protein contains 100% of UPR0564/PF10595. Regions of proteins that align with UPR0564/PF10595 are indicated by gray shading, with limits of alignment indicated by wavy lines. Note that alignment between proteins outside the identified domain is not shown.
	Table 1. Growth of numbers of sequences aligned to FAM161A in iterative searches. The numbers of hits with significant homologies (cut-off e-value â‰¤0.001) are shown for each iteration using four different strategies. 1/2: PSI-BLAST without a filter searching non-redundant databases of different sizes (nr50 and nr30 for #1 and #2 respectively); 3: PSI-BLAST with low complexity filter searching a comprehensive database (nr100); 4. HHblits searching a non-redundant database (nr30). Also showing the number of hits annotated as either titin for #1 and #2 or Tpx2 for #3 and #4 also showing e-value of hit. Red shading indicates multiple sequence alignment was too large for more iterations. Blue shading indicates convergence, with the next iteration identical.
	Figure 2. Fam161A and Tpx2 are homologous and contain repeats. (A) Results of pair-wise HHpred searches between FAM161A, Tpx2 and the â€œTpx2_importinâ€ domain (PF12214, which covers residues 362-498 of Tpx2- see part B). The probability of shared structure (pSS, as %) and the number of columns (in brackets below) are reported for all pairwise searches. Results in PDB are identical to the human proteome, because Tpx2 is present in PDB as 6BJC_T with no additional DSSP secondary structural information. NB: self-searches (long diagonal) have pSS=100% and include all columns. (B) Alignment of FAM161A with Tpx2. 320 residues of FAM161A (229â€“548) align with 228 residues of Tpx2 (474-701), which spans repeats 5 to 8. The probability of shared structure (pSS) =96%. Two adjacent repeats in FAM161A (79 and 88 residues, 239-317 and 318-395, both within the portion homologous to UPF0564) also align (pSS=96%, shaded semi-circle). Tpx2 repeats are coloured in a rainbow from violet to red. The region included in a cryo-EM structure with tubulin (residues 300-341) is shown by the dotted purple box. The region included in PF12214 is shown by the grey transparent box from repeat 3 to 6. (C) pSS of pairwise comparisons of each repeat in FAM161A and Tpx2, where repeat boundaries are 25 residues down-stream of those in B (arrow). Shading of each cell represents the average pSS of two searches on a grey scale with pSS from 10-100% repesented by grey 10-100%, with FAM161A as query (bottom left); and with Tpx2 as query (top right). The structures labelled R and W in repeat 2 of Tpx2 are the ridge and wedge that bind tubulin (see Figure 3).
	Figure 3. FAM161A has elements like the ridge and wedge of Tpx2. (A) Left: scheme of binding of one copy of Tpx2 residues 300-341 (rainbow blueâ†’red) binding to four tubulin monomers. Right: close up of the surfaces of the tubulins and Tpx2, shown as a ribbon, with the ridge (300-311) and wedge (323-341), but residues 312-322 modelled as a straight rod (length 2.4 nm) as they not seen in the cryo-EM structure. Conserved side-chains in Tpx2 (F307 F334: black, H335: blue) bind into pockets formed at tubulin interfaces. Image derived from PDB 6BJC chains A-D and P (Zhang et al., 2017). The asterisk indicates space at the C-terminus of the wedge (see Discussion). (B) FAM161A and Tpx2 contain repeated ridge-like motifs. Left: Ungapped alignment of extended loops from repeats 1â€“3 of FAM161A and repeats 1â€“8 of Tpx2, indicating PFÂ±Ã˜ motifs. Pink box indicates the ridge sequence in repeat 2. Sequence directly after repeat 2 is also included, showing the linker (green letters) and wedge (red letters, helix in blue box). Centre: yellow boxes indicate charged predicted helices (average length 25 aa, numbers of missing residues shown). Right: Extended loops following the helices, containing FKAÂ±PxP motifs. The analogous sequences in repeat 3 of both proteins do not form motifs, but are included for comparison (grey letters). Shading is according to the Clustal scheme. Numbers on the right indicate omitted residues between repeats. (C) Comparison of the wedge helix in Tpx2 with the amphipathic helix in FAM161A. Both the wedge of Tpx2 (blue box, left) and the predicted amphipathic helix of FAM161A (green box, right) are accompanied by logos made from 40 diverse sequences, and by a consensus in which bold lettering indicates a strong conservation, Ã˜ is hydrophobic (black), Â± is charged or polar, and other colouring is: D/E-red, KRH-blue, Q-purple, A-black.
	Figure 4. Structural and functional elements in FAM161A and Tpx2. Sequence and structural motifs as shown in key: prism = loop similar to ridge with PFÂ±Ã˜; ovoid = loop with FKAÂ±PxP; half cylinder = amphipathic helix: wedgeâ€“blue, FAM161Aâ€“green; yellow spheres: NLS; zig-zag = all charged helix. Repeat boundaries shown as in Figure 2C, with two known binding sites in Tpx2: aurora kinase A at N-terminus (black) and kinesin at C-terminus (red).

References [+] :

Alfaro-Aco, Structural analysis of the role of TPX2 in branching microtubule nucleation. 2017, Pubmed, Xenbase