Structure of the frequency-interacting RNA helicase: a protein interaction hub for the circadian clock

Abstract

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ-interacting RNA Helicase (FRH) rhythmically represses gene expression by the white-collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ-interacting region at the FRH N-terminus has variable structure in the absence of FRQ A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ A V142G substitution near the N-terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N-terminus and KOW module.

Keywords: chaperone; circadian clock; protein interactions; protein structure; transcriptional repressor.

Figure 1. Function and structure of FRH

1. In the Neurospora crassa circadian core clock oscillator, FRH associates with FRQ and CK1a to form a repressor complex (FFC) that inhibits the positive‐acting white‐collar transcription factors (WC‐1 and WC‐2) which form the white‐collar complex (WCC). Stoichiometries of the components are not represented in the schematic. FFC composition has been estimated as 1 FRH molecule: 2 FRQ molecules (Cheng et al, 2005; Hurley et al, 2013), or 2 FRH molecules: 2 FRQ molecules (Lauinger et al, 2014).

2. Structure of FRH from the large crystallographic cell with an accordingly colored domain map below.

Figure EV1. Primary sequence comparison of FRH and Mtr4

Sequence alignment of FRH and Mtr4 with secondary structure and corresponding domain identifiers colored in accordance with Fig 1. Relevant mutation sites are boxed in gray and starred.

Figure 2. Comparison of FRH and Mtr4 structures

1. FRH large cell (cyan), Mtr4 (pdb 2XGJ, green, RNA backbone in orange), and FRH small alternate cell (blue) differ primarily in the orientation of the arch domain. The FRH RNA‐bound structure also crystallizes in the small‐cell configuration. The two different FRH structures have an overall RMSD of 3.9 Å, and the RMSD between Mtr4 and FRH is 2.9 Å (over 734 residues, for large‐cell FRH).

2. SAXS data indicate that FRH‐Δ114 is monomeric and flexible. The crystal structure of the large‐cell structure predicts the solution scattering curve (left, χ² = 0.92) and fits well to the calculated molecular envelope (right).

Figure EV2. Conformational variability of FRH

Structural overlays of large (cyan) and small (marine) cell FRH structures shown from different perspectives and emphasizing the differences in the conformations of the N‐terminus and the KOW modules.

Figure 3. The N‐terminal region of FRH is unstructured in the absence of FRQ

1. Comparison of the FRH N‐terminus (large‐cell structure, magenta) to that of Mtr4 (green) aligned on their RecA domains. Only the additional elements of FRH are shown (cyan). The FRH N‐terminal extension has little ordered secondary structure, although regions 143–147 and 129–135 form small turns that interact with the helicase core.

2. The very N‐terminus of FRH extends away from the RecA‐2 domain to interact with a symmetry‐related molecule in the crystal (below), whereas the Mtr4 N‐terminus turns back to form an outer β‐strand. Center panel shows the crystal contacts, with close‐ups for each region highlighted as insets on the left and right.

Figure 4. The RecA and arch domains of FRH compared to Mtr4

1. The two RecA domains are rotated relative to one another in FRH (cyan, large cell) compared to Mtr4 (green, RNA backbone in orange). The change in positioning of RecA‐2 with respect to RecA‐1 results in an overall displacement from the Mtr4 domain juxtaposition that ranges from 1.1 to 4.0 Å throughout RecA‐2 with the largest differences at the external helices.

2. Comparison of arch domains conformations from Mtr4 (green) and FRH (large cell in cyan, small cell in marine) after superposition of the most N‐terminal helical region. The isolated KOW modules of FRH, Mtr4 (with FRH superimposed), and Ski2 have considerably different loop configurations (below).

Figure 5. Interactions of FRH with RNA and ADP

1. 3.8 Å resolution mF_o‐DF_c omits map density (green, calculated with sharpened B‐factors and contoured at 2.0σ), reveals the presence of ADP and a short segment of RNA bound to FRH.

