Our structural studies in combination with sequence analyses suggest that the H2+S2 domain of Brr2 resembles the entire Hel308 and that the full length Brr2 protein is composed of an N-terminal domain and two consecutive Hel308-like modules (Hel308-I and II). A recent structure of another Ski2-type DNA helicase Hjm also revealed a similar domain structure and organization as Hel308 40
. This raises the possibility that many Ski2-type helicases may share structural (and potentially mechanistic) similarities in regions beyond the helicase domains, in spite of the lack of recognizable sequence similarities in these regions.
The structural resemblance between Brr2 and Hel308 suggests possible helicase mechanisms for Brr2. Our brr2-3GS
mutant suggests that the putative β-hairpin region in Brr2 Hel308-I is structurally and/or functionally important. Further structural and mutational analyses will reveal whether Brr2 indeed contains a β-hairpin and utilizes an unwinding mechanism similar to Hel308 30
. Furthermore, our structural mapping and biochemical analyses of the brr2-1
mutants support the structural and mechanistic similarity between Brr2 and Hel308. These results suggest the possibility that Brr2 is more processive than other DExD/H-box proteins involved in pre-mRNA splicing. This potential processivity is attractive considering that yBrr2 needs to unwind U4/U6, which contains long stem regions and is highly stable in yeast 41
Brr2 is the only known helicase that contains two helicase-like modules. The function of the second helicase-like module has long been elusive and intriguing. There are several substantial deviations between the putative helicase motifs in Hel308-II and the canonical helicase motifs. For example, the typical DExD/H residues in motif II are replaced with DDAH in Hel308-II (Supplementary Fig. 4
). The Glu of motif II (DExD/H) has been postulated to be the key catalytic residue which activates a water molecule to hydrolyze ATP in DExD/H-box proteins and other helicases 5,30,42
. Likewise, the Ser-Ala-Thr (SAT) residues in motif III are replaced with SNC in Hel308-II (Supplementary Fig. 4
). The SAT residues in motif III do not interact with ATP or RNA but participate in inter-domain interactions between the N-terminal and C-terminal domains upon ATP and RNA binding and are thought to be important for the unwinding activity of DExD/H-box proteins 30
. Hel308-II also lacks obvious motifs IV – VI (Supplementary Fig. 4
). Substantial deviations from the canonical helicase motifs in Hel308-II probably have led to the lack of ATPase and helicase activities of this module.
We demonstrated that Hel308-II interacts with Prp8 and Snu114 in vitro
and in vivo
(, 7). We do not rule out the possibility that Prp8 and Snu114 also interact with the Hel308-I module, although we cannot yet test this hypothesis since Hel308-I alone is unstable. It is worth noting that protein-tag labeling and antibody recognition approaches have mapped the C-terminus of Brr2 to be somewhat distant from the C-terminus of Prp8 in the EM projection structure of the yeast tri-snRNP, while the C-termini of Prp8 and Snu114 are in close proximity to each other 43
. However, these labeling methods are designed to map the extreme C-terminus of a protein. The longest dimension of Hel308 is about 80Å and the Prp8-CTR can reach similar dimensions depending on the relative orientation of its C-terminal and β-finger domains 37,44–47
. This dimension can easily span the distance between the C-termini of Brr2 and Prp8 observed in the EM structure 43
. Therefore, the main body of Brr2 Hel308-II and Prp8-CTR can overlap and interact with each other even if the extreme C-termini of Brr2 and Prp8 are far from each other. Our results, in general, represent the first example of a helicase-like structural fold serving as a major protein-interaction platform.
The Hel308-II module can potentially play a role in mediating the regulation of Brr2 activity. Recently, it was shown that the C-terminal fragment of Prp8 (residues 1806–2413) greatly stimulates Brr2’s helicase activity but inhibits its ATPase in vitro
. Here we showed that the deletion of S2 results in a dramatic reduction of Brr2’s ATPase and helicase activity (). We also found that the deletion of Hel308-II drastically destabilizes the protein (). Both results suggest that the S2 domain as well as the entire Hel308-II module interact and communicate with Hel308-I. It is foreseeable that the interaction between Prp8 and Hel308-II can potentially affect the structure and/or stability of Hel308-I and consequently the ATPase and helicase activity of Brr2. We do not rule out the possibility that Prp8 may directly interact with Hel308-I to modulate Brr2’s activity. Either directly (through Hel308-I) or indirectly (through Hel308-II), Prp8 can potentially affect Brr2’s ATPase and helicase activities through increasing RNA binding affinity, stabilizing a favorable conformational change, increasing processivity, or a combination of the above. In analogous situations, detailed kinetic analyses suggest that eIf4B, eIF4H, and eIF4G stimulate eIF4A’s ATPase and helicase activity through one or more of the above mechanisms 48
. Likewise, Ntr1 was recently shown to stimulate the activity of Prp43 (another DExD/H-box protein involved in spliceosome disassembly) and was thought to affect Prp43’s processivity 49
Our observation of the effect of Prp8-CTR on Brr2’s RNA binding property raises another interesting possibility for the regulation of Brr2’s activity. We showed that Prp8-CTR binds U4/U6 with a much higher affinity than arbitrary 13nt ss or ds RNAs (). When Prp8-CTR and Brr2 (Hel308-II or full-length protein) are combined, the complex binds U4/U6 much better than Brr2 alone (). We cannot differentiate at this point whether Prp8-CTR increases Brr2’s intrinsic affinity to U4/U6 or the increased binding is solely attributed to Prp8-CTR in the complex. However, in either case, the interaction between Prp8-CTR and Brr2 clearly helps the complex bind U4/U6 better. This increased affinity provides a possible mechanism for how Prp8-CTR helps Brr2 to confer specificity toward U4/U6. Further studies will reveal the extent of this specificity, such as how stringent the Prp8-CTR is toward the sequence and/or structure of U4/U6. It is worth noting that Ritchie et al. found that the N-terminal RNase H domain of the human Prp8-CTR has a much higher binding affinity with a U2/U6 mimic than other RNAs 46
, consistent with the possibility that Prp8 may also help Brr2 confer specificity toward U2/U6 to facilitate Brr2’s role in U2/U6 unwinding. The higher local concentration of U4/U6 brought to Brr2 by Prp8-CTR could also serve as an additional mechanism for the stimulation effect by Prp8-CTR we observed in vitro
. There are other enzymes (such as collagen prolyl 4-hydroxylase, HIV integrase, and endonuclease NaeI) that have separate substrate binding and catalytic domains, especially when the substrate is a polypeptide or oligonucleotide 50–52
. Prp8 can potentially serve as an auxiliary substrate-binding and specificity domain for Brr2.
Our results also lead to reflections on the function of the Sec63 domain in general. Our structural result shows that the so-called Sec63 domain is in fact made of three domains, two helical and one all-beta Fn3 domains. The Sec63 domain has at least two functions. It can serve as a major RNA binding and processivity domain as in the case of Hel308-I. It can also serve as a major protein interaction domain as in the case of Hel308-II. The Fn3 fold in the Sec63 domain belongs to the immunoglobulin-like superfamily whose members are almost always involved in binding functions 53
. Deletion of the Sec63 domain in the Sec63 protein leads to impaired higher-order complex formation 54
. This Sec63 domain may also play a role in protein interaction, indicating the generality of the Sec63 domain serving as a protein interaction domain.