Torus Domain

•         Envelops Zinc finger domain, stabilises it further
•         Scaffolds Zinc finger (ZnF), RNA recognition motif (RRM) and connector

Figure 1. Torus domain (4 a-helices (a1–a3, a5) and two 310 helices (Z1, Z2), all connected by long loops) coloured in red with Zinc finger domain (in yellow) nestled in between (only one subunit shown)

•       Connects 2 distinct RNA-binding domains ~ functional benefits (below)
•        As a complex, RRM binds RNA more efficiently than isolated RRM

            Figure 2. RRM domain in green and key contacting amino acid residues highlighted

  

            The difference in efficiency of RRM in Cwc2 and free RRM was shown in the paper. This
            could be due to sequence and slight structural difference between RRM in Cwc2 and other
            RRM. This is further explored using Fox1 RNA-binding protein RRM domain.

  

            EMBOSS Water algorithm alignment was carried out to determine sequence homology.   

            Length of coinciding sequence: 60

            Identity:      18/60 (30.0%)

            Similarity:    29/60 (48.3%)

            Gaps:           9/60 (15.0%)

            Score: 54.5

            

We see that the score is not particularly high, but there is much conserved homology in terms of     amino acid properties. There are a number of key conserved hydrophobic and charged residues, which might be essential to its RNA recognition function. This homology translates to a structural similarities as well. Figure 3 shows RNA bound to Fox1 RRM domain and Figure 4 shows it aligned to Cwc2 RRM. 

                       

          Figure 3. RNA bound to Fox1 RRM (key RNA contacting residues highlighted)

          

           Figure 4. Alignment of Cwc2 RRM and Fox1 RRM with SSRNA shown in its binding site

We can see that the structure is largely conserved. Yet we can see that there are constraints to the secondary structure components of Cwc2 RRM due to surrounding protein domains. The conformations (such as angling of alpha helices in the middle of the picture) do differ and this might suggest better complementary fit with RNA, thus enabling more efficient binding (i.e. higher affinity).

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•            Frames depression on surface (a1 helix and adjacent loop form front rim)
•               Depression characterised and could be binding pocket for RNA

       

          Figure 5. a1 helix in orange and top view of Cwc2 showing the possible depression location

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•             Evolved from ZnF domain for new functions eg. Jab-MPN of Prp8 spliceosomal protein  

Yeast Prp8p has a conserved MPN (Mpr-1, Pad-1, N-terminal) domain at its C terminus (2178–2310).  This family of domains is used in different contexts such as in proteasome regulatory subunits, eukaryotic initiation factor 3 (eIF3) subunits, the signalosome and as transcription factor regulators. It typically has five conserved residues that coordinate a Zn²⁺ ion. It has conserved glutamate (E) and a JAMM motif that includes a conserved H-x-H-x[7]-S-xx-D sequence. The Prp8 MPN domain has the consensus H-x-Q-x[7]-S-xx-D has a conserved glutamine instead. Due to these differences, it may not bind zinc. However we can see from Figure 6 that it has a similar overall fold to Cwc2, suggesting similar evolutionary paths. The key beta strands in the Prp8 MPN domain is colored red and form a pocket that possibly binds RNA. Thus the novel structure of Cwc2 could be said to confer evolutionary advantage in terms of RNA binding and spliceosomal functionality. Another protein that has a similar structure is explored in the section under MBNL1.  

Figure 6. Structure of Jab-MPN of Prp8 spliceosomal protein of Yeast

•                     ZnF-Torus and RRM conserved contacts in homologue hRBM22 ~ importance of residues (despite low sequence homology in Torus and connector element, correlation of mechanism with Cwc2)

Movie 1. hRBM22 aligned to Cwc2 and contacts shown

•            Intra-molecular interactions involving Torus domain

Torus and Znf - zinc coordination and hydrophobic interface (aromatic residues)

Torus and RNA recognition motif - salt bridges and hydrophobic interface