Fig 1.
Expression and Purification of the Cuz1 AN1 Zinc Finger Domain.
A) Schematic diagram of the Cuz1 protein. UBL, ubiquitin-like domain. B) Purified Cuz1 AN1 ZnF protein (15 μg) as analyzed by SDS-PAGE followed by Coomassie staining. C) Size exclusion chromatography of purified Cuz1 AN1 ZnF protein. Molecular weight size standards are shown for reference. D) 15N-HSQC spectrum of the 15N-labeled Cuz1 AN1 ZnF domain sample showing assignments of Cuz1 residue resonance peaks. Backbone amide and sidechain peaks are labeled in red and green, respectively, and peaks from the cloning tag are marked with a “x” sign. An expanded view of the central spectral region is shown at the lower right corner.
Table 1.
NMR statistics of 15 solution structures of Cuz1 AN1 ZnF*.
Fig 2.
Solution Structure of the AN1 ZnF Showing a Compact Fold.
A) Ribbon diagram of the Cuz1 AN1 ZnF domain. Beta strands 1–4 and helices 1–3 are indicated, as well as the two zinc atoms as green spheres. B) Top view (rotated 90° about the X-axis) showing the compactness of the Cuz1 AN1 ZnF domain. Note that the scales are the same for panels A and B. C) Sequence alignment of the Cuz1 AN1 ZnF domain showing secondary structure: blue arrows, beta strands; red cylinders, short alpha helices. Residues coordinating the first zinc cluster are colored in red, and those coordinating the second zinc cluster in green. D) Central position of Phe38 at the core of AN1 ZnF domain between the two zinc chelating clusters. The dotted blue line indicates a hydrogen bond between His48 and His42 which further bridges the two zinc clusters. E) Additional polar and hydrophobic interactions that contribute to the stability of the compact Cuz1 AN1 ZnF domain.
Fig 3.
Rigidity of the AN1-ZnF Domain.
A) 15N-HMQC spectrum of histidine residue side-chain correlations showing the different tautomeric state patterns of zinc-coordinating His42 (blue) and His48 (red). Note the appearance of the His48 Hδ1 resonance peak that is protected from rapid exchanging with H2O by a side-chain-to-main-chain hydrogen bond. B) Atomic model of the zinc-coordinating histidine tautomeric states. The black dotted line indicates the side-chain-to-main-chain hydrogen bond involving His42 and His48. C) 2D-NOESY spectra of the Cuz1 ZnF showing chemical exchange cross-peaks between Hδ1/Hδ2 and Hε1/Hε2 spins of Phe38 due to the slow ring-flipping rate, marked by the red dotted lines. Note that the peaks are more broadened at 45°C than at 25°C due to faster chemical exchange, consistent with increasing ring-flipping rates.
Fig 4.
Identification of a Conserved LDFLP Sequence in the Cuz1 AN1 ZnF.
A) Sequence alignment of full-length Cuz1 with three human AN1 proteins: AIRAP (Zfand2A), AIRAP-L (Zfand2B), and Zfand1. Zinc chelating residues are highlighted in yellow. The LDFLP motif is highlighted in cyan. Asterisks, identical residues; double dots, highly similar residues; single dots, similar residues. B) Position of the LDFLP motif within the AN1 ZnF. The left panel shows the largely surface exposed LDFLP residues with their side chains. The right panel indicates hydrophobicity in green. Note the hydrophobic patch (in dark green) associated with the LDFLP motif, whereas the remainder of the structure shows a largely unremarkable hydrophobicity distribution.
Fig 5.
The LDFLP Motif Appears Dispensable for Cdc48- and Proteasome-Binding.
A) Binding of GST-tagged Cuz1 or the Cuz1LDFL→AAAA mutant to 12xHis-Sumo-tagged Cdc48 or to beads alone. Binding reactions were visualized by SDS-PAGE followed by immunoblot with anti-Cuz1 antibody (upper panel). Cdc48 levels were analyzed by SDS-PAGE followed by Coomassie staining (lower panel). Input levels of GST-Cuz1 species were visualized to ensure that equivalent amounts of protein were added to the binding assay, and to confirm the assignment of the bands attributed to bound GST-Cuz1. B) Binding of purified proteasome to GST-tagged Cuz1 or the Cuz1LDFL→AAAA mutant, as visualized by SDS-PAGE followed by immunoblot. Upper panel, anti-proteasome subunit Rpn8. Lower panel, anti-Cuz1 immunoblot. Metal chelated GST-Cuz1 serves as a negative control. C) Superimposed 15N-HSQC spectra of the AN1 ZnF domains from wild-type Cuz1 (blue) and Cuz1LDFL→AAAA mutant (red). The resonance peaks from mutated residues L24, D25, and L27 are clearly missing (F26 overlaps with another peak).