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. 2017 Jul 28;12(7):e0181551.
doi: 10.1371/journal.pone.0181551. eCollection 2017.

Solution NMR structure of the TRIM21 B-box2 and identification of residues involved in its interaction with the RING domain

Amélie Wallenhammar  1 , Madhanagopal Anandapadamanaban  1 , Alexander Lemak  2 , Claudio Mirabello  3 , Patrik Lundström  1 , Björn Wallner  3 , Maria Sunnerhagen  1
Affiliations

Solution NMR structure of the TRIM21 B-box2 and identification of residues involved in its interaction with the RING domain

Amélie Wallenhammar et al. PLoS One. .

Abstract

Tripartite motif-containing (TRIM) proteins are defined by the sequential arrangement of RING, B-box and coiled-coil domains (RBCC), where the B-box domain is a unique feature of the TRIM protein family. TRIM21 is an E3 ubiquitin-protein ligase implicated in innate immune signaling by acting as an autoantigen and by modifying interferon regulatory factors. Here we report the three-dimensional solution structure of the TRIM21 B-box2 domain by nuclear magnetic resonance (NMR) spectroscopy. The structure of the B-box2 domain, comprising TRIM21 residues 86-130, consists of a short α-helical segment with an N-terminal short β-strand and two anti-parallel β-strands jointly found the core, and adopts a RING-like fold. This ββαβ core largely defines the overall fold of the TRIM21 B-box2 and the coordination of one Zn2+ ion stabilizes the tertiary structure of the protein. Using NMR titration experiments, we have identified an exposed interaction surface, a novel interaction patch where the B-box2 is likely to bind the N-terminal RING domain. Our structure together with comparisons with other TRIM B-box domains jointly reveal how its different surfaces are employed for various modular interactions, and provides extended understanding of how this domain relates to flanking domains in TRIM proteins.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structural ensemble and secondary structure of TRIM21 B-box2.
(A) The backbone trace of the structural ensembles of the 20 best-fit NMR structures of no Zn2+ (grey), 1 Zn2+ (blue) and 2 Zn2+ (dark grey). The ensemble is aligned based on the ordered residues as reported by PSVS (Zn0: residues 91–117, 125–128; Zn1: residues 91–117, 125–128; Zn2: residues 90–118; 122–128). (B) Stereo view of the tertiary structure of TRIM21 Bbox2. Zinc atoms are shown as grey spheres and zinc-coordinating residues in Zn2+ site1 are highlighted as sticks. (C) The amino acid sequence of TRIM21 B-box286-130. Zinc-coordinating residues in Zn2+ site1 are highlighted in bold face. (D) 15N-HSQC spectrum of the TRIM21 B-box2, displaying a well-folded domain. (E) Identification of secondary structure elements by the Chemical Shift Index (CSI) per residue. (F) Secondary structure elements assigned by DSSP.
Fig 2
Fig 2. Relaxation data for TRIM21 B-box2.
(A) R1 relaxation rate constants, (B) R2 relaxation rate constants and (C) the heteronuclear NOE. Residues involved in Zn2+ coordination are highlighted in blue (site I) and red (site II). In site I, one zinc atom is coordinated by three cysteines and one histidine. In site II, one cysteine, one aspartic acid and two histidines would coordinate a second zinc atom.
Fig 3
Fig 3. Structural analysis of TRIM family type2 B-boxes.
(A) Sequence alignments of TRIM family showing type2 B-boxes region. Residues with a fractional surface accessibility side chain score <0.15 in the TRIM21 B-box2 sequence are marked with a red asterix. The zinc coordinating residues in site I in TRIM21 B-box2 are marked with a dark blue asterix. Conserved core residues are highlighted in blue. (B) HMM logo of the conservation of residues among TRIM type2 B-boxes. (C) Frequency of the conserved ββα core fold among TRIM B-box2 structures. (D) Structure alignment of the core residues of TRIM type2 B-boxes. (E) Highlight of conserved hydrophobic core residues (green) and the zinc coordinating residues in site I (dark blue) on the TRIM21 B-box2 structure. (F) Cartoon representation of TRIM21 B-box2 and the conserved core residues (blue).
Fig 4
Fig 4. TRIM21 RING1-91 titration of TRIM21 B-box2.
(A) Normalized chemical shift perturbations (CSP) upon addition of unlabeled TRIM21 RING1-91 to 15N-labeled TRIM21 B-box2. The CSP cut-off was set to 0.10. Residues are colored as blue (cluster1; V94, H95, G96) and red (cluster2; W112 side-chain, V113, C114, Q116, S117). (B) Mapping of residues on the B-box2 structure affected by the addition of unlabeled TRIM21 RING1-91. (C) Highlight of spectral perturbations of N-H shifts of the affected residues V94, H95, G96, W112 side-chain, V113, C114, Q116 and S117.
Fig 5
Fig 5. Evidence for a novel RING interaction patch.
(A) Superposition of TRIM5α BCC (PDB ID: 4TN3) chain A (cyan), chain B (grey) and TRIM21 B-box2 (dark grey). Residues in TRIM21 B-box2 affected by TRIM21 RING1-91 are highlighted in blue (V94, H95, G96; cluster 1) and red (V113, C114, Q116, S117; cluster 2) in this entire figure. (B) Structural alignment of TRIM18/MID1 tandem Bbox1-Bbox2 (grey; PDB ID: 2DQ5) and TRIM21 B-box2 (dark grey). Residues involved in TRIM18 Bbox1-2 tandem assembly do not interfere with a tentative RING interaction site as predicted from TRIM21. (C) Structural alignment of TRIM63 B-box2 dimer (ice blue; PDB ID: 3DDT), TRIM5α B-box2 dimer (grey; PDB ID: 5K3Q) and TRIM21 B-box2 (dark grey). In TRIM63, S143 in B-box1 and B-box2 (pink) stack together to form the dimer interface. In TRIM21, there is a tryptophan (green) in this position (W112). The bulky side-chain structure of W112 makes it unlikely that TRIM21 B-box forms a dimer due to steric clashes. In TRIM5α, the dimer is hold together by a salt bridge between R121 and E120. TRIM21 poses an alanine and a glutamine in these positions. The tryptophan W116 is conserved in TRIM21 (W112).
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This project was supported by the Swedish Science Council (BW, MS), the Swedish Cancer Foundation (MS) and the Swedish Foundation for International Cooperation in Research and Higher Education (BW, MS).