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Supplementary Figure 1.
To obtain a more quantitative perspective on the sequence relationships between
ubiquitin variants of different species, we used this phylogram to compare the protein
sequences of UbS27a with the ribosomal S27a domain and with homologs to the ubiquitinlike modifier SUMO1 as a reference. The UPGMA algorithm assumes same rates of
evolution; therefore the branch lengths correspond to amino acid substitutions per time
unit. Evidently, UbS27a domains are less conserved than ribosomal S27a domains or
homologs to SUMO1, despite the presence of a hypervariable loop at the N-terminus of
SUMO1. Particularly indicative of a high rate of mutations or polymorphisms is the
relatively large divergence between species that are closely related, such as A. ceylanicum
and C. elegans or L. major and T. cruzi. The ubiquitin variant GlUbS27a of G. lamblia is
indicated with an arrow.
Supplementary Figure 2.
15 species (see Figure 1 and Supplementary Figure 1) were analyzed for their pair-wise
distances within UbS27a domains, S27a domains and SUMO1 homologs, based on the
Jones-Taylor-Thornton matrix (values on the x-axis). The white rectangles represent the
range of matrix scores and the black boxes represent the arithmetic mean plus/minus
standard deviation. Ubiquitin variants show significantly higher sequence distances than
the other two protein domains, even though SUMO1 homologs carry hypervariable
domains at their N-termini.
Supplementary Figure 3.
Multiple sequence alignment of 39 human ubiquitin-like domains, of GlUbS27a, and of
the bacterial protein MoaD (Staphylococcus carnosus). This comparison contains
classical ubiquitin-like domains, as well as Band 4.1 domains, Ras-associating domains,
and Ras-binding domains (Kiel and Serrano, 2006). Despite the low conservation of
amino acid residues, a similar alternating pattern of hydrophobic and polar side chains is
evident. The secondary structure of ubiquitin is indicated at the bottom. GI sequence
identifier numbers of the proteins by the order of appearance are: 71068970 (GlUbS27a),
56078799 (Ubiquitin), 4503659 (FUBI), 52001470 (ISG15), 52000648 (FAT10),
2833270 (NEDD8), 52783799 (SUMO1), 50400081 (SUMO4), 48928058 (SUMO3),
113430052 (SUMO2), 13937891 (HUB1), 68565265 (URM1), 7705300 (UFM1),
38261969 (ATG12), 44888808 (GABARAP-L2), 44887916 (GABARAP), 44887972
(GABARAP-L3), 14424562 (GABARAP-L1), 51972260 (MAP1-LC3_C), 17433141
(MAP1-LC3_B), 31563518 (MAP1-LC3_A), 3955207 (MoaD), 5730868 (Merlin),
110430928 (EMP4.1), 119587533 (Radixin), 14625824 (Moesin), 46249758 (Ezrin),
14549162 (RalGDS), 12653099 (RGL2), 46276893 (Elongin B), 125651 (Raf), 6226835
(p59 OASL), 58530859 (HERP), 124056593 (Ubiquilin-2/hPLIC-2), 10998427 (Grb7),
19855059 (RGS14), 4758884 (Parkin 1), 1729927 (USP14), 119631035 (p47_UBX),
62089006 (hRAD23A), 4506387 (hRAD23B).
Supplementary Figure 4.
A. Phylogenetic comparison of 13 E2 conjugating enzymes in budding yeast and
homologs in Giardia lamblia. For each of the yeast conjugating enzymes, we could find a
nearest neighbor in Giardia lamblia, including the RUB1/Nedd8- and SUMO1-specific
E2s (indicated with asterisk). We also found orthologs to E1 activating enzymes, to E3
ubiquitin ligases, and to the ubiquitin-like modifiers RUB1, SUMO1, URM1, and Ufm1
(not shown).
B. Table showing proteasome subunits in budding yeast and homologous proteins in
Giardia lamblia. Alignment scores, as calculated with ClustalX, are indicated at the right.
C. Table representing homologs to ubiquitin-specific cysteine proteases found in the
genome of Giardia lamblia.
Supplementary Figure 5.
Phylogenetic analysis using Maximum Likelihood, based on the alignment shown in
Supplementary Figure 3. The unrooted consensus tree is based on 100 bootstrap
iterations. GlUbS27a does not cluster with ubiquitin or with other eukaryotic ubiquitinlike modifiers.
Supplementary Figure 6.
Structural comparison of GlUbS27a, ubiquitin (1ubi.pdb) (Ramage et al., 1994), MoaD
(1fm0.pdb) (Rudolph et al., 2001), Moesin (1sgh.pdb) (Finnerty et al., 2004), Grb7
(1wgr.pdb) (DOI 10.2210/pdb1wgr/pdb, to be published), and cRaf-1 (1rfa.pdb)
(Emerson et al., 1995). GlUbS27a represents the simplest form of a ubiquitin fold,
featuring only five -strands and one central -helix. Additions to this basic domain are
highlighted in green: ubiquitin, Ras-binding (Raf) and Ras-associating domains (Grb7)
contain an additional helix in the C-terminal portion, Band 4.1 proteins (Moesin) feature
an inserted -strand at this position, and the prokaryotic MoaD polypeptide possesses one
additional N-terminal and one additional central -helix. The main -helix of Raf is
distorted compared to other ubiquitin structures.