2. 3.8 Å resolution 2mF_o‐DF_c calculated without ADP or RNA contributing to the model. Green electron density shown for ADP and RNA (0.6σ); orange density shown for surrounding protein (0.6σ).

3. Comparison of Mtr4 and FRH ADP binding (FRH in cyan with ADP, RNA, and F179 in orange bonds; Mtr4 in green with ADP, RNA, and F148 in yellow bonds).

4. Unwound DNA from Hel308 (pdb 2PR6; magenta, DNA in orange bonds) overlaid in FRH with RNA and ADP (FRH in cyan with yellow RNA and cyan ADP in stick) after superposition of the respective RecA domains. See also Appendix Fig S4. The superposition indicates that the KOW module is positioned to interact with double stranded RNA that is being unwound by the RecA and DHSCT domains.

Figure 6. Features of FRH critical for helicase and clock function

1. Key residues for FRH function shown on the large‐cell structure: K208 and S324 compose the ATP‐binding site; E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712, and K766 contribute to a positively charged surface on the KOW domain module. R806H disrupts the clock. Q131, G132, and V142 in the N‐terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig 1, and ADP and RNA are in orange bonds).

2. Same as (A) after rotation about a horizontal axis.

3. Close‐up of the ADP‐binding pocket and N‐terminal interaction for the large‐cell structure (cyan ribbons) and the small‐cell structure (blue ribbons).

4. Electrostatic potential surface of FRH (blue positive, red negative) shown with the same orientation as in (B).

5. Close‐up of the KOW module showing the large number of basic (aqua sticks) and acidic side chains (yellow sticks) on the surface that also includes the mutational sites R806, R712, K811, and K766 (gray sticks)

Figure 7. FRH variant interactions with the core clock components examined by race tube and immunoprecipitation assays

The assays probe the interactions between the core clock components in strains with a wild‐type copy of FRH and FRH with several different residue substitutions.

1. Race tube assays with FRH mutants. Every circadian cycle N. crassa lays down aerial hyphae that appear as fluffy yellow/orange conidial bands. The distance between the bands reflects the clock period. The strains were grown in constant darkness, and growth fronts were labeled every 24 h (black vertical lines), rhythmicity on right (τ = period in hours, σ = standard deviation, n = number of race tubes; ARR, arrhythmic). The race tubes shown here are representative samples from three replicate tubes.

2. Pull‐down assays evaluating interactions among FRH, FRQ, WC‐1, and WC‐2. Ratio of FRQ, WC‐1, and WC‐2 binding to the V5 tagged FRH mutants as compared to the inter‐strain variations in FRH levels, error bars represent standard deviation for the assay run in triplicate. *P < 0.05 (unpaired two‐tailed t‐test with Gaussian distribution assumption). Representative immunoblots are shown in Appendix Fig S5.

Figure EV3. Putative TRAMP complex interactions sites

Structural comparison of the Mtr4 TRAMP complex (pdb 4U4C) with FRH (FRH in gray, Mtr4 in green with N‐terminus (added from pdb 2XGJ) in gold, Air2 in blue, Trf4 in magenta). Trf4 lies along RecA‐2, Air2 contacts the DSHCT, RecA‐2, and the KOW domain of a symmetry‐related molecule. The Trf4 and Air2 interfaces with Mtr4 are in regions well conserved by FRH. Although there is currently no data on an N.c. TRAMP complex, a Neurospora homolog of Trf4 (GenBank: EAA31314.2; NCU05588, 38% sequence identity) conserves residues that could potentially interact with α9 of FRH. A direct homolog of Air2 is even less obvious in the genome of N.c., although the hypothetical protein NcHyp (GenBank: CAD37057; NCU04617) has modest sequence similarity (33%) to yeast Air2. This putative Air2 homolog also maintains some residues that could interact with conserved residues in the FRH DSHCT domain (K1048, M1049, and E1054). Segments of Trf4 and Air2 that contact Mtr4 are aligned below with the related regions of the respective N.c. homologs; residues that make contacts are highlighted.