A structural investigation of NRZ mediated apoptosis regulation in zebrafish

Abstract

Bcl-2 family proteins play a crucial role in regulating apoptosis, a process critical for development, eliminating damaged or infected cells, host-pathogen interactions and in disease. Dysregulation of Bcl-2 proteins elicits an expansive cell survival mechanism promoting cell migration, invasion and metastasis. Through a network of intra-family protein-protein interactions Bcl-2 family members regulate the release of cell death factors from mitochondria. NRZ is a novel zebrafish pro-survival Bcl-2 orthologue resident on mitochondria and the endoplasmic reticulum (ER). However, the mechanism of NRZ apoptosis inhibition has not yet been clarified. Here we examined the interactions of NRZ with pro-apoptotic members of the Bcl-2 family using a combination of isothermal calorimetry and mutational analysis of NRZ. We show that NRZ binds almost all zebrafish pro-apoptotic proteins and displays a broad range of affinities. Furthermore, we define the structural basis for apoptosis inhibition of NRZ by solving the crystal structure of both apo-NRZ and a holo form bound to a peptide spanning the binding motif of the pro-apoptotic zBad, a BH3-only protein orthologous to mammalian Bad. The crystal structure of NRZ revealed that it adopts the conserved Bcl-2 like fold observed for other cellular pro-survival Bcl-2 proteins and employs the canonical ligand binding groove to bind Bad BH3 peptide. NRZ engagement of Bad BH3 involves the canonical ionic interaction between NRZ R86 and Bad D104 and an additional ionic interaction between NRZ D79 and Bad R100, and substitution of either NRZ R86 or D79 to Ala reduces the binding to Bad BH3 tenfold or more. Our findings provide a detailed mechanistic understanding for NRZ mediated anti-apoptotic activity in zebrafish by revealing binding to both Bad and Noxa, suggesting that NRZ is likely to occupy a unique mechanistic role in zebrafish apoptosis regulation by acting as a highly promiscuous pro-apoptotic Bcl-2 binder.

Fig. 1. Titration curves showing the raw heats of titration for ITC measurements of NRZ: BH3 motif interactions.

NRZ interacts with Bax as well as all other BH3-only proteins but not Bmf or Beclin-1. Affinities are summarized in Table 1

Fig. 2. Crystal structures of NRZ, NRZ: Bad BH3 complex and comparison with closely related complexes.

Cartoon representation of (a) apo NRZ (light blue). Helices are labelled as α1–8, with the view being into the hydrophobic groove formed by α2–5. b Cartoon representation of Mcl-1 (olive). c CNP058 (orange), the closest structural homolog from viral pro-survival Bcl-2 proteins for NRZ. d NRZ (green) in complex with zBad BH3 motif (hot pink). e Bcl-x_L (blue) in complex with Bad BH3 motif (yellow), f Mcl-1 (brown) in complex with Bim (magenta) BH3 motif. All views in b–f are as in a

Fig. 3. Superimposition of apo NRZ with NRZ: zBad BH3 complex.

Comparison of the backbone structures of NRZ and NRZ: zBad BH3 complex. a Cartoon representation of apo NRZ (light blue) superimposed onto NRZ: zBad BH3 complex (green). The view is into the canonical hydrophobic binding groove formed by α2–5. b Close up view of NRZ helix α4, which is shifted by 3 Å from its original position in the apo NRZ structure upon zBad BH3 motif binding, thus enlarging the binding groove

Fig. 4. Binding of BH3 motif peptides to NRZ result in local reorganization of the canonical NRZ binding groove.

 a Detailed view of the NRZ: zBad BH3 motif interface. The NRZ surface, backbone and floor of the binding groove are shown in grey, green and orange, respectively, while zBad BH3 is shown in hot pink. The four key hydrophobic residues of zBad (Y95, L99, M102, F106) protruding into the binding groove, and the conserved salt-bridge formed by NRZ D79 and zBad BH3 R100 are labelled, as well as all other residues involved in additional ionic interactions and hydrogen bonds. Interactions are denoted as dashed black lines. b Surface view of the conserved residues that are involved in NRZ-zBad BH3 interactions. Residues shown in blue are residues within canonical binding groove of NRZ (K49, E56, E75, L76, D79 and R86) that are conserved in NRZ-like proteins amongst different fish species

Fig. 5. Sequence alignment of NRZ with Bcl-2 homologs from other organisms.

The sequence alignment of Bcl-2 family proteins was generated with MUSCLE using sequences from zebrafish NRZ (Uniprot Accession number Q8UWD5), human Bcl-B (Q9HD36), mouse Boo (Q9Z0F3), chicken NR-13 (Q90ZN1) and herpes virus vNR-13 (Q9DH00). The α-helical secondary structure elements (α1–8) are marked as grey helices and loop regions are indicated as grey lines based on the crystal structure of NRZ. The boxed regions of the sequences are denoting the Bcl-2 homology motifs (BH motifs 1–4) and trans-membrane domains (TM) at the end of the sequences. Conserved identical residues between sequences are denoted as “*”, similar residues are denoted as “:” and semi conserved residues denoted as “.”

Fig. 6. Comparison of the BH3 motif binding profile of NRZ with Mcl-1, Bcl-x_L and CNP058.

a Binding profile of zebrafish NRZ with BH3 motif peptides (b) binding profile of human Mcl-1 (c) binding profile of human Bcl-x_L and (d) binding profile of canarypox virus CNP058. BH3 motif peptides used in (b) and (c) are of human origin, whereas peptides in (d) are from chicken. Bars indicate binding affinity ranges from <50 nM, <500 nM, <1000 nM, and >1000 nM as shown in inset