The C-terminal cassette as an intramolecular cofactor

Brr2 is one of the few known helicases (together with Slh1 (Martegani et al., 1997) and ASCC3 (Dango et al., 2011)) that contain two helicase-like modules. The function of the second helicase-like module has long been elusive and intriguing. The C-terminal cassette displays several substantial deviations in the canonical helicase motifs. The glutamate of the typical motif II, DExD/H, is replaced with an aspartate (DDAH) in the C-terminal cassette yBrr2. This glutamate has been postulated to be the key catalytic residue that activates a water molecule to hydrolyze NTP in DExD/H-box proteins and other helicases (Cordin et al., 2006;

Sengoku et al., 2006; Caruthers and McKay, 2002).Likewise, the Ser-Ala-Thr (SAT) residues in motif III are replaced with SNC or SSS in the case of yeast and human sequences, respectively. The SAT residues are thought to participate in interdomain interactions between

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the two RecA domains upon ATP and RNA binding and to play a role during unwinding by DExD/H-box proteins (Sengoku et al., 2006; Zhang et al., 2009). The C-terminal cassette also lacks obvious motifs IVa and V.

Most enzyme families contain inactive members thought to have arisen by gene duplication events followed by subsequent accumulation of inactivating mutations.

Evolutionary conservation suggests that such pseudoenzymes are functionally important.

However, in most cases, their functions are unknown (Adrain and Freeman, 2012). During this work, we have shown that the C-terminal cassette of Brr2 is a pseudo-helicase that has been converted into an intramolecular regulator of a neighboring, similarly structured active helicase. This enzymatic role of the C-terminal cassette is in agreement with its non-canonical ATPase and helicase motifs (Noble and Guthrie, 1996; Lauber et al., 1996) and with previous genetics analyses showing that mutations in putative active site residues of the C-terminal cassette still support splicing (Kim and Rossi, 1999). However, our results additionally show that the C-terminal cassette can still bind ATP but specifically lost its ability to hydrolyze the nucleotide. Furthermore, we have been able to show that, apart from the substantial deviations in the canonical helicase motifs, the C-terminal cassette exhibits an increased barrier to adopt a hydrolytic conformation which seems to further consolidate the inactivity of this cassette.

Our modeling studies corroborated by mutational analyses suggest that the C-terminal cassette most likely also does not bind RNA during duplex unwinding. Our findings are consistent with recent results from (Hahn et al., 2012). In agreement with this finding, the C-terminal cassette does not seem to rely on specific sequences or structures of its RNA substrate to modulate the N-terminal cassette activity since such stimulatory effect due to the presence of the C-terminal cassette was not only observed with U4/U6 di-snRNA as a substrate but also with a simple model duplex (Fig. 3.17).

Furthermore, our results identify a number of features that are required for the cofactor function of the C-terminal cassette and suggest mechanisms by which it may act. Our mutational studies show that direct inter-cassette contacts are essential for cassette communication. Due to their large contact area, the cassettes most likely mutually stabilize the specific conformational states they adopt in the apo form of hBrr2^HR. We suggest that crystal crosslinking has cemented these conformations considering that we do not observe any significant conformational changes in the nucleotide-bound states. Our nucleotide preparations obviously contained both ATP (or analog) and ADP. The outcome of our soaking experiments, therefore, indicates the nucleotide preference of the cassette

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conformations in the apo form (ADP at the N-terminal cassette, Mg²⁺-ATP at the C-terminal cassette). Bound ADP suggests that we observed the post-ATP hydrolysis and post-phosphate release state at the N-terminal cassette in agreement with the enhanced N-terminal ATPase activity in the presence of the C-terminal cassette. Thus, the C-terminal cassette may drive ATP hydrolysis and/or phosphate release at the N-terminal cassette, thereby facilitating associated changes in nucleic acid binding and unwinding.

Our structural analyses show that the C-terminal cassette preferentially binds Mg²⁺-ATP in the presence of the N-terminal cassette, but we did not indicate whether it cycles between nucleotide-bound and free states during RNA unwinding. However, our preliminary pre-steady state kinetic studies on nucleotide binding to hBrr2 indicated that nucleotide release from the C-terminal cassette binding pocket is very slow suggesting that ATP is stably bound to the C-terminal cassette when Brr2 is not in the presence of RNA. Since the adoption of the hydrolytic conformation is hindered at the C-terminal cassette, it seems to be conformationally more restricted than the N-terminal cassette and may remain stably associated with the nucleotide. The function of the nucleotide at the C-terminal cassette may thus be to rigidify its structure and allow it to act as a scaffold on which the N-terminal cassette could efficiently undergo conformational changes required for duplex unwinding.

The C-terminal cassette may also exploit inter-cassette contacts to directly influence the positioning of active site domains in the N-terminal cassette. Interactions between the HLH and HB domains are important for duplex unwinding in the related Hel308 (Richards et al., 2008; Woodman et al., 2007). In Brr2, the N-terminal IG domain intervenes between the HLH and HB domains and is connected to the upper part of the inter-cassette linker.

Mutations in the linker affected Brr2^HR activity both negatively and positively (Fig. 3.21). It is conceivable that different functional states (such as ATP bound, ATP hydrolyzed, ADP bound) are associated with different relative orientations of the cassettes and that such conformational changes may be transmitted via the linker and the IG domain to the N-terminal HLH and HB domains, thereby modulating the activity of the N-N-terminal helicase.

It has been suggested that Brr2 may acquire processivity by oligomerization of its ATPase units in a fashion analogous to ring-forming helicases (Staley and Guthrie, 1998). While our data indeed indicate that Brr2's full helicase activity only unfolds given a direct interaction of the N- and C-terminal RecA domains, the linear disposition of these four domains in Brr2 is decisively different from the ring-like arrangement of the corresponding domains in hexameric helicases. Furthermore, our results also suggest that the C-terminal RecA domains do not engage RNA during unwinding. Rather, the dependence of the stimulation on

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cassette RecA contacts may be explained by stabilization of the N-terminal RecA and WH domains and by their positioning relative to other active site elements.