Armadillo motifs involved in vesicular transport

Abstract

Armadillo (ARM) repeat proteins function in various cellular processes including vesicular transport and membrane tethering. They contain an imperfect repeating sequence motif that forms a conserved three-dimensional structure. Recently, structural and functional insight into tethering mediated by the ARM-repeat protein p115 has been provided. Here we describe the p115 ARM-motifs for reasons of clarity and nomenclature and show that both sequence and structure are highly conserved among ARM-repeat proteins. We argue that there is no need to invoke repeat types other than ARM repeats for a proper description of the structure of the p115 globular head region. Additionally, we propose to define a new subfamily of ARM-like proteins and show lack of evidence that the ARM motifs found in p115 are present in other long coiled-coil tethering factors of the golgin family.

Figure 1. ARM-motif consensus and structure-based sequence alignment of human p115^GHR.

On the top of the alignment the cartoon and helical wheel representation of isolated ARM-repeat helices are shown. Each repeat is composed of three helices that are displayed in green (H1), blue (H2), and yellow (H3). The universal ARM-repeat consensus sequence derived from the alignment of five ARM-repeat proteins is shown beneath as well as the consensus sequence for β-catenin followed by the single ARM-repeat sequences of p115^GHR and, at the bottom of the alignment, the consensus sequence for p115^GHR. Residues comprising H1, H2 and H3 in each repeat are separated by their connecting loop regions. Italicized residues are not present in the X-ray structure of human p115^GHR and derived from the structure of the bovine homolog . Residues shown in green are missing from both the human and bovine p115^GHR structure. Conserved residues that define the ARM-consensus motif are highlighted in red. Structural positions with strong preferences for a given amino acid or group of amino acids are shaded with the following symbols: half-closed box = general hydrophobic; open box = small hydrophobic; diagonal-filled box = hydrophilic; closed box = large hydrophobic; (+) = basic. In the consensus sequence, the single-letter code is listed at the bottom if the residue is present in at least six of twelve repeats. Residues that mediate contacts (hydrogen bond or salt bridge) between the USO repeat and the USO-domain helix H3 are highlighted in blue.

Figure 2. Crystal structure of human p115^GHR .

The color scheme of the ARM helices is the same as that in Figure 1. (A) The protein is composed of 11 ARM repeats and the USO element (repeat numbers are shown next to the repeats, in red for the USO1 head domain, in black for the armadillo helical domain). ARM1 is not visible in the structure of human p115^GHR but is partially resolved in the bovine p115^GHR structure. (B) A superimposition of the ARM repeats of human p115^GHR (excluding ARM2 due to a disordered H1) is shown on the left. For comparison, the ARM repeats of murine β-catenin are superimposed on the right.

Figure 3. Comparison of repeat motif structures.

(A) A comparison of p115^GHR ARM8/10 with β-catenin ARM11/5. The backbones are superimposed on the right. The individual repeats are shown on the left, with the side chains of the conserved consensus residues shown as sticks. (B) The C-terminal non-canonical USO element. Key residues that mediate interactions of the USO element with the superhelical groove are shown in stick representation on the right.

Figure 4. Dimeric organization of the USO element.

The dimeric orientation of human p115^GHR is shown on the left, the orientation of the USO element on the right side accordingly.

Figure 5. Search for alpha-rod repeats in p115, GM130 and homologs.

Human sequences and representative orthologs were aligned, and the multiple sequence alignment (MSA) and hits to alpha-rods (from ARD) were represented using BiasViz . Basically, gaps are represented in red and aligned sequence in black unless a significant match to an alpha-rod was recorded (white). Top: MSA of human GM130 and orthologs from 12 species. Only three scattered matches are observed, and most sequences (including the human) did not have any significant match. Bottom: MSA of human p115 and orthologs from 7 species. Significant matches were observed in all but one ortholog for four ARM repeats (the human sequence showing the four of them).