Ubiquitin interactions of NZF zinc fingers

Abstract

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.

Figure 1

Secondary structures and intermolecular contacts in the Npl4 NZF/Ub complex. (A) Sequences, numbering schemes, and secondary structures of the rat Npl4 NZF and human Ub proteins. The Ub-binding TF/M motif of Npl4 NZF is highlighted in red. Note that rat and human Npl4 NZF proteins differ by a single S-to-A substitution at residue 4. (B) Matched pairs of unfiltered and half-filtered (HF) NOESY strips, aligned to illustrate reciprocal intermolecular NOE contacts between NZF residue Phe14 (strip 1) and Ub residues Ile44, Lys48, and Gln49 (strips 2–5). Resonances on the diagonal are denoted with solid black dots, and their identities are given below each strip. Dashes represent intermolecular NOEs, and arrows denote forward and return NOEs. The intermolecular NOEs from NZF F14 to Ub are labeled at left and the proton indirect chemical shift scale is shown at right (ppm).

Figure 2

Structure of the Npl4 NZF/Ub complex. (A) Stereoview showing an overlay of the 20 lowest penalty complexes of Npl4 NZF and Ub. Ub is shown in red, the NZF domain is shown in blue, and the Zn²⁺ ion in purple. (B) Ribbon diagram of the Npl4 NZF/Ub complex. Color coding is the same as in (A), with the secondary structural elements in both proteins labeled, and key interface residues from both proteins shown explicitly. The Ub Lys48 residue is also shown to help orient the reader.

Figure 3

Npl4 NZF/Ub interaction surfaces. (A) Stereoview of Npl4 NZF (ribbon) bound to Ub (surface). Ub residue numbers are shown. (B) Stereoview of Ub (ribbon) bound to Npl4 NZF (surface). NZF residue numbers are shown. (C) Expanded stereoview showing the key interface residues (numbered) from Npl4 NZF (blue) and Ub (red).

Figure 4

Transferability of the NZF ₁₃TF₁₄/M₂₅ Ub-binding motif. (A) Schematic alignment of the Npl4 and RanBP2 NZF sequences. Dark blue: highly conserved NZF residues (>50%); light blue: moderately conserved NZF residues (>20%); yellow: residues that contact Ub; and green: residues that are both highly conserved and contact Ub. NZF residue conservation was defined as in Wang et al (2003). (B) Ub binding by wt (inset) and mutant RanBP2 (L13T,V14F,A25M) NZF domains. Ub was injected in triplicate at concentrations of 0–1500 μM over GST-RanBP2 NZF proteins captured on anti-GST surfaces. Note that Ub binding by the wt RanBP2 NZF domain (inset) was negligible, even at 1500 μM Ub. (C) Binding isotherms for the NZF domains of Npl4 (positive control, black), wt RanBP2 (negative control, purple), RanBP2 (L13T,V14F) (blue), RanBP2 (L13T,V14F,A25M) (green), and RanBP2 (L13T,V14F,A24E,A25M) (red). K_d values are given in Table II.

Figure 5

Ub binding by Vps36 NZF domains. (A) Surface plasmon resonance biosensor analyses of the wt Vps36-1 NZF/Ub interaction. Ub was injected in triplicate at concentrations of 0–1500 μM over GST-Vps36-1 NZF captured on an anti-GST surface. The inset depicts the much weaker binding responses obtained for the same concentrations of Ub injected over a mutant GST-Vps36-1 (₁₃TF₁₄ to ₁₃GS₁₄) captured on an anti-GST surface. (B) Isotherms for Ub binding to wt and mutant NZF domains from Vps36p: wt Vps36-1 NZF (black), Vps36-1 (T13G) NZF mutant (red), Vps36-1 (₁₃TF₁₄ to ₁₃GS₁₄) NZF mutant (blue), and wt Vps36-2 NZF (green). Full sequences of the NZF domains and estimated dissociation constants are given in Table II.

Figure 6

A Vps36 NZF mutant is defective in Ub binding and MVB sorting. (A) Extracts were prepared from vps36Δ(MBY30) yeast cells expressing the indicated VPS36 alleles and incubated with immobilized GST (negative control) or Ub-GST. Bound, Myc-tagged Vps36 proteins were visualized by Western blotting with anti-Myc antisera. (B) Extracts were prepared from E. coli expressing all three ESCRT-II genes (pMB202) and incubated with immobilized GST (negative control) or Ub-GST. Bound, HA-tagged Vps22 protein (ESCRT-II complex) was visualized by Western blotting with anti-HA antisera. (C) Normarski optics (bottom panels) and fluorescence localization of GFP-CPS (top panels) and FM 4-64 (middle panels) in vps36Δ cells expressing either wt Vps36, Vps36^T187G,F186S, or no Vps36 (vps36Δ) at 37°C. Class E compartments are indicated by arrowheads.